Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option.

PubMed

Stamm, Hauke; Klingler, Felix; Grossjohann, Eva-Maria; Muschhammer, Jana; Vettorazzi, Eik; Heuser, Michael; Mock, Ulrike; Thol, Felicitas; Vohwinkel, Gabi; Latuske, Emily; Bokemeyer, Carsten; Kischel, Roman; Dos Santos, Cedric; Stienen, Sabine; Friedrich, Matthias; Lutteropp, Michael; Nagorsen, Dirk; Wellbrock, Jasmin; Fiedler, Walter

2018-05-31

Immune checkpoints are promising targets in cancer therapy. Recently, poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2) have been identified as novel immune checkpoints. In this investigation we show that acute myeloid leukemia (AML) cell lines and AML patient samples highly express the T-cell immunoreceptor with Ig and ITIM domains (TIGIT) ligands PVR and PVRL2. Using two independent patient cohorts, we could demonstrate that high PVR and PVRL2 expression correlates with poor outcome in AML. We show for the first time that antibody blockade of PVR or PVRL2 on AML cell lines or primary AML cells or TIGIT blockade on immune cells increases the anti-leukemic effects mediated by PBMCs or purified CD3 + cells in vitro. The cytolytic activity of the BiTE® antibody construct AMG 330 against leukemic cells could be further enhanced by blockade of the TIGIT-PVR/PVRL2 axis. This increased immune reactivity is paralleled by augmented secretion of Granzyme B by immune cells. Employing CRISPR/Cas9-mediated knockout of PVR and PVRL2 in MV4-11 cells, the cytotoxic effects of antibody blockade could be recapitulated in vitro. In NSG mice reconstituted with human T cells and transplanted with either MV4-11 PVR/PVRL2 knockout or wildtype cells, prolonged survival was observed for the knockout cells. This survival benefit could be further extended by treating the mice with AMG 330. Therefore, targeting the TIGIT-PVR/PVRL2 axis with blocking antibodies might represent a promising future therapeutic option in AML.

Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein

PubMed Central

Fiedler, Nicola; Quant, Ellen; Fink, Ludger; Sun, Jianguang; Schuster, Ralph; Gerlich, Wolfram H; Schaefer, Stephan

2006-01-01

AIM: Hepatitis B virus protein X (HBx) has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type. METHODS: We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation. RESULTS: The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21waf/cip/sdi which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2. In AML-HBx9 cells, p21waf/cip/sdi-protein expression and p21waf/cip/sdi transcription were deregulated. Furthermore, the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation. Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines. CONCLUSION: Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis. PMID:16937438

Combination screening in vitro identifies synergistically acting KP372-1 and cytarabine against acute myeloid leukemia.

PubMed

Österroos, A; Kashif, M; Haglund, C; Blom, K; Höglund, M; Andersson, C; Gustafsson, M G; Eriksson, A; Larsson, R

2016-10-15

Cytogenetic lesions often alter kinase signaling in acute myeloid leukemia (AML) and the addition of kinase inhibitors to the treatment arsenal is of interest. We have screened a kinase inhibitor library and performed combination testing to find promising drug-combinations for synergistic killing of AML cells. Cytotoxicity of 160 compounds in the library InhibitorSelect™ 384-Well Protein Kinase Inhibitor I was measured using the fluorometric microculture cytotoxicity assay (FMCA) in three AML cell lines. The 15 most potent substances were evaluated for dose-response. The 6 most cytotoxic compounds underwent combination synergy analysis based on the FMCA readouts after either simultaneous or sequential drug addition in AML cell lines. The 4 combinations showing the highest level of synergy were evaluated in 5 primary AML samples. Synergistic calculations were performed using the combination interaction analysis package COMBIA, written in R, using the Bliss independence model. Based on obtained results, an iterative combination search was performed using the therapeutic algorithmic combinatorial screen (TACS) algorithm. Of 160 substances, cell survival was ⩽50% at <0.5μM for Cdk/Crk inhibitor, KP372-1, synthetic fascaplysin, herbimycin A, PDGF receptor tyrosine kinase inhibitor IV and reference-drug cytarabine. KP372-1, synthetic fascaplysin or herbimycin A obtained synergy when combined with cytarabine in AML cell lines MV4-11 and HL-60. KP372-1 added 24h before cytarabine gave similar results in patient cells. The iterative search gave further improved synergy between cytarabine and KP372-1. In conclusion, our in vitro studies suggest that combining KP372-1 and cytarabine is a potent and synergistic drug combination in AML. Copyright © 2016 Elsevier Inc. All rights reserved.

AML1/ETO induces self-renewal in hematopoietic progenitor cells via the Groucho-related amino-terminal AES protein.

PubMed

Steffen, Björn; Knop, Markus; Bergholz, Ulla; Vakhrusheva, Olesya; Rode, Miriam; Köhler, Gabriele; Henrichs, Marcel-Philipp; Bulk, Etmar; Hehn, Sina; Stehling, Martin; Dugas, Martin; Bäumer, Nicole; Tschanter, Petra; Brandts, Christian; Koschmieder, Steffen; Berdel, Wolfgang E; Serve, Hubert; Stocking, Carol; Müller-Tidow, Carsten

2011-04-21

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia.

PubMed

Lee, Chin-Cheng; Lin, Che-Pin; Lee, Yueh-Lun; Wang, Giueng-Chueng; Cheng, Yuan-Chih; Liu, H Eugene

2010-05-01

Meisoindigo, a derivative of Indigo naturalis, has been used in China for chronic myeloid leukemia. In vitro cell line studies have shown that this agent might induce apoptosis and myeloid differentiation of acute myeloid leukemia (AML). In this study, we explored its mechanisms and potential in AML. NB4, HL-60, and U937 cells and primary AML cells were used to examine its effects and the NOD/SCID animal model was used to evaluate its in vivo activity. Meisoindigo inhibited the growth of leukemic cells by inducing marked apoptosis and moderate cell-cycle arrest at the G(0)/G(1) phase. It down-regulated anti-apoptotic Bcl-2, and up-regulated pro-apoptotic Bak and Bax and cell-cycle related proteins, p21and p27. Furthermore, it induced myeloid differentiation, as demonstrated by morphologic changes, up-regulation of CD11b, and increased nitroblue tetrazolium reduction activity in all cell lines tested. In addition, meisoindigo down-regulated the expression of human telomerase reverse transcriptase and enhanced the cytotoxicity of conventional chemotherapeutic agents, cytarabine and idarubicin. As with the results from cell lines, meisoindigo also induced apoptosis, up-regulated p21 and p27, and down-regulated Bcl-2 in primary AML cells. The in vivo anti-leukemic activity of meisoindigo was also demonstrated by decreased spleen size in a dose-dependent manner. Taking these results together, meisoindigo is a potential agent for AML.

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody–Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML

PubMed Central

Pereira, Daniel S.; Guevara, Claudia I.; Jin, Liqing; Mbong, Nathan; Verlinsky, Alla; Hsu, Ssucheng J.; Aviña, Hector; Karki, Sher; Abad, Joseph D.; Yang, Peng; Moon, Sung-Ju; Malik, Faisal; Choi, Michael Y.; An, Zili; Morrison, Kendall; Challita-Eid, Pia M.; Doñate, Fernando; Joseph, Ingrid B.J.; Kipps, Thomas J.; Dick, John E.; Stover, David R.

2015-01-01

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody–drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent mono-methyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34+CD38− leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML. PMID:25934707

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells.

PubMed

Cardona, Maria E; Simonson, Oscar E; Oprea, Iulian I; Moreno, Pedro M D; Silva-Lara, Maria F; Mohamed, Abdalla J; Christensson, Birger; Gahrton, Gösta; Dilber, M Sirac; Smith, C I Edvard; Arteaga, H Jose

2016-01-01

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

Somatic mutations in the transcriptional corepressor gene BCORL1 in adult acute myelogenous leukemia.

PubMed

Li, Meng; Collins, Roxane; Jiao, Yuchen; Ouillette, Peter; Bixby, Dale; Erba, Harry; Vogelstein, Bert; Kinzler, Kenneth W; Papadopoulos, Nickolas; Malek, Sami N

2011-11-24

To further our understanding of the genetic basis of acute myelogenous leukemia (AML), we determined the coding exon sequences of ∼ 18 000 protein-encoding genes in 8 patients with secondary AML. Here we report the discovery of novel somatic mutations in the transcriptional corepressor gene BCORL1 that is located on the X-chromosome. Analysis of BCORL1 in an unselected cohort of 173 AML patients identified a total of 10 mutated cases (6%) with BCORL1 mutations, whereas analysis of 19 AML cell lines uncovered 4 (21%) BCORL1 mutated cell lines. The majority (87%) of the mutations in BCORL1 were predicted to inactivate the gene product as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. These results indicate that BCORL1 by genetic criteria is a novel candidate tumor suppressor gene, joining the growing list of genes recurrently mutated in AML.

Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia

PubMed Central

Folkerts, Hendrik; Hilgendorf, Susan; Wierenga, Albertus T J; Jaques, Jennifer; Mulder, André B; Coffer, Paul J; Schuringa, Jan Jacob; Vellenga, Edo

2017-01-01

Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34+ AML cells with a large variability in basal autophagy between AMLs observed. The autophagy flux was higher in AMLs classified as poor risk, which are frequently associated with TP53 mutations (TP53mut), compared with favorable- and intermediate-risk AMLs. In addition, the higher flux was associated with a higher expression level of several autophagy genes, but was not affected by alterations in p53 expression by knocking down p53 or overexpression of wild-type p53 or p53R273H. AML CD34+ cells were more sensitive to the autophagy inhibitor hydroxychloroquine (HCQ) than normal bone marrow CD34+ cells. Similar, inhibition of autophagy by knockdown of ATG5 or ATG7 triggered apoptosis, which coincided with increased expression of p53. In contrast to wild-type p53 AML (TP53wt), HCQ treatment did not trigger a BAX and PUMA-dependent apoptotic response in AMLs harboring TP53mut. To further characterize autophagy in the leukemic stem cell-enriched cell fraction AML CD34+ cells were separated into ROSlow and ROShigh subfractions. The immature AML CD34+-enriched ROSlow cells maintained higher basal autophagy and showed reduced survival upon HCQ treatment compared with ROShigh cells. Finally, knockdown of ATG5 inhibits in vivo maintenance of AML CD34+ cells in NSG mice. These results indicate that targeting autophagy might provide new therapeutic options for treatment of AML since it affects the immature AML subfraction. PMID:28703806

Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and novel therapeutic target in acute myeloid leukemia

PubMed Central

Porter, Christopher C.; Kim, Jihye; Fosmire, Susan; Gearheart, Christy M.; van Linden, Annemie; Baturin, Dmitry; Zaberezhnyy, Vadym; Patel, Purvi R.; Gao, Dexiang; Tan, Aik Choon; DeGregori, James

2011-01-01

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide shRNA screens to identify proteins that mediate AML cell fate after cytarabine exposure, gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context, and examination of existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. PMID:22289989

AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells

PubMed Central

Saia, Marco; Termanini, Alberto; Rizzi, Nicoletta; Mazza, Massimiliano; Barbieri, Elisa; Valli, Debora; Ciana, Paolo; Gruszka, Alicja M.; Alcalay, Myriam

2016-01-01

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression. PMID:27713544

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress

PubMed Central

Sriskanthadevan, Shrivani; Jeyaraju, Danny V.; Chung, Timothy E.; Prabha, Swayam; Xu, Wei; Skrtic, Marko; Jhas, Bozhena; Hurren, Rose; Gronda, Marcela; Wang, Xiaoming; Jitkova, Yulia; Sukhai, Mahadeo A.; Lin, Feng-Hsu; Maclean, Neil; Laister, Rob; Goard, Carolyn A.; Mullen, Peter J.; Xie, Stephanie; Penn, Linda Z.; Rogers, Ian M.; Dick, John E.; Minden, Mark D.

2015-01-01

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy. PMID:25631767

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo.

PubMed

Schnerch, Dominik; Schüler, Julia; Follo, Marie; Felthaus, Julia; Wider, Dagmar; Klingner, Kathrin; Greil, Christine; Duyster, Justus; Engelhardt, Monika; Wäsch, Ralph

2017-03-28

Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv(16) subtype.

PubMed

Estécio, Marcos R H; Maddipoti, Sirisha; Bueso-Ramos, Carlos; DiNardo, Courtney D; Yang, Hui; Wei, Yue; Kondo, Kimie; Fang, Zhihong; Stevenson, William; Chang, Kun-Sang; Pierce, Sherry A; Bohannan, Zachary; Borthakur, Gautam; Kantarjian, Hagop; Garcia-Manero, Guillermo

2015-05-01

Correlative and functional studies support the involvement of the RUNX gene family in haematological malignancies. To elucidate the role of epigenetics in RUNX inactivation, we evaluated promoter DNA methylation of RUNX1, 2, and 3 in 23 leukaemia cell lines and samples from acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL) and myelodysplatic syndromes (MDS) patients. RUNX1 and RUNX2 gene promoters were mostly unmethylated in cell lines and clinical samples. Hypermethylation of RUNX3 was frequent among cell lines (74%) and highly variable among patient samples, with clear association to cytogenetic status. High frequency of RUNX3 hypermethylation (85% of the 20 studied cases) was found in AML patients with inv(16)(p13.1q22) compared to other AML subtypes (31% of the other 49 cases). RUNX3 hypermethylation was also frequent in ALL (100% of the six cases) but low in MDS (21%). In support of a functional role, hypermethylation of RUNX3 was correlated with low levels of protein, and treatment of cell lines with the DNA demethylating agent, decitabine, resulted in mRNA re-expression. Furthermore, relapse-free survival of non-inv(16)(p13.1q22) AML patients without RUNX3 methylation was significantly better (P = 0·016) than that of methylated cases. These results suggest that RUNX3 silencing is an important event in inv(16)(p13.1q22) leukaemias. © 2015 John Wiley & Sons Ltd.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

PubMed

Liyanage, Sanduni U; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D; Bader, Gary D; Laposa, Rebecca; Schimmer, Aaron D

2017-05-11

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. © 2017 by The American Society of Hematology.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML

PubMed Central

Liyanage, Sanduni U.; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P.; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D.; Bader, Gary D.; Laposa, Rebecca

2017-01-01

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2′3′-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2′3′-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. PMID:28283480

Targeting chemotherapy-resistant leukemia by combining DNT cellular therapy with conventional chemotherapy.

PubMed

Chen, Branson; Lee, Jong Bok; Kang, Hyeonjeong; Minden, Mark D; Zhang, Li

2018-04-24

While conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease. Drug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity. Here, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity. Our results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.

SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Li; Weng, Wei; Sun, Zhi-Xin

Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, andmore » concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent. - Highlights: • SKI-II inhibits proliferation and survival of primary and transformed AML cells. • SKI-II induces apoptotic death of AML cells, but is safe to normal PBMCs. • SKI-II is more efficient than two known SphK1 inhibitors in inhibiting AML cells. • SKI-II inhibits SphK1 activity, while increasing ceramide production in AML cells. • SKI-II dose-dependently inhibits U937 xenograft growth in SCID mice.« less

Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2.

PubMed

Karathedath, Sreeja; Rajamani, Bharathi M; Musheer Aalam, Syed Mohammed; Abraham, Ajay; Varatharajan, Savitha; Krishnamurthy, Partha; Mathews, Vikram; Velayudhan, Shaji Ramachandran; Balasubramanian, Poonkuzhali

2017-01-01

Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.

Somatic mutations in the transcriptional corepressor gene BCORL1 in adult acute myelogenous leukemia

PubMed Central

Li, Meng; Collins, Roxane; Jiao, Yuchen; Ouillette, Peter; Bixby, Dale; Erba, Harry; Vogelstein, Bert; Kinzler, Kenneth W.

2011-01-01

To further our understanding of the genetic basis of acute myelogenous leukemia (AML), we determined the coding exon sequences of ∼ 18 000 protein-encoding genes in 8 patients with secondary AML. Here we report the discovery of novel somatic mutations in the transcriptional corepressor gene BCORL1 that is located on the X-chromosome. Analysis of BCORL1 in an unselected cohort of 173 AML patients identified a total of 10 mutated cases (6%) with BCORL1 mutations, whereas analysis of 19 AML cell lines uncovered 4 (21%) BCORL1 mutated cell lines. The majority (87%) of the mutations in BCORL1 were predicted to inactivate the gene product as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. These results indicate that BCORL1 by genetic criteria is a novel candidate tumor suppressor gene, joining the growing list of genes recurrently mutated in AML. PMID:21989985

Effects of MERTK Inhibitors UNC569 and UNC1062 on the Growth of Acute Myeloid Leukaemia Cells.

PubMed

Koda, Yuki; Itoh, Mai; Tohda, Shuji

2018-01-01

MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase that affects cancer cell proliferation. This study evaluated the effects of the synthetic MERTK inhibitors UNC569 and UNC1062 on in vitro growth of acute myeloid leukaemia (AML) cells. Four AML cell lines expressing MERTK were treated with UNC569 and UNC1062 and analyzed for cell proliferation, immunoblotting, and gene expression. The effects of MERTK knockdown were also evaluated. Treatment with the inhibitors suppressed cell growth and induced apoptosis in all cell lines. OCI/AML5 and TMD7 cells, in which MERTK was constitutively phosphorylated by autocrine mechanisms, were highly susceptible to these inhibitors. The treatment reduced the phosphorylation of MERTK and its down-stream signalling molecules, v-akt murine thymoma viral oncogene homolog 1 (AKT) and extracellular signal-regulated kinase (ERK). Similar effects were observed after MERTK knockdown. The inhibitors and the knockdown caused similar changes in mRNA expression. These MERTK inhibitors are potential molecular-targeted drugs for treating AML expressing constitutively phosphorylated MERTK. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.

PubMed

Pillinger, Genevra; Abdul-Aziz, Amina; Zaitseva, Lyubov; Lawes, Matthew; MacEwan, David J; Bowles, Kristian M; Rushworth, Stuart A

2015-08-21

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

Tunneling nanotube (TNT) formation is downregulated by cytarabine and NF-κB inhibition in acute myeloid leukemia (AML)

PubMed Central

Omsland, Maria; Bruserud, Øystein; Gjertsen, Bjørn T; Andresen, Vibeke

2017-01-01

Acute myeloid leukemia (AML) is a bone marrow derived blood cancer where intercellular communication in the leukemic bone marrow participates in disease development, progression and chemoresistance. Tunneling nanotubes (TNTs) are intercellular communication structures involved in transport of cellular contents and pathogens, also demonstrated to play a role in both cell death modulation and chemoresistance. Here we investigated the presence of TNTs by live fluorescent microscopy and identified TNT formation between primary AML cells and in AML cell lines. We found that NF-κB activity was involved in TNT regulation and formation. Cytarabine downregulated TNTs and inhibited NF-κB alone and in combination with daunorubicin, providing additional support for involvement of the NF-κB pathway in TNT formation. Interestingly, daunorubicin was found to localize to lysosomes in TNTs connecting AML cells indicating a novel function of TNTs as drug transporting devices. We conclude that TNT communication could reflect important biological features of AML that may be explored in future therapy development. PMID:27974700

The TPO/c-MPL pathway in the bone marrow may protect leukemia cells from chemotherapy in AML Patients.

PubMed

Dong-Feng, Zeng; Ting, Liu; Yong, Zhang; Cheng, Chang; Xi, Zhang; Pei-Yan, Kong

2014-04-01

Accumulating evidence indicates that the interaction of human LSCs (leukemic stem cells) with the hematopoietic microenvironment, mediated by the thrombopoietin (TPO)/c-MPL pathway, may be an underlying mechanism for resistance to cell cycle-dependent cytotoxic chemotherapy. However, the role of TPO/c-MPL signaling in AML (acute myelogenous leukemia) chemotherapy resistance hasn't been fully understood. The c-MPL and TPO levels in different AML samples were measured by flow cytometry and ELISA. We also assessed the TPO levels in the osteoblasts derived from bone mesenchymal stem cells (BMSCs). The survival rate of an AML cell line that had been co-cultured with different BMSC-derived osteoblasts was measured to determine the IC50 of an AML chemotherapy drug daunorubicin (DNR). The levels of TPO/c-MPL in the initial and relapse AML patients were significantly higher than that in the control (P < 0.05). The osteoblasts derived from AML patients' BMSCs secreted more TPO than the osteoblasts derived from normal control BMSCs (P < 0.05). A strong positive correlation between the TPO level and c-MPL expression was found in the bone marrow mononuclear cells of the relapse AML patients. More importantly, the IC50 of DNR in the HEL + AML-derived osteoblasts was the highest among all co-culture systems. High level of TPO/c-MPL signaling may protect LSCs from chemotherapy in AML. The effects of inhibition of the TPO/c-MPL pathway on enhancing the chemotherapy sensitivity of AML cells, and on their downstream effector molecules that direct the interactions between patient-derived blasts and leukemia repopulating cells need to be further studied.

Correlation between p65 and TNF-α in patients with acute myelocytic leukemia.

PubMed

Dong, Qiao-Mei; Ling, Chun; Zhu, Jun-Fang; Chen, Xuan; Tang, Yan; Zhao, L I

2015-11-01

The correlation between the expression levels of p65 and TNF-α in patients with acute myelocytic leukemia (AML) and AML cell lines were investigated. The bone marrow samples of 30 AML patients and 10 non-leukemia controls were studied. The mRNA expression levels of p65 and TNF-α were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Pearson's Correlation test was used to demonstrate the correlation between TNF-α and p65 expression levels in AML specimens. Receiver operating characteristic (ROC) curves were plotted to determine whether TNF-α and p65 expression levels could be used to differentiate AML samples from non-leukemia samples. MG132 and anti-TNF-α antibody were used to inhibit the expression of p65 and TNF-α in the AML cell line, HL-60. The expression of p65 and TNF-α were detected by RT-qPCR and western blot analysis. The mRNA expression levels of p65 and TNF-α were significantly increased in AML patients compared with non-leukemia control bone marrow samples by RT-qPCR, and the two molecules expression pattern's exhibited sufficient predictive power to distinguish AML patients from non-leukemia control samples. Pearson's correlation analysis demonstrated that TNF-α expression was strongly correlated with p65 expression in AML bone marrow samples. In HL-60 cells, inhibition of TNF-α reduced the expression of p65; in addition, inhibition of p65 reduced the expression of TNF-α as assessed by RT-qPCR and western blot analysis. p65 and TNF-α were highly expressed in AML patients, and these 2 molecules were strongly correlated. The present study indicates that p65 and TNF-α have potential as molecular markers to distinguish AML patients from non-leukemia control samples, and that these 2 molecules may be useful prognostic factor for patients with AML.

Enhanced sensitivity to glucocorticoids in cytarabine-resistant AML.

PubMed

Malani, D; Murumägi, A; Yadav, B; Kontro, M; Eldfors, S; Kumar, A; Karjalainen, R; Majumder, M M; Ojamies, P; Pemovska, T; Wennerberg, K; Heckman, C; Porkka, K; Wolf, M; Aittokallio, T; Kallioniemi, O

2017-05-01

We sought to identify drugs that could counteract cytarabine resistance in acute myeloid leukemia (AML) by generating eight resistant variants from MOLM-13 and SHI-1 AML cell lines by long-term drug treatment. These cells were compared with 66 ex vivo chemorefractory samples from cytarabine-treated AML patients. The models and patient cells were subjected to genomic and transcriptomic profiling and high-throughput testing with 250 emerging and clinical oncology compounds. Genomic profiling uncovered deletion of the deoxycytidine kinase (DCK) gene in both MOLM-13- and SHI-1-derived cytarabine-resistant variants and in an AML patient sample. Cytarabine-resistant SHI-1 variants and a subset of chemorefractory AML patient samples showed increased sensitivity to glucocorticoids that are often used in treatment of lymphoid leukemia but not AML. Paired samples taken from AML patients before treatment and at relapse also showed acquisition of glucocorticoid sensitivity. Enhanced glucocorticoid sensitivity was only seen in AML patient samples that were negative for the FLT3 mutation (P=0.0006). Our study shows that development of cytarabine resistance is associated with increased sensitivity to glucocorticoids in a subset of AML, suggesting a new therapeutic strategy that should be explored in a clinical trial of chemorefractory AML patients carrying wild-type FLT3.

Enhanced sensitivity to glucocorticoids in cytarabine-resistant AML

PubMed Central

Malani, D; Murumägi, A; Yadav, B; Kontro, M; Eldfors, S; Kumar, A; Karjalainen, R; Majumder, M M; Ojamies, P; Pemovska, T; Wennerberg, K; Heckman, C; Porkka, K; Wolf, M; Aittokallio, T; Kallioniemi, O

2017-01-01

We sought to identify drugs that could counteract cytarabine resistance in acute myeloid leukemia (AML) by generating eight resistant variants from MOLM-13 and SHI-1 AML cell lines by long-term drug treatment. These cells were compared with 66 ex vivo chemorefractory samples from cytarabine-treated AML patients. The models and patient cells were subjected to genomic and transcriptomic profiling and high-throughput testing with 250 emerging and clinical oncology compounds. Genomic profiling uncovered deletion of the deoxycytidine kinase (DCK) gene in both MOLM-13- and SHI-1-derived cytarabine-resistant variants and in an AML patient sample. Cytarabine-resistant SHI-1 variants and a subset of chemorefractory AML patient samples showed increased sensitivity to glucocorticoids that are often used in treatment of lymphoid leukemia but not AML. Paired samples taken from AML patients before treatment and at relapse also showed acquisition of glucocorticoid sensitivity. Enhanced glucocorticoid sensitivity was only seen in AML patient samples that were negative for the FLT3 mutation (P=0.0006). Our study shows that development of cytarabine resistance is associated with increased sensitivity to glucocorticoids in a subset of AML, suggesting a new therapeutic strategy that should be explored in a clinical trial of chemorefractory AML patients carrying wild-type FLT3. PMID:27833094

Niclosamide suppresses acute myeloid leukemia cell proliferation through inhibition of CREB-dependent signaling pathways

PubMed Central

Chae, Hee-Don; Cox, Nick; Dahl, Gary V.; Lacayo, Norman J.; Davis, Kara L.; Capolicchio, Samanta; Smith, Mark; Sakamoto, Kathleen M.

2018-01-01

CREB (cAMP Response Element Binding protein) is a transcription factor that is overexpressed in primary acute myeloid leukemia (AML) cells and associated with a decreased event-free survival and increased risk of relapse. We recently reported a small molecule inhibitor of CREB, XX-650-23, which inhibits CREB activity in AML cells. Structure-activity relationship analysis for chemical compounds with structures similar to XX-650-23 led to the identification of the anthelminthic drug niclosamide as a potent anti-leukemic agent that suppresses cell viability of AML cell lines and primary AML cells without a significant decrease in colony forming activity of normal bone marrow cells. Niclosamide significantly inhibited CREB function and CREB-mediated gene expression in cells, leading to apoptosis and G1/S cell cycle arrest with reduced phosphorylated CREB levels. CREB knockdown protected cells from niclosamide treatment-mediated cytotoxic effects. Furthermore, treatment with a combination of niclosamide and CREB inhibitor XX-650-23 showed an additive anti-proliferative effect, consistent with the hypothesis that niclosamide and XX-650-23 regulate the same targets or pathways to inhibit proliferation and survival of AML cells. Niclosamide significantly inhibited the progression of disease in AML patient-derived xenograft (PDX) mice, and prolonged survival of PDX mice. Niclosamide also showed synergistic effects with chemotherapy drugs to inhibit AML cell proliferation. While chemotherapy antagonized the cytotoxic potential of niclosamide, pretreatment with niclosamide sensitized cells to chemotherapeutic drugs, cytarabine, daunorubicin, and vincristine. Therefore, our results demonstrate niclosamide as a potential drug to treat AML by inducing apoptosis and cell cycle arrest through inhibition of CREB-dependent pathways in AML cells. PMID:29435104

Acute myeloid leukemia-targeted toxin activates both apoptotic and necroptotic death mechanisms.

PubMed

Horita, Henrick; Frankel, Arthur E; Thorburn, Andrew

2008-01-01

Acute myelogenous leukemia (AML) is the second most common leukemia with approximately 13,410 new cases and 8,990 deaths annually in the United States. A novel fusion toxin treatment, diphtheria toxin GM-CSF (DT-GMCSF) has been shown to selectively eliminate leukemic repopulating cells that are critical for the formation of AML. We previously showed that DT-GMCSF treatment of U937 cells, an AML cell line, causes activation of caspases and the induction of apoptosis. In this study we further investigate the mechanisms of cell death induced by DT-GMCSF and show that, in addition to the activation of caspase-dependent apoptosis, DT-GMCSF also kills AML cells by simultaneously activating caspase-independent necroptosis. These mechanisms depend on the ability of the targeted toxin to inhibit protein synthesis, and are not affected by the receptor that is targeted or the mechanism through which protein synthesis is blocked. We conclude that fusion toxin proteins may be effective for treating AML cells whether or not they are defective in apoptosis.

Reovirus-Mediated Cytotoxicity and Enhancement of Innate Immune Responses Against Acute Myeloid Leukemia

PubMed Central

Hall, Kathryn; Scott, Karen J.; Rose, Ailsa; Desborough, Michael; Harrington, Kevin; Pandha, Hardev; Parrish, Christopher; Vile, Richard; Coffey, Matt; Bowen, David; Errington-Mais, Fiona

2012-01-01

Abstract Reovirus is a naturally occurring oncolytic virus that has shown preclinical efficacy in the treatment of a wide range of tumor types and has now reached phase III testing in clinical trials. The anti-cancer activity of reovirus has been attributed to both its direct oncolytic activity and the enhancement of anti-tumor immune responses. In this study, we have investigated the direct effect of reovirus on acute myeloid leukemia (AML) cells and its potential to enhance innate immune responses against AML, including the testing of primary samples from patients. Reovirus was found to replicate in and kill AML cell lines, and to reduce cell viability in primary AML samples. The pro-inflammatory cytokine interferon alpha (IFNα) and the chemokine (C-C motif) ligand 5 (known as RANTES [regulated upon activation, normal T-cell expressed, and secreted]) were also secreted from AML cells in response to virus treatment. In addition, reovirus-mediated activation of natural killer (NK) cells, within the context of peripheral blood mononuclear cells, stimulated their anti-leukemia response, with increased NK degranulation and IFNγ production and enhanced killing of AML targets. These data suggest that reovirus has the potential as both a direct cytotoxic and an immunotherapeutic agent for the treatment of AML. PMID:23515241

IL-8 as mediator in the microenvironment-leukaemia network in acute myeloid leukaemia.

PubMed

Kuett, Alexander; Rieger, Christina; Perathoner, Deborah; Herold, Tobias; Wagner, Michaela; Sironi, Silvia; Sotlar, Karl; Horny, Hans-Peter; Deniffel, Christian; Drolle, Heidrun; Fiegl, Michael

2015-12-17

The bone marrow microenvironment is physiologically hypoxic with areas being as low as 1% O2, e.g. the stem cell niche. Acute myeloid leukaemia (AML) blasts misuse these bone marrow niches for protection by the local microenvironment, but also might create their own microenvironment. Here we identify IL-8 as a hypoxia-regulated cytokine in both AML cell lines and primary AML samples that is induced within 48 hours of severe hypoxia (1% O2). IL-8 lacked effects on AML cells but induced migration in mesenchymal stromal cells (MSC), an integral part of the bone marrow. Accordingly, MSC were significantly increased in AML bone marrow as compared to healthy bone marrow. Interestingly, mononuclear cells obtained from healthy bone marrow displayed both significantly lower endogenous and hypoxia-induced production of IL-8. IL-8 mRNA expression in AML blasts from 533 patients differed between genetic subgroups with significantly lower expression of IL-8 in acute promyelocytic leukaemia (APL), while in non APL-AML patients with FLT ITD had the highest IL-8 expression. In this subgroup, high IL-8 expression was also prognostically unfavourable. In conclusion, hypoxia as encountered in the bone marrow specifically increases IL-8 expression of AML, which in turn impacts niche formation. High IL-8 expression might be correlated with poor prognosis in certain AML subsets.

CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia.

PubMed

Wang, Jinghua; Chen, Siyu; Xiao, Wei; Li, Wende; Wang, Liang; Yang, Shuo; Wang, Weida; Xu, Liping; Liao, Shuangye; Liu, Wenjian; Wang, Yang; Liu, Nawei; Zhang, Jianeng; Xia, Xiaojun; Kang, Tiebang; Chen, Gong; Cai, Xiuyu; Yang, Han; Zhang, Xing; Lu, Yue; Zhou, Penghui

2018-01-10

Acute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment. We tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells. The CLL-1 CAR-T cells specifically lysed CLL-1 + cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1 + myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression. CLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.

Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia.

PubMed

Alachkar, Houda; Mutonga, Martin B G; Metzeler, Klaus H; Fulton, Noreen; Malnassy, Gregory; Herold, Tobias; Spiekermann, Karsten; Bohlander, Stefan K; Hiddemann, Wolfgang; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2014-12-15

Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.

Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia

PubMed Central

Alachkar, Houda; Mutonga, Martin B.G.; Metzeler, Klaus H.; Fulton, Noreen; Malnassy, Gregory; Herold, Tobias; Spiekermann, Karsten; Bohlander, Stefan K.; Hiddemann, Wolfgang; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2014-01-01

Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients. PMID:25365263

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

PubMed

Kurata, Morito; Rathe, Susan K; Bailey, Natashay J; Aumann, Natalie K; Jones, Justine M; Veldhuijzen, G Willemijn; Moriarity, Branden S; Largaespada, David A

2016-11-03

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Protein Profiling Identifies mTOR Pathway Modulation and Cytostatic Effects of Pim Kinase Inhibitor, AZD1208, in Acute Myeloid Leukemia

PubMed Central

Chen, Lisa S.; Yang, Ji-Yeon; Liang, Han; Cortes, Jorge E.; Gandhi, Varsha

2017-01-01

Pim kinases phosphorylate and regulate a number of key AML cell survival proteins, and Pim inhibitors have recently entered clinical trial for hematological malignancies. AZD1208 is a small molecule pan-Pim kinase inhibitor and AZD1208 treatment resulted in growth inhibition and cell size reduction in AML cell lines including FLT3-WT (OCI-AML-3, KG-1a, MOLM-16) and FLT3-ITD mutated (MOLM-13, MV-4-11). There was limited apoptosis induction (<10% increase) in the AML cell lines evaluated with up to 3 μM AZD1208 for 24h, suggesting that growth inhibition is not through apoptosis induction. Using reverse phase protein array (RPPA) and immunoblot analysis, we identified that AZD1208 resulted in suppression of mTOR signaling, including inhibition of protein phosphorylation of mTOR(Ser2448), p70S6K(Thr389), S6(Ser235/236) and 4E-BP1(Ser65). Consistent with mTOR inhibition, there was also a reduction in protein synthesis that correlated with cell size reduction and growth inhibition with AZD1208; our study provide insights into the mechanism of AZD1208. PMID:27054578

miR-181a promotes G1/S transition and cell proliferation in pediatric acute myeloid leukemia by targeting ATM.

PubMed

Liu, Xiaodan; Liao, Wang; Peng, Hongxia; Luo, Xuequn; Luo, Ziyan; Jiang, Hua; Xu, Ling

2016-01-01

Abnormal expression of miRNAs is intimately related to a variety of human cancers. The purpose of this study is to confirm the expression of miR-181a and elucidate its physiological function and mechanism in pediatric acute myeloid leukemia (AML). Pediatric AML patients and healthy controls were enrolled, and the expression of miR-181a and ataxia telangiectasia mutated (ATM) in tissues were examined using quantitative PCR. Moreover, cell proliferation and cell cycle were evaluated in several cell lines (HL60, NB4 and K562) by using flow cytometry after transfected with miR-181a mimics and inhibitors, or ATM siRNA and control siRNA. Finally, ATM as the potential target protein of miR-181a was examined. We found that miR-181a was significantly increased in pediatric AML, which showed an inverse association with ATM expression. Overexpressed miR-181a in cell lines significantly enhanced cell proliferation, as well as increased the ratio of S-phase cells by miR-181a mimics transfection in vitro. Luciferase activity of the reporter construct identified ATM as the direct molecular target of miR-181a. ATM siRNA transfection significantly enhanced cell proliferation and increased the ratio of S-phase cells in vitro. The results revealed novel mechanism through which miR-181a regulates G1/S transition and cell proliferation in pediatric AML by regulating the tumor suppressor ATM, providing insights into the molecular mechanism in pediatric AML.

Dual mTORC2/mTORC1 targeting results in potent suppressive effects on acute myeloid leukemia (AML) progenitors*

PubMed Central

Altman, Jessica K.; Sassano, Antonella; Kaur, Surinder; Glaser, Heather; Kroczynska, Barbara; Redig, Amanda J.; Russo, Suzanne; Barr, Sharon; Platanias, Leonidas C.

2011-01-01

Purpose To determine whether mTORC2 and RI-mTORC1 complexes are present in AML cells and to examine the effects of dual mTORC2/mTORC1 inhibition on primitive AML leukemic progenitors. Experimental Design Combinations of different experimental approaches were used, including immunoblotting to detect phosphorylated/activated forms of elements of the mTOR pathway in leukemic cell lines and primary AML blasts; cell proliferation assays; direct assessment of mRNA translation in polysomal fractions of leukemic cells; and clonogenic assays in methylcellulose to evaluate leukemic progenitor colony formation. Results mTORC2 complexes are active in AML cells and play critical roles in leukemogenesis. Rapamycin insensitive (RI) mTORC1 complexes are also formed and regulate the activity of the translational repressor 4E-BP1 in AML cells. OSI-027, blocks mTORC1 and mTORC2 activities and suppresses mRNA translation of cyclin D1 and other genes that mediate proliferative responses in AML cells. Moreover, OSI-027 acts as a potent suppressor of primitive leukemic precursors from AML patients and is much more effective than rapamycin in eliciting antileukemic effects in vitro. Conclusions Dual targeting of mTORC2 and mTORC1 results in potent suppressive effects on primitive leukemic progenitors from AML patients. Inhibition of the mTOR catalytic site with OSI-027 results in suppression of both mTORC2 and RI-mTORC1 complexes and elicits much more potent antileukemic responses than selective mTORC1 targeting with rapamycin. PMID:21415215

Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

PubMed

Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

2015-07-01

We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

CAR T-cells targeting FLT3 have potent activity against FLT3-ITD+ AML and act synergistically with the FLT3-inhibitor crenolanib.

PubMed

Jetani, Hardikkumar; Garcia-Cadenas, Irene; Nerreter, Thomas; Thomas, Simone; Rydzek, Julian; Meijide, Javier Briones; Bonig, Halvard; Herr, Wolfgang; Sierra, Jordi; Einsele, Hermann; Hudecek, Michael

2018-05-01

FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We engineered CD8 + and CD4 + T-cells expressing a FLT3-specific chimeric antigen receptor (CAR) and demonstrate they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3 with internal tandem duplication (FLT3-ITD). We also show that treatment with the FLT3-inhibitor crenolanib leads to increased surface expression of FLT3 specifically on FLT3-ITD + AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells in vitro and in vivo. As anticipated, we found that FLT3-CAR T-cells recognize normal HSCs in vitro and in vivo, and disrupt normal hematopoiesis in colony-formation assays, suggesting that adoptive therapy with FLT3-CAR T-cells will require subsequent CAR T-cell depletion and allogeneic HSC transplantation to reconstitute the hematopoietic system. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD + AML. Further, our data provide the first proof-of-concept that CAR T-cell immunotherapy and small molecule inhibition can be used synergistically, as exemplified by our data showing superior antileukemia efficacy of FLT3-CAR T-cells in combination with crenolanib.

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy

PubMed Central

Ichim, CV; Atkins, HL; Iscove, NN; Wells, RA

2016-01-01

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. PMID:21637284

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML.

PubMed

Morgado-Palacin, Isabel; Day, Amanda; Murga, Matilde; Lafarga, Vanesa; Anton, Marta Elena; Tubbs, Anthony; Chen, Hua Tang; Ergan, Aysegul; Anderson, Rhonda; Bhandoola, Avinash; Pike, Kurt G; Barlaam, Bernard; Cadogan, Elaine; Wang, Xi; Pierce, Andrew J; Hubbard, Chad; Armstrong, Scott A; Nussenzweig, André; Fernandez-Capetillo, Oscar

2016-09-13

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias. Copyright © 2016, American Association for the Advancement of Science.

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13

PubMed Central

Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2016-01-01

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2−), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2− production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2− is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2−. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment. PMID:26655501

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13.

PubMed

Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2016-04-12

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2-), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2- production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2- is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2-. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment.

Proteolytic systems and AMP-activated protein kinase are critical targets of acute myeloid leukemia therapeutic approaches

PubMed Central

Pereira, Olga; Sampaio-Marques, Belém; Paiva, Artur; Correia-Neves, Margarida; Castro, Isabel; Ludovico, Paula

2015-01-01

The therapeutic strategies against acute myeloid leukemia (AML) have hardly been modified over four decades. Although resulting in a favorable outcome in young patients, older individuals, the most affected population, do not respond adequately to therapy. Intriguingly, the mechanisms responsible for AML cells chemoresistance/susceptibility are still elusive. Mounting evidence has shed light on the relevance of proteolytic systems (autophagy and ubiquitin-proteasome system, UPS), as well as the AMPK pathway, in AML biology and treatment, but their exact role is still controversial. Herein, two AML cell lines (HL-60 and KG-1) were exposed to conventional chemotherapeutic agents (cytarabine and/or doxorubicin) to assess the relevance of autophagy and UPS on AML cells’ response to antileukemia drugs. Our results clearly showed that the antileukemia agents target both proteolytic systems and the AMPK pathway. Doxorubicin enhanced UPS activity while drugs’ combination blocked autophagy specifically on HL-60 cells. In contrast, KG-1 cells responded in a more subtle manner to the drugs tested consistent with the higher UPS activity of these cells. In addition, the data demonstrates that autophagy may play a protective role depending on AML subtype. Specific modulators of autophagy and UPS are, therefore, promising targets for combining with standard therapeutic interventions in some AML subtypes. PMID:25537507

CXCR4 Chemokine Receptor Signaling Induces Apoptosis in Acute Myeloid Leukemia Cells via Regulation of the Bcl-2 Family Members Bcl-XL, Noxa, and Bak*

PubMed Central

Kremer, Kimberly N.; Peterson, Kevin L.; Schneider, Paula A.; Meng, X. Wei; Dai, Haiming; Hess, Allan D.; Smith, B. Douglas; Rodriguez-Ramirez, Christie; Karp, Judith E.; Kaufmann, Scott H.; Hedin, Karen E.

2013-01-01

The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted. PMID:23798675

Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Lin; Shaoyang Central Hospital, Hunan Province; Zhang, Yanan

Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AMLmore » progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings. - Highlights: • A-674563 is cytotoxic and anti-proliferative in U937 and AML progenitor cells. • A-674563 activates caspase-3/9 and apoptosis in U937 and AML progenitor cells. • Whiling blocking Akt, A-674563 manipulates other signalings in AML cells. • A-674563 inhibits SphK1 activity in AML cells, independent of Akt blockage. • A-674563 injection inhibits U937 xenograft in vivo growth, and improves mice survival.« less

Direct and indirect targeting of MYC to treat acute myeloid leukemia.

PubMed

Brondfield, Sam; Umesh, Sushma; Corella, Alexandra; Zuber, Johannes; Rappaport, Amy R; Gaillard, Coline; Lowe, Scott W; Goga, Andrei; Kogan, Scott C

2015-07-01

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is often resistant to conventional therapies. The MYC oncogene is commonly overexpressed in AML but has remained an elusive target. We aimed to examine the consequences of targeting MYC both directly and indirectly in AML overexpressing MYC/Myc due to trisomy 8/15 (human/mouse), FLT3-ITD mutation, or gene amplification. We performed in vivo knockdown of Myc (shRNAs) and both in vitro and in vivo experiments using four drugs with indirect anti-MYC activity: VX-680, GDC-0941, artemisinin, and JQ1. shRNA knockdown of Myc in mice prolonged survival, regardless of the mechanism underlying MYC overexpression. VX-680, an aurora kinase inhibitor, demonstrated in vitro efficacy against human MYC-overexpressing AMLs regardless of the mechanism of MYC overexpression, but was weakest against a MYC-amplified cell line. GDC-0941, a PI3-kinase inhibitor, demonstrated efficacy against several MYC-overexpressing AMLs, although only in vitro. Artemisinin, an antimalarial, did not demonstrate consistent efficacy against any of the human AMLs tested. JQ1, a bromodomain and extra-terminal bromodomain inhibitor, demonstrated both in vitro and in vivo efficacy against several MYC-overexpressing AMLs. We also confirmed a decrease in MYC levels at growth inhibitory doses for JQ1, and importantly, sensitivity of AML cell lines to JQ1 appeared independent of the mechanism of MYC overexpression. Our data support growing evidence that JQ1 and related compounds may have clinical efficacy in AML treatment regardless of the genetic abnormalities underlying MYC deregulation.

APG-1252-12A induces mitochondria-dependent apoptosis through inhibiting the antiapoptotic proteins Bcl-2/Bcl-xl in HL-60 cells.

PubMed

Wang, Jing; Yang, Dajun; Luo, Qiuyun; Qiu, Miaozhen; Zhang, Lin; Li, Baoxia; Chen, Haibo; Yi, Hanjie; Yan, Xianglei; Li, Shuxia; Sun, Jian

2017-08-01

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Despite improved remission rates, current treatment regimens for AML are often associated with a very poor prognosis and adverse effects, necessitating more effective and safer agents. B-cell leukemia/lymphoma 2 (Bcl-2) family proteins regulate apoptotic pathway that can be targeted with small molecule inhibitors. APG-1252-12A is a Bcl-2 homology (BH)-3 mimetic that specifically binds to Bcl-2 and Bcl-xl, which has shown efficacy in some Bcl-2 dependent hematological cancers. In this study, we investigated whether APG-1252-12A inhibits the growth of five leukemia cell lines in a concentration- or time-dependent manner by MTS assay. Following treatment of AML cell line HL-60 with this compound, cell apoptosis was detected using flow cytometry and nuclear condensation was observed after Hoechst 33258 dye. Immunoblotting for cytochrome c, cleaved caspase-3 and PARP-1 cleavage was used to demonstrate the mechanism of inducing mitochondria-dependent apoptosis by APG-1252-12A. Our findings showed that this new compound inhibited cell proliferation in five leukemia cell lines and induced apoptotic death. There was a link between the level of Bcl-2 protein and IC50. APG-1252-12A targeted mitochondria and induced caspase-dependent apoptosis by inducing the HL-60 cell cytochrome c released, PARP cleavage and caspase activation. These data suggested that APG-1252-12A is a candidate drug for the in vivo analysis and clinical evaluation in AML.

Azacitidine for Front-Line Therapy of Patients with AML: Reproducible Efficacy Established by Direct Comparison of International Phase 3 Trial Data with Registry Data from the Austrian Azacitidine Registry of the AGMT Study Group

PubMed Central

Pleyer, Lisa; Döhner, Hartmut; Dombret, Hervé; Seymour, John F.; Schuh, Andre C.; Beach, CL; Swern, Arlene S.; Burgstaller, Sonja; Stauder, Reinhard; Girschikofsky, Michael; Sill, Heinz; Schlick, Konstantin; Thaler, Josef; Halter, Britta; Machherndl Spandl, Sigrid; Zebisch, Armin; Pichler, Angelika; Pfeilstöcker, Michael; Autzinger, Eva M.; Lang, Alois; Geissler, Klaus; Voskova, Daniela; Sperr, Wolfgang R.; Hojas, Sabine; Rogulj, Inga M.; Andel, Johannes; Greil, Richard

2017-01-01

We recently published a clinically-meaningful improvement in median overall survival (OS) for patients with acute myeloid leukaemia (AML), >30% bone marrow (BM) blasts and white blood cell (WBC) count ≤15 G/L, treated with front-line azacitidine versus conventional care regimens within a phase 3 clinical trial (AZA-AML-001; NCT01074047; registered: February 2010). As results obtained in clinical trials are facing increased pressure to be confirmed by real-world data, we aimed to test whether data obtained in the AZA-AML-001 trial accurately represent observations made in routine clinical practice by analysing additional AML patients treated with azacitidine front-line within the Austrian Azacitidine Registry (AAR; NCT01595295; registered: May 2012) and directly comparing patient-level data of both cohorts. We assessed the efficacy of front-line azacitidine in a total of 407 patients with newly-diagnosed AML. Firstly, we compared data from AML patients with WBC ≤ 15 G/L and >30% BM blasts included within the AZA-AML-001 trial treated with azacitidine (“AML-001” cohort; n = 214) with AAR patients meeting the same inclusion criteria (“AAR (001-like)” cohort; n = 95). The current analysis thus represents a new sub-analysis of the AML-001 trial, which is directly compared with a new sub-analysis of the AAR. Baseline characteristics, azacitidine application, response rates and OS were comparable between all patient cohorts within the trial or registry setting. Median OS was 9.9 versus 10.8 months (p = 0.616) for “AML-001” versus “AAR (001-like)” cohorts, respectively. Secondly, we pooled data from both cohorts (n = 309) and assessed the outcome. Median OS of the pooled cohorts was 10.3 (95% confidence interval: 8.7, 12.6) months, and the one-year survival rate was 45.8%. Thirdly, we compared data from AAR patients meeting AZA-AML-001 trial inclusion criteria (n = 95) versus all AAR patients with World Health Organization (WHO)-defined AML (“AAR (WHO-AML)” cohort; n = 193). Within the registry population, median OS for AAR patients meeting trial inclusion criteria versus all WHO-AML patients was 10.8 versus 11.8 months (p = 0.599), respectively. We thus tested and confirmed the efficacy of azacitidine as a front-line agent in patients with AML, >30% BM blasts and WBC ≤ 15 G/L in a routine clinical practice setting. We further show that the efficacy of azacitidine does not appear to be limited to AML patients who meet stringent clinical trial inclusion criteria, but instead appears efficacious as front-line treatment in all patients with WHO-AML. PMID:28212292

Allium compounds, dipropyl and dimethyl thiosulfinates as antiproliferative and differentiating agents of human acute myeloid leukemia cell lines.

PubMed

Merhi, Faten; Auger, Jacques; Rendu, Francine; Bauvois, Brigitte

2008-12-01

Epidemiologic studies support the premise that Allium vegetables may lower the risk of cancers. The beneficial effects appear related to the organosulfur products generated upon processing of Allium. Leukemia cells from patients with acute myeloid leukemia (AML) display high proliferative capacity and have a reduced capacity of undergoing apoptosis and maturation. Whether the sulfur-containing molecules thiosulfinates (TS), diallyl TS (All(2)TS), dipropyl TS (Pr(2)TS) and dimethyl TS (Me(2)TS), are able to exert chemopreventative activity against AML is presently unknown. The present study was an evaluation of proliferation, cytotoxicity, differentiation and secretion of AML cell lines (U937, NB4, HL-60, MonoMac-6) in response to treatment with these TS and their related sulfides (diallylsulfide, diallyl disulfide, dipropyl disulfide, dimethyl disulfide). As assessed by flow cytometry, ELISA, gelatin zymogaphy and RT-PCR, we showed that Pr(2)TS and Me(2)TS, but not All(2)TS and sulfides, 1) inhibited cell proliferation in dose- and time-dependent manner and this process was neither due to cytotoxicity nor apoptosis, 2) induced macrophage maturation, and 3) inhibited the levels of secreted MMP-9 (protein and activity) and TNF-alpha protein, without altering mRNA levels. By establishing for the first time that Pr(2)TS and Me(2)TS affect proliferation, differentiation and secretion of leukemic cell lines, this study provides the opportunity to explore the potential efficiency of these molecules in AML.

Matrine induces Akt/mTOR signalling inhibition-mediated autophagy and apoptosis in acute myeloid leukaemia cells.

PubMed

Wu, Junqing; Hu, Gang; Dong, Yuqing; Ma, Ruye; Yu, Zhijie; Jiang, Songfu; Han, Yixiang; Yu, Kang; Zhang, Shenghui

2017-06-01

Pharmacological modulation of autophagy has been referred to as a promising therapeutic strategy for cancer. Matrine, a main alkaloid extracted from Sophora flavescens Ait, has antitumour activity against acute myelocytic leukaemia (AML). Whether autophagy is involved in antileukaemia activity of matrine remains unobvious. In this study, we demonstrated that matrine inhibited cell viability and colony formation via inducing apoptosis and autophagy in AML cell lines HL-60, THP-1 and C1498 as well as primary AML cells. Matrine promoted caspase-3 and PARP cleavage dose-dependently. Matrine up-regulated the level of LC3-II and down-regulated the level of SQSTM1/p62 in a dose-dependent way, indicating that autophagy should be implicated in anti-AML effect of matrine. Furthermore, the autophagy inhibitor bafilomycin A1 relieved the cytotoxicity of matrine by blocking the autophagic flux, while the autophagy promoter rapamycin enhanced the cytotoxicity of matrine. Additionally, matrine inhibited the phosphorylation of Akt, mTOR and their downstream substrates p70S6K and 4EBP1, which led to the occurrence of autophagy. In vivo study demonstrated that autophagy was involved in antileukaemia effect of matrine in C57BL/6 mice bearing murine AML cell line C1498, and the survival curves showed that mice did benefit from treatment with matrine. Collectively, our findings indicate that matrine exerts antitumour effect through apoptosis and autophagy, and the latter one might be a potential therapeutic strategy for AML. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies.

PubMed

Ricciardi, Maria Rosaria; Scerpa, Maria Cristina; Bergamo, Paola; Ciuffreda, Ludovica; Petrucci, Maria Teresa; Chiaretti, Sabina; Tavolaro, Simona; Mascolo, Maria Grazia; Abrams, Stephen L; Steelman, Linda S; Tsao, Twee; Marchetti, Antonio; Konopleva, Marina; Del Bufalo, Donatella; Cognetti, Francesco; Foà, Robin; Andreeff, Michael; McCubrey, James A; Tafuri, Agostino; Milella, Michele

2012-10-01

In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70 % of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML.

Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Feng; Wang, Lingling; Shen, Yunfeng

Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk)more » attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML. - Highlights: • WYE-687 inhibits survival and proliferation of human AML cells/progenitor cells. • WYE-687 induces apoptotic death of human AML cells/progenitor cells. • WYE-687 inhibits mTORC1/2 activation in human AML cells/progenitor cells. • WYE-687 inhibits U937 xenograft growth in SCID mice.« less

Inhibition of FLT3 Expression by Green Tea Catechins in FLT3 Mutated-AML Cells

PubMed Central

Ly, Bui Thi Kim; Chi, Hoang Thanh; Yamagishi, Makoto; Kano, Yasuhiko; Hara, Yukihiko; Nakano, Kazumi; Sato, Yuko; Watanabe, Toshiki

2013-01-01

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a block in differentiation and uncontrolled proliferation. FLT3 is a commonly mutated gene found in AML patients. In clinical trials, the presence of a FLT3-ITD mutation significantly correlates with an increased risk of relapse and dismal overall survival. Therefore, activated FLT3 is a promising molecular target for AML therapies. In this study, we have shown that green tea polyphenols including (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), and (−)-epicatechin-3-gallate (ECG) suppress the proliferation of AML cells. Interestingly, EGCG, EGC and ECG showed the inhibition of FLT3 expression in cell lines harboring FLT3 mutations. In the THP-1 cells harboring FLT3 wild-type, EGCG showed the suppression of cell proliferation but did not suppress the expression of FLT3 even at the concentration that suppress 100% cell proliferation. Moreover, EGCG-, EGC-and ECG-treated cells showed the suppression of MAPK, AKT and STAT5 phosphorylation. Altogether, we suggest that green tea polyphenols could serve as reagents for treatment or prevention of leukemia harboring FLT3 mutations. PMID:23840454

Antagonism of SET using OP449 enhances the efficacy of tyrosine kinase inhibitors and overcome drug resistance in myeloid leukemia

PubMed Central

Agarwal, Anupriya; MacKenzie, Ryan J.; Pippa, Raffaella; Eide, Christopher A.; Oddo, Jessica; Tyner, Jeffrey W.; Sears, Rosalie; Vitek, Michael P.; Odero, María D.; Christensen, Dale; Druker, Brian J.

2014-01-01

Purpose The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A. Experimental Design In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis and colonogenic assays. Efficacy of target inhibition by OP449 is evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model. Results We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34+ CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1-mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells. Conclusions We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML. PMID:24436473

Effective non-viral delivery of siRNA to acute myeloid leukemia cells with lipid-substituted polyethylenimines.

PubMed

Landry, Breanne; Aliabadi, Hamidreza Montazeri; Samuel, Anuja; Gül-Uludağ, Hilal; Jiang, Xiaoyan; Kutsch, Olaf; Uludağ, Hasan

2012-01-01

Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ≈ 0.5 and led to siRNA/polymer complexes of ≈ 100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia.

Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines

PubMed Central

Landry, Breanne; Aliabadi, Hamidreza Montazeri; Samuel, Anuja; Gül-Uludağ, Hilal; Jiang, Xiaoyan; Kutsch, Olaf; Uludağ, Hasan

2012-01-01

Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ∼0.5 and led to siRNA/polymer complexes of ∼100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia. PMID:22952927

Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

PubMed Central

Zhou, Fuling; Pan, Yunbao; Wei, Yongchang; Zhang, Ronghua; Bai, Gaigai; Shen, Qiuju; Meng, Shan; Le, Xiao-Feng; Andreeff, Michael; Claret, Francois X.

2018-01-01

Purpose High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway. Experimental Design We studied ROS levels and conducted JAB1/COPS5 and TRX gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1’s regulation of Trx in leukemia cell lines. Results Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of thioredoxin (TRX) and c-Jun activation domain-binding protein-1 (JAB1/COPS5) were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivo. Conclusions We identified a novel Jab1-Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. PMID:28270496

Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-06-05

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.

MK-2206 induces apoptosis of AML cells and enhances the cytotoxicity of cytarabine.

PubMed

Lu, Jeng-Wei; Lin, Yu-Min; Lai, Yen-Ling; Chen, Chien-Yuan; Hu, Chung-Yi; Tien, Hwei-Fang; Ou, Da-Liang; Lin, Liang-In

2015-07-01

Genetic alterations in the PI3K/AKT cascade have been linked to various human cancers including acute myeloid leukemia (AML) and have emerged to be promising targets for treatment. In this study, we explored the molecular mechanism and clinical implication of a specific allosteric AKT inhibitor, MK-2206, in the treatment of AML. Four leukemia cell lines, MV-4-11, MOLM-13, OCI/AML3, and U937, were used. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Expression of anti-apoptotic protein family and glycogen synthase kinase 3β (GSK3β) signaling was determined by western blotting. Drug combination effects of MK-2206 with cytarabine were evaluated by cell proliferation assay, and the combination index values were calculated by CompuSyn software. MK-2206 had no effect on normal peripheral blood mononuclear cells, but induced G1-phase arrest and apoptosis in leukemia cells. Among anti-apoptotic Bcl-2 family members, only myeloid cell leukemia-1 (Mcl-1) was significantly suppressed. Mcl-1 suppression by MK-2206 was closely associated with decreased GSK3β phosphorylation at Ser9, an event leads to GSK3β activation. Furthermore, the effect of MK-2206 on Mcl-1 downregulation was abolished by GSK3β inhibitor, lithium chloride and proteasome inhibitor, MG-132, suggesting that MK-2206 acted through a GSK3β-mediated, proteasome-dependent protein degradation. In addition, co-administration of MK-2206 with cytarabine could enhance the cytotoxic efficacy of cytarabine in leukemia cell lines. In conclusion, we have demonstrated that MK-2206 is an active agent in AML and its efficacy as in combination with cytarabine is implicated.

Panobinostat Enhances Cytarabine and Daunorubicin Sensitivities in AML Cells through Suppressing the Expression of BRCA1, CHK1, and Rad51

PubMed Central

Edwards, Holly; Caldwell, J. Timothy; Chen, Wei; Inaba, Hiroto; Xu, Xuelian; Buck, Steven A.; Taub, Jeffrey W.; Baker, Sharyn D.; Ge, Yubin

2013-01-01

Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples in vitro and in vivo. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγnull mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of BRCA1, CHK1, and RAD51 through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of BRCA1, CHK1, and RAD51 to enhance cytarabine and daunorubicin sensitivities in AML cells. PMID:24244429

INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia.

PubMed

Jin, Hongjun; Yang, Liyuan; Wang, Lu; Yang, Zailin; Zhan, Qian; Tao, Yao; Zou, Qin; Tang, Yuting; Xian, Jingrong; Zhang, Shuaishuai; Jing, Yipei; Zhang, Ling

2018-01-17

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been recognized as a distinct leukemia entity in the 2016 World Health Organization (WHO) classification. The genetic events underlying oncogenesis in NPM1-mutated AML that is characterized by a normal karyotype remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the phosphoinositide-3 kinase (PI3K) pathway-associated cancers, has been recently found a clinically relevant role in AML. However, little is known about the specific mechanistic function of INPP4B in NPM1-mutated AML. The INPP4B expression levels in NPM1-mutated AML primary blasts and AML OCI-AML3 cell lines were determined by qRT-PCR and western blotting. The effect of INPP4B knockdown on OCI-AML3 leukemia cell proliferation was evaluated, using the Cell Counting Kit-8 and colony formation assay. After INPP4B overexpression or knockdown, the activation of serum and glucocorticoid-regulated kinase 3 (SGK3) and AKT was assessed. The effects of PI3K signaling pathway inhibitors on the levels of p-SGK3 in OCI-AML3 cells were tested. The mass of PI (3,4) P 2 and PI (3) P was analyzed by ELISA upon INPP4B overexpression. Knockdown of SGK3 by RNA interference and a rescue assay were performed to confirm the critical role of SGK3 in INPP4B-mediated cell survival. In addition, the molecular mechanism underlying INPP4B expression in NPM1-mutated leukemia cells was explored. Finally, Kaplan-Meier survival analysis was conducted on the NPM1-mutated AML cohort stratified into quartiles for INPP4B expression in The Cancer Genome Atlas (TCGA) dataset. High expression of INPP4B was observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro. Mechanically, INPP4B enhanced phosphorylated SGK3 (p-SGK3) status, but did not affect AKT activation. SGK3 was required for INPP4B-induced cell proliferation in OCI-AML3 cells. High levels of INPP4B were at least partially caused by the NPM1 mutant via ERK/Ets-1 signaling. Finally, high expression of INPP4B showed a trend towards lower overall survival and event-free survival in NPM1-mutated AML patients. Our results indicate that INPP4B promotes leukemia cell survival via SGK3 activation, and INPP4B might be a potential target in the treatment of NPM1-mutated AML.

The novel compound OSI-461 induces apoptosis and growth arrest in human acute myeloid leukemia cells.

PubMed

Singh, Raminder; Fröbel, Julia; Cadeddu, Ron-Patrick; Bruns, Ingmar; Schroeder, Thomas; Brünnert, Daniela; Wilk, Christian Matthias; Zerbini, Luiz Fernando; Haas, Rainer; Czibere, Akos

2012-02-01

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy. Treatment of patients suffering from high-risk AML as defined by clinical parameters, cytogenetics, and/or molecular analyses is often unsuccessful. OSI-461 is a pro-apoptotic compound that has been proposed as a novel therapeutic option for patients suffering from solid tumors like prostate or colorectal carcinoma. But little is known about its anti-proliferative potential in AML. Hence, we treated bone marrow derived CD34(+) selected blast cells from 20 AML patients and the five AML cell lines KG-1a, THP-1, HL-60, U-937, and MV4-11 with the physiologically achievable concentration of 1 μM OSI-461 or equal amounts of DMSO as a control. Following incubation with OSI-461, we found a consistent induction of apoptosis and an accumulation of cells in the G2/M phase of the cell cycle. In addition, we demonstrate that the OSI-461 mediated anti-proliferative effects observed in AML are associated with the induction of the pro-apoptotic cytokine mda-7/IL-24 and activation of the growth arrest and DNA-damage inducible genes (GADD) 45α and 45γ. Furthermore, OSI-461 treated leukemia cells did not regain their proliferative potential for up to 8 days after cessation of treatment following the initial 48 h treatment period with 1 μM OSI-461. This indicates sufficient targeting of the leukemia-initiating cells in our in vitro experiments through OSI-461. The AML samples tested in this study included samples from patients who were resistant to conventional chemotherapy and/or had FLT3-ITD mutations demonstrating the high potential of OSI-461 in human AML.

Deferasirox has strong anti-leukemia activity but may antagonize theanti-leukemia effect of doxorubicin.

PubMed

Chang, Yu-Chien; Lo, Wen-Jyi; Huang, Yu-Ting; Lin, Chaio-Lin; Feng, Chiu-Che; Lin, Hsin-Ting; Cheng, Hsu-Chen; Yeh, Su-Peng

2017-09-01

Deferasirox (DFX), in addition to its iron-chelation property, has marked anti-proliferative effects on cancer cells. However, the activity and mechanism by which DFX inhibits acute myeloid leukemia (AML) cells remain to be elucidated. Furthermore, the anti-leukemia effect of combining DFX with currently recommended agents doxorubicin (DOX) and cytosine arabinoside (Ara-C) has not been studied. In this study, we show that DFX significantly reduces the viability of three AML cell lines, HL60, THP1, and WEHI3 and two primary leukemic cells harvested from AML patients. DFX induces cell cycle arrest at G1 phase and apoptosis and inhibits phosphorylation of ERK. We also showed that DFX antagonizes the anti-leukemic effect of DOX. On the contrary, combining DFX with Ara-C created a synergistic effect. Our study confirms the anti-leukemia activity of DFX and provides important information on how to select a partner drug for DFX for the treatment of AML in future clinical trials.

Isocitrate dehydrogenase 1 mutations prime the all-trans retinoic acid myeloid differentiation pathway in acute myeloid leukemia

PubMed Central

Boutzen, Héléna; Saland, Estelle; Larrue, Clément; de Toni, Fabienne; Gales, Lara; Castelli, Florence A.; Cathebas, Mathilde; Zaghdoudi, Sonia; Stuani, Lucille; Kaoma, Tony; Riscal, Romain; Yang, Guangli; Hirsch, Pierre; David, Marion; De Mas-Mansat, Véronique; Delabesse, Eric; Vallar, Laurent; Delhommeau, François; Jouanin, Isabelle; Ouerfelli, Ouathek; Le Cam, Laurent; Linares, Laetitia K.; Junot, Christophe; Portais, Jean-Charles; Vergez, François; Récher, Christian

2016-01-01

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD–Scid–IL2rγnull mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies. PMID:26951332

Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite.

PubMed

Ricciardi, M R; Licchetta, R; Mirabilii, S; Scarpari, M; Parroni, A; Fabbri, A A; Cescutti, P; Reverberi, M; Fanelli, C; Tafuri, A

2017-01-01

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor , on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

PubMed Central

Scarpari, M.; Parroni, A.; Fabbri, A. A.; Cescutti, P.; Reverberi, M.; Fanelli, C.

2017-01-01

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity. PMID:29270245

Clinical significance of In vivo Cytarabine Induced Gene Expression Signature in AML

PubMed Central

Lamba, Jatinder K.; Pounds, Stanley; Cao, Xueyuan; Crews, Kristine R.; Cogle, Christopher R.; Bhise, Neha; Raimondi, Susana C.; Downing, James R.; Baker, Sharyn D.; Ribeiro, Raul C.; Rubnitz, Jeffrey E.

2016-01-01

Despite initial remission, approximately 60-70% of adult and 30% of pediatric patients experience relapse or refractory AML. Studies so far have identified base line gene expression profiles of pathogenic and prognostic significance in AML, however extent of change in gene expression post-initiation of treatment has not been investigated. Exposure of leukemic cells to chemotherapeutic agents such as cytarabine, a mainstay of AML chemotherapy can trigger adaptive response by influencing leukemic cell transcriptome and hence development of resistance or refractory disease. It is however challenging to perform such a study due to lack of availability of specimens post-drug treatment. In this study our primary objective was to identify in vivo cytarabine induced changes in leukemia cell transcriptome and to evaluate their impact on clinical outcome. Our results highlight genes relevant to cytarabine resistance and support the concept of targeting cytarabine-induced genes as a means of improving response. PMID:26366682

Clinical significance of in vivo cytarabine-induced gene expression signature in AML.

PubMed

Lamba, Jatinder K; Pounds, Stanley; Cao, Xueyuan; Crews, Kristine R; Cogle, Christopher R; Bhise, Neha; Raimondi, Susana C; Downing, James R; Baker, Sharyn D; Ribeiro, Raul C; Rubnitz, Jeffrey E

2016-01-01

Despite initial remission, ∼60-70% of adult and 30% of pediatric patients experience relapse or refractory AML. Studies so far have identified base line gene expression profiles of pathogenic and prognostic significance in AML; however, the extent of change in gene expression post-initiation of treatment has not been investigated. Exposure of leukemic cells to chemotherapeutic agents such as cytarabine, a mainstay of AML chemotherapy, can trigger adaptive response by influencing leukemic cell transcriptome and, hence, development of resistance or refractory disease. It is, however, challenging to perform such a study due to lack of availability of specimens post-drug treatment. The primary objective of this study was to identify in vivo cytarabine-induced changes in leukemia cell transcriptome and to evaluate their impact on clinical outcome. The results highlight genes relevant to cytarabine resistance and support the concept of targeting cytarabine-induced genes as a means of improving response.

Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

PubMed Central

Park, Jung Eun; Yuen, Hiu Fung; Zhou, Jian Biao; Al-aidaroos, Abdul Qader O; Guo, Ke; Valk, Peter J; Zhang, Shu Dong; Chng, Wee Joo; Hong, Cheng William; Mills, Ken; Zeng, Qi

2013-01-01

FLT3-ITD mutations are prevalent mutations in acute myeloid leukaemia (AML). PRL-3, a metastasis-associated phosphatase, is a downstream target of FLT3-ITD. This study investigates the regulation and function of PRL-3 in leukaemia cell lines and AML patients associated with FLT3-ITD mutations. PRL-3 expression is upregulated by the FLT3-STAT5 signalling pathway in leukaemia cells, leading an activation of AP-1 transcription factors via ERK and JNK pathways. PRL-3-depleted AML cells showed a significant decrease in cell growth. Clinically, high PRL-3 mRNA expression was associated with FLT3-ITD mutations in four independent AML datasets with 1158 patients. Multivariable Cox-regression analysis on our Cohort 1 with 221 patients identified PRL-3 as a novel prognostic marker independent of other clinical parameters. Kaplan–Meier analysis showed high PRL-3 mRNA expression was significantly associated with poorer survival among 491 patients with normal karyotype. Targeting PRL-3 reversed the oncogenic effects in FLT3-ITD AML models in vitro and in vivo. Herein, we suggest that PRL-3 could serve as a prognostic marker to predict poorer survival and as a promising novel therapeutic target for AML patients. PMID:23929599

Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

PubMed Central

Hu, Shuiying; Niu, Hongmei; Inaba, Hiroto; Orwick, Shelley; Rose, Charles; Panetta, John C.; Yang, Shengping; Pounds, Stanley; Fan, Yiping; Calabrese, Christopher; Rehg, Jerold E.; Campana, Dario; Rubnitz, Jeffrey E.

2011-01-01

Background Acute myeloid leukemia (AML) is a genetically heterogeneous cancer that frequently exhibits aberrant kinase signaling. We investigated a treatment strategy combining sorafenib, a multikinase inhibitor with limited single-agent activity in AML, and cytarabine, a key component of AML chemotherapy. Methods Using 10 human AML cell lines, we determined the effects of sorafenib (10 μM) on antileukemic activity by measuring cell viability, proliferation, ERK1/2 signaling, and apoptosis. We also investigated the effects of sorafenib treatment on the accumulation of cytarabine and phosphorylated metabolites in vitro. A human equivalent dose of sorafenib in nontumor-bearing NOD-SCID-IL2Rγnull mice was determined by pharmacokinetic studies using high performance liquid chromatography with tandem mass spectrometric detection, and steady-state concentrations were estimated by the fit of a one-compartment pharmacokinetic model to concentration–time data. The antitumor activity of sorafenib alone (60 mg/kg) twice daily, cytarabine alone (6.25 mg/kg administered intraperitoneally), or sorafenib once or twice daily plus cytarabine was evaluated in NOD-SCID-IL2Rγnull mice bearing AML xenografts. Results Sorafenib at 10 μM inhibited cell viability, proliferation and ERK1/2 signaling, and induced apoptosis in all cell lines studied. Sorafenib also increased the cellular accumulation of cytarabine and metabolites resulting in additive to synergistic antileukemic activity. A dose of 60 mg/kg in mice produced a human equivalent sorafenib steady-state plasma exposure of 10 μM. The more dose-intensive twice-daily sorafenib plus cytarabine (n = 15) statistically significantly prolonged median survival in an AML xenograft model compared with sorafenib once daily plus cytarabine (n = 12), cytarabine alone (n = 26), or controls (n = 27) (sorafenib twice daily plus cytarabine, median survival = 46 days; sorafenib once daily plus cytarabine, median survival = 40 days; cytarabine alone, median survival = 36 days; control, median survival = 19 days; P < .001 for combination twice daily vs all other treatments listed). Conclusions Sorafenib in combination with cytarabine resulted in strong anti-AML activity in vitro and in vivo. These results warrant clinical evaluation of sorafenib with cytarabine-based regimens in molecularly heterogeneous AML. PMID:21487100

The homeobox gene CDX2 is aberrantly expressed in most cases of acute myeloid leukemia and promotes leukemogenesis

PubMed Central

Scholl, Claudia; Bansal, Dimple; Döhner, Konstanze; Eiwen, Karina; Huntly, Brian J.P.; Lee, Benjamin H.; Rücker, Frank G.; Schlenk, Richard F.; Bullinger, Lars; Döhner, Hartmut; Gilliland, D. Gary; Fröhling, Stefan

2007-01-01

The homeobox transcription factor CDX2 plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. We have found that CDX2 is expressed in leukemic cells of 90% of patients with acute myeloid leukemia (AML) but not in hematopoietic stem and progenitor cells derived from normal individuals. Stable knockdown of CDX2 expression by RNA interference inhibited the proliferation of various human AML cell lines and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity, were able to be continuously propagated in liquid culture, generated fully penetrant and transplantable AML in BM transplant recipients, and displayed dysregulated expression of Hox family members in vitro and in vivo. These results demonstrate that aberrant expression of the developmental regulatory gene CDX2 in the adult hematopoietic compartment is a frequent event in the pathogenesis of AML; suggest a role for CDX2 as part of a common effector pathway that promotes the proliferative capacity and self-renewal potential of myeloid progenitor cells; and support the hypothesis that CDX2 is responsible, in part, for the altered HOX gene expression that is observed in most cases of AML. PMID:17347684

Jab1/Csn5-Thioredoxin Signaling in Relapsed Acute Monocytic Leukemia under Oxidative Stress.

PubMed

Zhou, Fuling; Pan, Yunbao; Wei, Yongchang; Zhang, Ronghua; Bai, Gaigai; Shen, Qiuju; Meng, Shan; Le, Xiao-Feng; Andreeff, Michael; Claret, Francois X

2017-08-01

Purpose: High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway. Experimental Design: We studied ROS levels and conducted c-Jun activation domain-binding protein-1 ( JAB1/COPS5 ) and thioredoxin ( TRX ) gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1's regulation of Trx in leukemia cell lines. Results: Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of TRX and JAB1/COPS5 were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivo Conclusions: We identified a novel Jab1-Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. Clin Cancer Res; 23(15); 4450-61. ©2017 AACR . ©2017 American Association for Cancer Research.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells.

PubMed

Chen, Jian; Mu, Qitian; Li, Xia; Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-06-20

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells

PubMed Central

Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-01-01

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT. PMID:28454099

Targeting AMPK-ULK1-mediated autophagy for combating BET inhibitor resistance in acute myeloid leukemia stem cells.

PubMed

Jang, Ji Eun; Eom, Ju-In; Jeung, Hoi-Kyung; Cheong, June-Won; Lee, Jung Yeon; Kim, Jin Seok; Min, Yoo Hong

2017-04-03

Bromodomain and extraterminal domain (BET) inhibitors are promising epigenetic agents for the treatment of various subsets of acute myeloid leukemia (AML). However, the resistance of leukemia stem cells (LSCs) to BET inhibitors remains a major challenge. In this study, we evaluated the mechanisms underlying LSC resistance to the BET inhibitor JQ1. We evaluated the levels of apoptosis and macroautophagy/autophagy induced by JQ1 in LSC-like leukemia cell lines and primary CD34 + CD38 - leukemic blasts obtained from AML cases with normal karyotype without recurrent mutations. JQ1 effectively induced apoptosis in a concentration-dependent manner in JQ1-sensitive AML cells. However, in JQ1-resistant AML LSCs, JQ1 induced little apoptosis and led to upregulation of BECN1/Beclin 1, increased LC3 lipidation, formation of autophagosomes, and downregulation of SQSTM1/p62. Inhibition of autophagy by pharmacological inhibitors or knockdown of BECN1 using specific siRNA enhanced JQ1-induced apoptosis in resistant cells, indicating that prosurvival autophagy occurred in these cells. Independent of MTOR signaling, activation of the AMPK (p-Thr172)-ULK1 (p-Ser555) pathway was found to be associated with JQ1-induced autophagy in resistant cells. AMPK inhibition using the pharmacological inhibitor compound C or by knockdown of PRKAA/AMPKα suppressed autophagy and promoted JQ1-induced apoptosis in AML LSCs. These findings revealed that prosurvival autophagy was one of the mechanisms involved in the resistance of AML LSCs to JQ1. Targeting the AMPK-ULK1 pathway or inhibition of autophagy could be an effective therapeutic strategy for combating resistance to BET inhibitors in AML and other types of cancer.

Expression of myeloid differentiation antigens on normal and malignant myeloid cells.

PubMed Central

Griffin, J D; Ritz, J; Nadler, L M; Schlossman, S F

1981-01-01

A series of monoclonal antibodies have been characterized that define four surface antigens (MY3, MY4, MY7, and MY8) of human myeloid cells. They were derived from a fusion of the NS-1 plasmacytoma cell line with splenocytes from a mouse immunized with human acute myelomonocytic leukemia cells. MY3 and MY4 are expressed by normal monocytes and by greater than 90% of patients with acute monocytic leukemia or acute myelomonocytic leukemia, but are detected much less often on other types of myeloid leukemia. MY7 is expressed by granulocytes, monocytes, and 5% of normal bone marrow cells. 79% of all acute myeloblastic leukemia (AML) patients tested (72 patients) express MY7 without preferential expression by any AML subtype. MY8 is expressed by normal monocytes, granulocytes, all peroxidase-positive bone marrow cells, and 50% of AML patients. MY3, MY4, and MY8 define myeloid differentiation antigens in that they are not detected on myeloid precursor cells and appear at discrete stages of differentiation. These antigens are not expressed by lymphocytes, erythrocytes, platelets, or lymphoid malignancies. The monoclonal antisera defining these antigens have been used to study differentiation of normal myeloid cells and malignant cell lines. Images PMID:6945311

Preclinical characterization of AMG 330, a CD3/CD33-bispecific T-cell-engaging antibody with potential for treatment of acute myelogenous leukemia.

PubMed

Friedrich, Matthias; Henn, Anja; Raum, Tobias; Bajtus, Monika; Matthes, Katja; Hendrich, Larissa; Wahl, Joachim; Hoffmann, Patrick; Kischel, Roman; Kvesic, Majk; Slootstra, Jerry W; Baeuerle, Patrick A; Kufer, Peter; Rattel, Benno

2014-06-01

There is high demand for novel therapeutic options for patients with acute myelogenous leukemia (AML). One possible approach is the bispecific T-cell-engaging (BiTE, a registered trademark of Amgen) antibody AMG 330 with dual specificity for CD3 and the sialic acid-binding lectin CD33 (SIGLEC-3), which is frequently expressed on the surface of AML blasts and leukemic stem cells. AMG 330 binds with low nanomolar affinity to CD33 and CD3ε of both human and cynomolgus monkey origin. Eleven human AML cell lines expressing between 14,400 and 56,700 CD33 molecules per cell were all potently lysed with EC(50) values ranging between 0.4 pmol/L and 3 pmol/L (18-149 pg/mL) by previously resting, AMG 330-redirected T cells. Complete lysis was achieved after 40 hours of incubation. In the presence of AML cells, AMG 330 specifically induced expression of CD69 and CD25 as well as release of IFN-γ, TNF, interleukin (IL)-2, IL-10, and IL-6. Ex vivo, AMG 330 mediated autologous depletion of CD33-positive cells from cynomolgous monkey bone marrow aspirates. Soluble CD33 at concentrations found in bone marrow of patients with AML did not significantly affect activities of AMG 330. Neoexpression of CD33 on newly activated T cells was negligible as it was limited to 6% of T cells in only three out of ten human donors tested. Daily intravenous administration with as low as 0.002 mg/kg AMG 330 significantly prolonged survival of immunodeficient mice adoptively transferred with human MOLM-13 AML cells and human T cells. AMG 330 warrants further development as a potential therapy for AML. ©2014 American Association for Cancer Research.

Expression of the transcription factor Evi-1 in human erythroleukemia cell lines and in leukemias.

PubMed

Fontenay-Roupie, M; Bouscary, D; Melle, J; Viguié, F; Picard, F; Guesnu, M; Dreyfus, F

1997-02-01

The Evi-1 proto-oncogene is a zinc finger DNA binding protein. Although activation of the Evi-1 gene has been associated with chromosomal rearrangements of the 3q25-q28 region, ectopic expression of Evi-1 could also be observed in acute myelogenous leukemias and myelodysplastic syndromes without cytogenetic abnormalities of the 3q26 locus. In this study, human erythroleukemic cell lines were screened for the expression of Evi-1 mRNA by northern blotting. Evi-1 was expressed in all the erythroid cell lines, whether undifferentiated (K 562, HEL, LAMA 84) or exhibiting spontaneous terminal erythroid differentiation (KU 812, JK-1). Evi-1 mRNA levels were constant or elevated in hemoglobin-synthesizing KU 812 or K 562 cells in response to erythropoietin or hemin treatment, respectively. In human acute myeloblastic leukemias (AML), 11/30 expressed Evi-1 by RT-PCR. Among these cases, 4/6 erythroleukemias without abnormalities of the 3q25-q28 region were found positive. The presence of acidophilic erythroblasts (15-47% of bone marrow cells) accounted for the existence of a terminal erythroid differentiation in all Evi-1-positive AML M6, whereas one negative case was poorly differentiated and referred to as AML M6 variant. These results suggest that Evi-1 mRNA expression can coexist with erythroid differentiation.

Association of a cytarabine chemosensitivity related gene expression signature with survival in cytogenetically normal acute myeloid leukemia.

PubMed

Yan, Han; Wen, Lu; Tan, Dan; Xie, Pan; Pang, Feng-Mei; Zhou, Hong-Hao; Zhang, Wei; Liu, Zhao-Qian; Tang, Jie; Li, Xi; Chen, Xiao-Ping

2017-01-03

The prognosis of cytogenetically normal acute myeloid leukemia (CN-AML) varies greatly among patients. Achievement of complete remission (CR) after chemotherapy is indispensable for a better prognosis. To develop a gene signature predicting overall survival (OS) in CN-AML, we performed data mining procedure based on whole genome expression data of both blood cancer cell lines and AML patients from open access database. A gene expression signature including 42 probes was derived. These probes were significantly associated with both cytarabine half maximal inhibitory concentration values in blood cancer cell lines and OS in CN-AML patients. By using cox regression analysis and linear regression analysis, a chemo-sensitive score calculated algorithm based on mRNA expression levels of the 42 probes was established. The scores were associated with OS in both the training sample (p=5.13 × 10-4, HR=2.040, 95% CI: 1.364-3.051) and the validation sample (p=0.002, HR=2.528, 95% CI: 1.393-4.591) of the GSE12417 dataset from Gene Expression Omnibus. In The Cancer Genome Atlas (TCGA) CN-AML patients, higher scores were found to be associated with both worse OS (p=0.013, HR=2.442, 95% CI: 1.205-4.950) and DFS (p=0.015, HR=2.376, 95% CI: 1.181-4.779). Results of gene ontology (GO) analysis showed that all the significant GO Terms were correlated with cellular component of mitochondrion. In summary, a novel gene set that could predict prognosis of CN-AML was identified presently, which provided a new way to identify genes impacting AML chemo-sensitivity and prognosis.

Association of a cytarabine chemosensitivity related gene expression signature with survival in cytogenetically normal acute myeloid leukemia

PubMed Central

Yan, Han; Wen, Lu; Tan, Dan; Xie, Pan; Pang, Feng-mei; Zhou, Hong-hao; Zhang, Wei; Liu, Zhao-qian; Tang, Jie; Li, Xi; Chen, Xiao-ping

2017-01-01

The prognosis of cytogenetically normal acute myeloid leukemia (CN-AML) varies greatly among patients. Achievement of complete remission (CR) after chemotherapy is indispensable for a better prognosis. To develop a gene signature predicting overall survival (OS) in CN-AML, we performed data mining procedure based on whole genome expression data of both blood cancer cell lines and AML patients from open access database. A gene expression signature including 42 probes was derived. These probes were significantly associated with both cytarabine half maximal inhibitory concentration values in blood cancer cell lines and OS in CN-AML patients. By using cox regression analysis and linear regression analysis, a chemo-sensitive score calculated algorithm based on mRNA expression levels of the 42 probes was established. The scores were associated with OS in both the training sample (p=5.13 × 10−4, HR=2.040, 95% CI: 1.364-3.051) and the validation sample (p=0.002, HR=2.528, 95% CI: 1.393-4.591) of the GSE12417 dataset from Gene Expression Omnibus. In The Cancer Genome Atlas (TCGA) CN-AML patients, higher scores were found to be associated with both worse OS (p=0.013, HR=2.442, 95% CI: 1.205-4.950) and DFS (p=0.015, HR=2.376, 95% CI: 1.181-4.779). Results of gene ontology (GO) analysis showed that all the significant GO Terms were correlated with cellular component of mitochondrion. In summary, a novel gene set that could predict prognosis of CN-AML was identified presently, which provided a new way to identify genes impacting AML chemo-sensitivity and prognosis. PMID:27903973

Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia.

PubMed

Swords, Ronan T; Kelly, Kevin R; Smith, Peter G; Garnsey, James J; Mahalingam, Devalingam; Medina, Ernest; Oberheu, Kelli; Padmanabhan, Swaminathan; O'Dwyer, Michael; Nawrocki, Steffan T; Giles, Francis J; Carew, Jennifer S

2010-05-06

NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models. MLN4924 induced cell death in AML cell lines and primary patient specimens independent of Fms-like tyrosine kinase 3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of nuclear factor-kappaB activity, DNA damage, and reactive oxygen species generation. Disruption of cellular redox status was shown to be a key event in MLN4924-induced apoptosis. Administration of MLN4924 to mice bearing AML xenografts led to stable disease regression and inhibition of NEDDylated cullins. Our findings indicate that MLN4924 is a highly promising novel agent that has advanced into clinical trials for the treatment of AML.

Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models.

PubMed

Lehmann, Christian; Friess, Thomas; Birzele, Fabian; Kiialainen, Anna; Dangl, Markus

2016-06-28

Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies. Our findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies.

SOX12: a novel potential target for acute myeloid leukaemia.

PubMed

Wan, Haixia; Cai, Jiayi; Chen, Fangyuan; Zhu, Jianyi; Zhong, Jihua; Zhong, Hua

2017-02-01

The role of SRY-related high-mobility-group box (SOX) 12 in leukaemia progression and haematopoiesis remains elusive. This study aimed to examine the expression and function of SOX12 in acute myeloid leukaemia (AML) using human myeloid leukaemia samples and the acute myeloid cell line THP1. Mononuclear cells were isolated from the bone marrow of AML patients and healthy donors. SOX12 expression in haematopoietic cells was evaluated by reverse transcription polymerase chain reaction (RT-PCR). SOX12 short hairpin RNAs (shRNAs) were transduced into THP1 cells, and gene knockdown was confirmed by quantitative RT-PCR and Western blot analysis. SOX12 was preferentially expressed in CD34 + cells in AML patients. The THP1 cells transduced with SOX12 shRNAs exhibited significantly reduced SOX12 expression and cell proliferation. SOX12 knockdown had no effect on apoptosis, but it induced cell cycle arrest at G1 phase and reduced the number of colonies. The transduced THP1 and primary AML cells were reconstituted in non-obese diabetic-severe combined immunodeficient (NOD/SCID) mice, and their numbers were significantly reduced 6-12 weeks after transplantation. The mRNA and protein levels of β-catenin were significantly diminished following SOX12 knockdown, accompanied by a decrease in TCF/Wnt activity. SOX12 may be involved in leukaemia progression by regulating the expression of β-catenin and then interfering with TCF/Wnt pathway, which may be a target for AML. © 2016 John Wiley & Sons Ltd.

Mechanistic insights into the antileukemic activity of hyperforin.

PubMed

Billard, C; Merhi, F; Bauvois, B

2013-01-01

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) - conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells - two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Gallic acid targets acute myeloid leukemia via Akt/mTOR-dependent mitochondrial respiration inhibition.

PubMed

Gu, Ruixin; Zhang, Minqin; Meng, Hu; Xu, Dandan; Xie, Yonghua

2018-06-05

Gallic acid is one of the many phenolic acids that can be found in dietary substances and traditional medicine herbs. The anti-cancer activities of gallic acid have been shown in various cancers but its underlying molecular mechanisms are not well understood. In this study, we show Akt/mammalian target of rapamycin (mTOR)-dependent inhibition of mitochondrial respiration as a mechanism of gallic acid's action in acute myeloid leukemia (AML). Gallic acid significantly induces apoptosis of AML cell lines, primary mononuclear cells (MNC) and CD34 stem/progenitors isolated form AML patients via caspase-dependent pathway. It also significantly enhances two standard AML chemotherapeutic agents' efficacy in vitro cell culture system and in vivo xenograft model. Gallic acid inhibits dose- and time-dependent mitochondrial respiration, leading to decreased ATP production and oxidative stress. Overexpression of constitutively active Akt restores gallic acid-mediated inhibition of mTOR signaling, mitochondrial dysfunction, energy crisis and apoptosis. Our results demonstrate that mitochondrial respiration inhibition by gallic acid is a consequence of Akt/mTOR signaling suppression. Our findings suggest that combination therapy with gallic acid may enhance antileukemic efficacy of standard chemotherapeutic agents in AML. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Discovery of a BTK/MNK Dual Inhibitor for Lymphoma and Leukemia

PubMed Central

Wu, Hong; Hu, Chen; Wang, Aoli; Weisberg, Ellen L.; Chen, Yongfei; Yun, Cai-Hong; Wang, Wenchao; Liu, Yan; Liu, Xiaochuan; Tian, Bei; Wang, Jinhua; Zhao, Zheng; Liang, Yanke; Li, Binhua; Wang, Li; Wang, Beilei; Chen, Cheng; Buhrlage, Sara J.; Nonami, Atsushi; Li, Yuyang; Fernandes, Stacey M.; Adamia, Sophia; Stone, Richard M.; Galinsky, Ilene A.; Wang, Xianhuo; Yang, Guang; Griffin, James D.; Brown, Jennifer R.; Eck, Michael J.; Liu, Jing; Gray, Nathanael S.; Liu, Qingsong

2016-01-01

BTK kinase is a member of the TEC kinase family and is a key regulator of the B-cell Receptor (BCR)-mediated signaling pathway. It is important for B-cell maturation, proliferation, survival and metastasis. Pharmacological inhibition of BTK is clinically effective against a variety of B-cell malignances, such as MCL, CLL and AML. MNK kinase is one of the key downstream regulators in the RAF-MEK-ERK signaling pathway and controls protein synthesis via regulating the activity of eIF4E. Inhibition of MNK activity has shown moderate efficacy for AML cell lines proliferation. Through a structure-based drug design approach, we have discovered a selective and potent BTK/MNK dual kinase inhibitor (QL-X-138), which exhibits covalent binding to BTK and non-covalent binding to MNK. Compared to the BTK kinase inhibitor (PCI-32765) and the MNK kinase inhibitor (cercosporamide), QL-X-138 displays a stronger anti-proliferative effect against a variety of B-cell cancer cell lines, as well as AML and CLL primary patient cells. The agent can effectively arrest the growth of lymphoma and leukemia cells at the G0–G1 stage and can induce strong apoptotic cell death. These results demonstrated that simultaneous inhibition of BTK and MNK kinase activity might be a new therapeutic strategy for B-cell malignances. PMID:26165234

The oral HDAC inhibitor pracinostat (SB939) is efficacious and synergistic with the JAK2 inhibitor pacritinib (SB1518) in preclinical models of AML

PubMed Central

Novotny-Diermayr, V; Hart, S; Goh, K C; Cheong, A; Ong, L-C; Hentze, H; Pasha, M K; Jayaraman, R; Ethirajulu, K; Wood, J M

2012-01-01

Acute myeloid leukemia (AML) is currently treated with aggressive chemotherapy that is not well tolerated in many elderly patients, hence the unmet medical need for effective therapies with less toxicity and better tolerability. Inhibitors of FMS-like tyrosine kinase 3 (FLT3), JAK2 and histone deacetylase inhibitors (HDACi) have been tested in clinical studies, but showed only moderate single-agent activity. High efficacy of the HDACi pracinostat treating AML and synergy with the JAK2/FLT3 inhibitor pacritinib is demonstrated. Both compounds inhibit JAK-signal transducer and activator of transcription (STAT) signaling in AML cells with JAK2V617F mutations, but also diminish FLT3 signaling, particularly in FLT3-ITD (internal tandem duplication) cell lines. In vitro, this combination led to decreased cell proliferation and increased apoptosis. The synergy translated in vivo in two different AML models, the SET-2 megakaryoblastic AML mouse model carrying a JAK2V617F mutation, and the MOLM-13 model of FLT3-ITD-driven AML. Pracinostat and pacritinib in combination showed synergy on tumor growth, reduction of metastases and synergistically decreased JAK2 or FLT signaling, depending on the cellular context. In addition, several plasma cytokines/growth factors/chemokines triggered by the tumor growth were normalized, providing a rationale for combination therapy with an HDACi and a JAK2/FLT3 inhibitor for the treatment of AML patients, particularly those with FLT3 or JAK2 mutations. PMID:22829971

FLT3-regulated antigens as targets for leukemia-reactive cytotoxic T lymphocytes

PubMed Central

Brackertz, B; Conrad, H; Daniel, J; Kast, B; Krönig, H; Busch, D H; Adamski, J; Peschel, C; Bernhard, H

2011-01-01

The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD+ myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. Antigens, such as PR-3, RHAMM, Survivin, WT-1 and PRAME, were upregulated by constitutively active FLT3-ITD as well as FLT3-WT activated by FLT3 ligand (FL). Cytotoxic T-cell (CTL) clones against PR-3, RHAMM, Survivin and an AML-directed CTL clone recognized AML cell lines and primary AML blasts expressing FLT3-ITD, as well as FLT3-WT+ myeloid dendritic cells in the presence of FL. Downregulation of FLT3 led to the abolishment of CTL recognition. Comparing our findings concerning LAA upregulation by the FLT3 kinase with those already made for the Bcr-Abl kinase, we found analogies in the LAA expression pattern. Antigens upregulated by both FLT3 and Bcr-Abl may be promising targets for the development of immunotherapeutical approaches against myeloid leukemia of different origin. PMID:22829124

Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells.

PubMed

Xu, Xuelian; Xie, Chengzhi; Edwards, Holly; Zhou, Hui; Buck, Steven A; Ge, Yubin

2011-02-16

Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML. Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis. Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis. Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.

Lectins interact differentially with purified human eosinophils, cultured cord blood-derived mast cells and the myeloid leukaemic cell line AML14.3D10: induction of interleukin-4 secretion is conserved among granulocytes, but is not proportional to agglutination or lectin-glycoprotein interaction.

PubMed

Hoffmann, H J; Dahl, C; Schiøtz, P O; Berglund, L; Dahl, R

2003-07-01

Atopy is closely associated with the cellular T helper type-2 (Th2) phenotype, that is dominated by the pleiotrophic cytokine IL-4. The cellular source of IL-4 has yet to be determined, although basophils have been proposed. Eosinophils and mast cells are likely contenders investigated here, and the eosinophil-like leukaemia line AML14.3D10 is compared to eosinophils as an in vitro culturable model for eosinophils. Lectins can cross-link-specific surface glycoproteins and are found in the ingested (processed foods) and inhaled (airborne pollen grains) human environment. Therefore it is of interest to determine whether lectins can elicit the release of IL-4 from Th2-associated granulocytes other than basophils. This study investigated the ability of eosinophils, AML14.3D10 and mast cells to secrete preformed IL-4 in response to stimulation with lectins, and explored molecular mechanisms underlying the interaction. Purified eosinophils and basophils, and cultured mast cells and AML14.3D10 cells were incubated with 1 micro m lectin. Agglutination was scored by microscopy. IL-4 secretion was measured by enzyme-linked immunosorbent assay. Biotinylated lectins were used to determine binding to cells by flow cytometry and in lectin blots of sodium dodecyl sulphate (SDS) gels. Purified human eosinophils, AML14.3D10 cells and cultured mast cells secrete IL-4 with a pattern similar to that found in basophils when stimulated with a panel of reactive and unreactive lectins. The lectin SNA induces IL-4 secretion from mast cells and basophils, but not from eosinophils or AML14.3D10. Eosinophils appear to secrete only pre-formed IL-4, whereas mast cells may synthesize IL-4 on ligation with the lectin LCA. Lectins that agglutinate the granulocytes investigated do not necessarily induce secretion of IL-4. Lectins that elicit secretion of IL-4 bind more to eosinophils than unreactive lectins as determined by flow cytometry and lectin blotting of SDS gels. As granulocytes with functions related to that of basophils, eosinophils, AML14.3D10 and cultured mast cells respond to stimulation with lectins similarly to basophils. This emphasizes the possibility that eosinophils and mast cells may be linked in their cellular heritage as the cellular partners, and lectins as ligands, may contribute to the maintenance of a Th2-favoured microenvironment that is thought to underlie the allergic march.

The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.

PubMed

Blanco, Teresa Mortera; Mantalaris, Athanasios; Bismarck, Alexander; Panoskaltsis, Nicki

2010-03-01

Acute myeloid leukaemia (AML) is a cancer of haematopoietic cells that develops in three-dimensional (3-D) bone marrow niches in vivo. The study of AML has been hampered by lack of appropriate ex vivo models that mimic this microenvironment. We hypothesised that fabrication and optimisation of suitable biomimetic scaffolds for culturing leukaemic cells ex vivo might facilitate the study of AML in its native 3-D niche. We evaluated the growth of three leukaemia subtype-specific cell lines, K-562, HL60 and Kasumi-6, on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials, such as poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate), poly (D, L-lactade), poly (caprolactone), and polystyrene. Our results show that PLGA and PU supported the best seeding efficiency and leukaemic growth. Furthermore, the PLGA and PU scaffolds were coated with extracellular matrix (ECM) proteins, collagen type I (62.5 or 125 microg/ml) and fibronectin (25 or 50 microg/ml) to provide biorecognition signals. The 3 leukaemia subtype-specific lines grew best on PU scaffolds coated with 62.5 microg/ml collagen type I over 6 weeks in the absence of exogenous growth factors. In conclusion, PU-collagen scaffolds may provide a practical model to study the biology and treatment of primary AML in an ex vivo mimicry. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

XPO1 Inhibition Using Selinexor Synergizes With Chemotherapy in Acute Myeloid Leukemia (AML) by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus

PubMed Central

Ranganathan, Parvathi; Kashyap, Trinayan; Yu, Xueyan; Meng, Xiaomei; Lai, Tzung-Huei; McNeil, Betina; Bhatnagar, Bhavana; Shacham, Sharon; Kauffman, Michael; Dorrance, Adrienne M.; Blum, William; Sampath, Deepa; Landesman, Yosef; Garzon, Ramiro

2016-01-01

Purpose Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore we asked whether adding already established effective drugs such as Topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. Experimental Design The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. Results Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared to each single therapy. Conclusions Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of AML patients that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression, and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. PMID:27358488

A non-canonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia (AML)

PubMed Central

Shanmugam, Rajasubramaniam; Sayar, Hamid; Suvannasankha, Attaya; Goswami, Chirayu; Li, Lang; Gupta, Sushil; Cardoso, Angelo A.; Baghdadi, Tareq Al; Sargent, Katie J.; Cripe, Larry D.; Kalvakolanu, Dhananjaya V.; Boswell, H. Scott

2014-01-01

Purpose DAPK1, a tumor suppressor, is a rate-limiting effector in an ER stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of AML with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB- and c- jun-responsive genes, was studied. RNAi knockdown studies were performed in Flt3ITD+ve cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were performed to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. Results AMLs characterized by normal karyotype with Flt3ITD were found to have 10-100-fold lower DAPK1 transcripts normalized to the expression of c-jun, a transcriptional activator of DAPK1, as compared to a heterogeneous cytogenetic category. Meis1, a c-jun-responsive adverse AML prognostic gene signature was also measured as control. These Flt3ITD+ve AMLs over-express relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter along with HDAC2 and HDAC6 in the Flt3ITD+ve human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NIK, de-repressed DAPK1. DAPK1-repressed primary Flt3ITD+ve AMLs had selective nuclear activation of p52NF-κB. Conclusions Flt3ITD promotes a non-canonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD+ve AML. PMID:22096027

shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

PubMed

Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

2015-03-01

EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

AML sensitivity to YM155 is modulated through AKT and Mcl-1

PubMed Central

de Necochea-Campion, Rosalia; Diaz Osterman, Carlos J.; Hsu, Heng-Wei; Fan, Junjie; Mirshahidi, Saied; Wall, Nathan R.; Chen, Chien-Shing

2015-01-01

HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound’s ability to downregulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells. PMID:26118775

Lanthanide-doped nanoparticles conjugated with an anti-CD33 antibody and a p53-activating peptide for acute myeloid leukemia therapy.

PubMed

Niu, Fan; Yan, Jin; Ma, Bohan; Li, Shichao; Shao, Yongping; He, Pengcheng; Zhang, Wanggang; He, Wangxiao; Ma, Peter X; Lu, Wuyuan

2018-06-01

Roughly one third of all human cancers are attributable to the functional inhibition of the tumor suppressor protein p53 by its two negative regulators MDM2 and MDMX, making dual-specificity peptide antagonists of MDM2 and MDMX highly attractive drug candidates for anticancer therapy. Two pharmacological barriers, however, remain a major obstacle to the development of peptide therapeutics: susceptibility to proteolytic degradation in vivo and inability to traverse the cell membrane. Here we report the design of a fluorescent lanthanide oxyfluoride nanoparticle (LONp)-based multifunctional peptide drug delivery system for potential treatment of acute myeloid leukemia (AML) that commonly harbors wild type p53, high levels of MDM2 and/or MDMX, and an overexpressed cell surface receptor, CD33. We conjugated to LONp via metal-thiolate bonds a dodecameric peptide antagonist of both MDM2 and MDMX, termed PMI, and a CD33-targeted, humanized monoclonal antibody to allow for AML-specific intracellular delivery of a stabilized PMI. The resultant nanoparticle antiCD33-LONp-PMI, while nontoxic to normal cells, induced apoptosis of AML cell lines and primary leukemic cells isolated from AML patients by antagonizing MDM2 and/or MDMX to activate the p53 pathway. Fluorescent antiCD33-LONp-PMI also enabled real-time visualization of a series of apoptotic events in AML cells, proving a useful tool for possible disease tracking and treatment response monitoring. Our studies shed light on the development of antiCD33-LONp-PMI as a novel class of antitumor agents, which, if further validated, may help targeted molecular therapy of AML. Copyright © 2018 Elsevier Ltd. All rights reserved.

Acute myeloid leukemia stem cell markers in prognosis and targeted therapy: potential impact of BMI-1, TIM-3 and CLL-1.

PubMed

Darwish, Noureldien H E; Sudha, Thangirala; Godugu, Kavitha; Elbaz, Osama; Abdelghaffar, Hasan A; Hassan, Emad E A; Mousa, Shaker A

2016-09-06

Acute myeloid leukemia (AML) patients show high relapse rates and some develop conventional chemotherapy resistance. Leukemia Stem Cells (LSCs) are the main player for AML relapses and drug resistance. LSCs might rely on the B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) in promoting cellular proliferation and survival. Growth of LSCs in microenvironments that are deprived of nutrients leads to up-regulation of the signaling pathways during the progression of the disease, which may illustrate the sensitivity of LSCs to inhibitors of those signaling pathways as compared to normal cells. We analyzed the expression of LSC markers (CD34, CLL-1, TIM-3 and BMI-1) using quantitative RT-PCR in bone marrow samples of 40 AML patients of different FAB types (M1, M2, M3, M4, M5, and M7). We also studied the expression of these markers in 2 AML cell lines (Kasumi-1 and KG-1a) using flow cytometry and quantitative RT-PCR. The overexpression of TIM-3, CLL-1, and BMI-1 was markedly correlated with poor prognosis in these patients. Our in vitro findings demonstrate that targeting BMI-1, which markedly increased in the leukemic cells, was associated with marked decrease in leukemic burden. This study also presents results for blocking LSCs' surface markers CD44, CLL-1, and TIM-3. These markers may play an important role in elimination of AML. Our study indicates a correlation between the expression of markers TIM-3, CLL-1, and especially of BMI-1 and the aggressiveness of AML and thus the potential impact of prognosis and therapies that target LSCs on improving the cure rates.

Inhibition of HOX/PBX dimer formation leads to necroptosis in acute myeloid leukemia cells.

PubMed

Alharbi, Raed A; Pandha, Hardev S; Simpson, Guy R; Pettengell, Ruth; Poterlowicz, Krzysztof; Thompson, Alexander; Harrington, Kevin; El-Tanani, Mohamed; Morgan, Richard

2017-10-27

The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9 ), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5 , HOXB2 , HOXB4 , HOXB9 , and HOXC9 , but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone.

Inhibition of HOX/PBX dimer formation leads to necroptosis in acute myeloid leukemia cells

PubMed Central

Alharbi, Raed A.; Pandha, Hardev S.; Simpson, Guy R.; Pettengell, Ruth; Poterlowicz, Krzysztof; Thompson, Alexander; Harrington, Kevin; El-Tanani, Mohamed; Morgan, Richard

2017-01-01

The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5, HOXB2, HOXB4, HOXB9, and HOXC9, but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone. PMID:29163771

Targeting CD123 in acute myeloid leukemia using a T-cell–directed dual-affinity retargeting platform

PubMed Central

Al-Hussaini, Muneera; Rettig, Michael P.; Ritchey, Julie K.; Karpova, Darja; Uy, Geoffrey L.; Eissenberg, Linda G.; Gao, Feng; Eades, William C.; Bonvini, Ezio; Chichili, Gurunadh R.; Moore, Paul A.; Johnson, Syd; Collins, Lynne

2016-01-01

T-cell–directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123+ AML. PMID:26531164

Induction of cancer testis antigen expression in circulating acute myeloid leukemia blasts following hypomethylating agent monotherapy

PubMed Central

Srivastava, Pragya; Paluch, Benjamin E.; Matsuzaki, Junko; James, Smitha R.; Collamat-Lai, Golda; Blagitko-Dorfs, Nadja; Ford, Laurie Ann; Naqash, Rafeh; Lübbert, Michael; Karpf, Adam R.; Nemeth, Michael J.; Griffiths, Elizabeth A.

2016-01-01

Cancer testis antigens (CTAs) are promising cancer associated antigens in solid tumors, but in acute myeloid leukemia, dense promoter methylation silences their expression. Leukemia cell lines exposed to HMAs induce expression of CTAs. We hypothesized that AML patients treated with standard of care decitabine (20mg/m2 per day for 10 days) would demonstrate induced expression of CTAs. Peripheral blood blasts serially isolated from AML patients treated with decitabine were evaluated for CTA gene expression and demethylation. Induction of NY-ESO-1 and MAGEA3/A6, were observed following decitabine. Re-expression of NY-ESO-1 and MAGEA3/A6 was associated with both promoter specific and global (LINE-1) hypomethylation. NY-ESO-1 and MAGEA3/A6 mRNA levels were increased irrespective of clinical response, suggesting that these antigens might be applicable even in patients who are not responsive to HMA therapy. Circulating blasts harvested after decitabine demonstrate induced NY-ESO-1 expression sufficient to activate NY-ESO-1 specific CD8+ T-cells. Induction of CTA expression sufficient for recognition by T-cells occurs in AML patients receiving decitabine. Vaccination against NY-ESO-1 in this patient population is feasible. PMID:26883197

Inhibition of Histone Deacetylases 1 and 6 Enhances Cytarabine-Induced Apoptosis in Pediatric Acute Myeloid Leukemia Cells

PubMed Central

Xu, Xuelian; Xie, Chengzhi; Edwards, Holly; Zhou, Hui; Buck, Steven A.; Ge, Yubin

2011-01-01

Background Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML. Methodology Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis. Results Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis. Conclusion Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs. PMID:21359182

PRL-3, a Metastasis Associated Tyrosine Phosphatase, Is Involved in FLT3-ITD Signaling and Implicated in Anti-AML Therapy

PubMed Central

Zhou, Jianbiao; Bi, Chonglei; Chng, Wee-Joo; Cheong, Lip-Lee; Liu, Shaw-Cheng; Mahara, Sylvia; Tay, Kian-Ghee; Zeng, Qi; Li, Jie; Guo, Ke; Tan, Cheng Peow Bobby; Yu, Hanry; Albert, Daniel H.; Chen, Chien-Shing

2011-01-01

Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription) pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation. PMID:21589872

Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

PubMed

Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

2016-05-01

All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR. ©2016 American Association for Cancer Research.

Inhibition of Mcl-1 enhances cell death induced by the Bcl-2-selective inhibitor ABT-199 in acute myeloid leukemia cells.

PubMed

Luedtke, Daniel A; Niu, Xiaojia; Pan, Yihang; Zhao, Jianyun; Liu, Shuang; Edwards, Holly; Chen, Kang; Lin, Hai; Taub, Jeffrey W; Ge, Yubin

2017-01-01

Acute myeloid leukemia (AML) is a serious disease. The 5-year survival rates remain frustratingly low (65% for children and 26% for adults). Resistance to frontline chemotherapy (usually cytarabine) often develops; therefore a new treatment modality is needed. Bcl-2 family proteins play an important role in balancing cell survival and apoptosis. The antiapoptotic Bcl-2 family proteins have been found to be dysregulated in AML. ABT-199, a BH3 mimetic, was developed to target antiapoptotic protein Bcl-2. Although ABT-199 has demonstrated promising results, resistance occurs. Previous studies in AML show that ABT-199 alone decreases the association of proapoptotic protein Bim with Bcl-2, but this is compensated by increased association of Bim with prosurvival protein Mcl-1, stabilizing Mcl-1, resulting in resistance to ABT-199. In this study, we investigated the antileukemic activity of the Mcl-1-selective inhibitor A-1210477 in combination with ABT-199 in AML cells. We found that A-1210477 synergistically induced apoptosis with ABT-199 in AML cell lines and primary patient samples. The synergistic induction of apoptosis was decreased upon Bak, Bax and Bim knockdown. While A-1210477 treatment alone also increased Mcl-1 protein levels, combination with ABT-199 reduced binding of Bim to Mcl-1. Our results demonstrate that sequestration of Bim by Mcl-1, a mechanism of ABT-199 resistance, can be abrogated by combined treatment with the Mcl-1 inhibitor A-1201477.

Arsenic disulfide induced apoptosis and concurrently promoted erythroid differentiation in cytokine-dependent myelodysplastic syndrome-progressed leukemia cell line F-36p with complex karyotype including monosomy 7.

PubMed

Hu, Xiao-mei; Tanaka, Sachiko; Onda, Kenji; Yuan, Bo; Toyoda, Hiroo; Ma, Rou; Liu, Feng; Hirano, Toshihiko

2014-05-01

Acute myeloid leukemia progressed from myelodysplastic syndrome (MDS/AML) is generally incurable with poor prognosis for complex karyotype including monosomy 7 (-7). Qinghuang Powder (, QHP), which includes Qing Dai (Indigo naturalis) and Xiong Huang (realgar) in the formula, is effective in treating MDS or MDS/AML even with the unfavorable karyotype, and its therapeutic efficacy could be enhanced by increasing the Xiong huang content in the formula, while Xiong huang contains > 90% arsenic disulfide (As2S2). F-36p cell line was established from a MDS/AML patient with complex karyotype including -7, and was in cytokine-dependent. The present study was to investigate the effects of As2S2 on F-36p cells. Cell proliferation was measured by an 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was identified by Annexin V-staining. Cell viability was determined by a propidium iodide (PI) exclusion. Erythroid differentiation was evaluated by the expression of cell surface antigen CD235a (GpA). After treatment with As2S2 at concentrations of 0.5 to 16 μmol/L for 72 h, As2S2 inhibited the proliferation of F-36p cells. The 50% inhibitory concentrations (IC50) of As2S2 against the proliferation of F-36p cells was 6 μmol/L. The apoptotic cells significantly increased in a dose-dependent mannar (P<0.05). The cell viabilities were significantly inhibited by As2S2 dose-dependent in a dose-dependent manner (P<0.05). Significant increases of CD235a-positive cells were concurrently observed (P<0.05) also in a dose-dependent manner. As2S2 could inhibit proliferation and viability, induce apoptosis, and concurrently promote erythroid differentiation dose-dependently in F-36p cells. As2S2 can inhibit proliferation and viability, induce apoptosis, and concurrently promote erythroid differentiation in cytokine-dependent MDS-progressed human leukemia cell line F-36p with complex karyotype including -7. The data suggest that QHP and/or As2S2 could be a potential candidate in the treatment of MDS or MDS/AML even with unfavorable cytogenetics.

Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

PubMed Central

Lee-Sherick, Alisa B.; Zhang, Weihe; Menachof, Kelly K.; Hill, Amanda A.; Rinella, Sean; Kirkpatrick, Gregory; Page, Lauren S.; Stashko, Michael A.; Jordan, Craig T.; Wei, Qi; Liu, Jing; Zhang, Dehui; DeRyckere, Deborah; Wang, Xiaodong; Frye, Stephen; Earp, H. Shelton; Graham, Douglas K.

2015-01-01

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML. PMID:25762638

A noncanonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia.

PubMed

Shanmugam, Rajasubramaniam; Gade, Padmaja; Wilson-Weekes, Annique; Sayar, Hamid; Suvannasankha, Attaya; Goswami, Chirayu; Li, Lang; Gupta, Sushil; Cardoso, Angelo A; Baghdadi, Tareq Al; Sargent, Katie J; Cripe, Larry D; Kalvakolanu, Dhananjaya V; Boswell, H Scott

2012-01-15

Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB. Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML. ©2011 AACR.

IMGN779, a Novel CD33-Targeting Antibody-Drug Conjugate with DNA-Alkylating Activity, Exhibits Potent Antitumor Activity in Models of AML.

PubMed

Kovtun, Yelena; Noordhuis, Paul; Whiteman, Kathleen R; Watkins, Krystal; Jones, Gregory E; Harvey, Lauren; Lai, Katharine C; Portwood, Scott; Adams, Sharlene; Sloss, Callum M; Schuurhuis, Gerrit Jan; Ossenkoppele, Gert; Wang, Eunice S; Pinkas, Jan

2018-06-01

The myeloid differentiation antigen CD33 has long been exploited as a target for antibody-based therapeutic approaches in acute myeloid leukemia (AML). Validation of this strategy was provided with the approval of the CD33-targeting antibody-drug conjugate (ADC) gemtuzumab ozogamicin in 2000; the clinical utility of this agent, however, has been hampered by safety concerns. Thus, the full potential of CD33-directed therapy in AML remains to be realized, and considerable interest exists in the design and development of more effective ADCs that confer high therapeutic indices and favorable tolerability profiles. Here, we describe the preclinical characterization of a novel CD33-targeting ADC, IMGN779, which utilizes a unique DNA-alkylating payload to achieve potent antitumor effects with good tolerability. The payload, DGN462, is prototypical of a novel class of purpose-created indolinobenzodiazeprine pseudodimers, termed IGNs. With low picomolar potency, IMGN779 reduced viability in a panel of AML cell lines in vitro Mechanistically, the cytotoxic activity of IMGN779 involved DNA damage, cell-cycle arrest, and apoptosis consistent with the mode of action of DGN462. Moreover, IMGN779 was highly active against patient-derived AML cells, including those with adverse molecular abnormalities, and sensitivity correlated to CD33 expression levels. In vivo , IMGN779 displayed robust antitumor efficacy in multiple AML xenograft and disseminated disease models, as evidenced by durable tumor regressions and prolonged survival. Taken together, these findings identify IMGN779 as a promising new candidate for evaluation as a novel therapeutic in AML. Mol Cancer Ther; 17(6); 1271-9. ©2018 AACR . ©2018 American Association for Cancer Research.

Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells.

PubMed

Nalaskowski, Marcus M; Ehm, Patrick; Rehbach, Christoph; Nelson, Nina; Täger, Maike; Modest, Kathrin; Jücker, Manfred

2018-05-28

The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1. In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of the mitochondrial protease, ClpP, as a therapeutic strategy for human acute myeloid leuekmia

PubMed Central

Cole, Alicia; Wang, Zezhou; Coyaud, Etienne; Voisin, Veronique; Gronda, Marcela; Jitkova, Yulia; Mattson, Rachel; Hurren, Rose; Babovic, Sonja; Maclean, Neil; Restall, Ian; Wang, Xiaoming; Jeyaraju, Danny V.; Sukhai, Mahadeo A.; Prabha, Swayam; Bashir, Shaheena; Ramakrishnan, Ashwin; Leung, Elisa; Qia, Yi Hua; Zhang, Nianxian; Combes, Kevin R.; Ketela, Troy; Lin, Fengshu; Houry, Walid A.; Aman, Ahmed; Al-awar, Rima; Zheng, Wei; Wienholds, Erno; Xu, Chang Jiang; Dick, John; Wang, Jean C.Y.; Moffat, Jason; Minden, Mark D.; Eaves, Connie J.; Bader, Gary D.; Hao, Zhenyue; Kornblau, Steven M.; Raught, Brian; Schimmer, Aaron D.

2015-01-01

Summary From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in the leukemic cells from approximately half of patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression, but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism. PMID:26058080

Binding of released Bim to Mcl-1 is a mechanism of intrinsic resistance to ABT-199 which can be overcome by combination with daunorubicin or cytarabine in AML cells

PubMed Central

Niu, Xiaojia; Zhao, Jianyun; Ma, Jun; Xie, Chengzhi; Edwards, Holly; Wang, Guan; Caldwell, J. Timothy; Xiang, Shengyan; Zhang, Xiaohong; Chu, Roland; Wang, Zhihong; Lin, Hai; Taub, Jeffrey W.; Ge, Yubin

2016-01-01

Purpose To investigate the molecular mechanism underlying intrinsic resistance to ABT-199. Experimental Design Western blots and real-time RT-PCR were used to determine levels of Mcl-1 after ABT-199 treatment alone or in combination with cytarabine or daunorubicin. Immunoprecipitation of Bim and Mcl-1 were used to determine the effect of ABT-199 treatment on their interactions with Bcl-2 family members. Lentiviral shRNA knockdown of Bim and CRISPR knockdown of Mcl-1 were used to confirm their role in resistance to ABT-199. JC-1 assays and flow cytometry were used to determine drug-induced apoptosis. Results Immunoprecipitation of Bim from ABT-199 treated cell lines and a primary patient sample demonstrated decreased association with Bcl-2, but increased association with Mcl-1 without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein, unchanged or decreased Mcl-1 transcript levels, and increased Mcl-1 protein half-life, suggesting that the association with Bim plays a role in stabilizing Mcl-1 protein. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage along with decreased Mcl-1 protein levels, compared to ABT-199 alone, and synergistic induction of cell death in both AML cell lines and primary patient samples obtained from AML patients at diagnosis. Conclusions Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance and supports the clinical development of ABT-199 in combination with cytarabine or daunorubicin for the treatment of AML. PMID:27103402

Binding of Released Bim to Mcl-1 is a Mechanism of Intrinsic Resistance to ABT-199 which can be Overcome by Combination with Daunorubicin or Cytarabine in AML Cells.

PubMed

Niu, Xiaojia; Zhao, Jianyun; Ma, Jun; Xie, Chengzhi; Edwards, Holly; Wang, Guan; Caldwell, J Timothy; Xiang, Shengyan; Zhang, Xiaohong; Chu, Roland; Wang, Zhihong J; Lin, Hai; Taub, Jeffrey W; Ge, Yubin

2016-09-01

To investigate the molecular mechanism underlying intrinsic resistance to ABT-199. Western blots and real-time RT-PCR were used to determine levels of Mcl-1 after ABT-199 treatment alone or in combination with cytarabine or daunorubicin. Immunoprecipitation of Bim and Mcl-1 were used to determine the effect of ABT-199 treatment on their interactions with Bcl-2 family members. Lentiviral short hairpin RNA knockdown of Bim and CRISPR knockdown of Mcl-1 were used to confirm their role in resistance to ABT-199. JC-1 assays and flow cytometry were used to determine drug-induced apoptosis. Immunoprecipitation of Bim from ABT-199-treated cell lines and a primary patient sample demonstrated decreased association with Bcl-2, but increased association with Mcl-1 without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein, unchanged or decreased Mcl-1 transcript levels, and increased Mcl-1 protein half-life, suggesting that the association with Bim plays a role in stabilizing Mcl-1 protein. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage along with decreased Mcl-1 protein levels, compared with ABT-199 alone, and synergistic induction of cell death in both AML cell lines and primary patient samples obtained from AML patients at diagnosis. Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance and supports the clinical development of ABT-199 in combination with cytarabine or daunorubicin for the treatment of AML. Clin Cancer Res; 22(17); 4440-51. ©2016 AACR. ©2016 American Association for Cancer Research.

Anti-leukemic effect of a synthetic compound, (±) trans-dihydronarciclasine (HYU-01) via cell-cycle arrest and apoptosis in acute myeloid leukemia.

PubMed

Kim, Seo Ju; Park, Hyun Ki; Kim, Ju Young; Yoon, Jin Sun; Kim, Eun Shil; Cho, Cheon-Gyu; Kim, Byoung Kook; Park, Byeong Bae; Lee, Young Yiul

2012-10-01

(±) trans-Dihydronarciclasine, isolated from Chinese medicinal plant Zephyranthes candida, has been shown to possess quite potent anti-tumoral effect against selected human cancer cell lines. However, little is known about the anti-tumoral effect of (±) trans-dihydronarciclasine in acute myeloid leukemia (AML). This study was performed to investigate the effect of a novel synthetic (±) trans-dihydronarciclasine (code name; HYU-01) in AML. The HYU-01 inhibited the proliferation of various AML cell lines including HL-60 as well as primary leukemic blasts in a dose-dependent manner. To investigate the mechanism of the anti-proliferative effect of HYU-01, cell-cycle analysis was attempted in HL-60 cells, resulting in G1 arrest. The expression levels of CDK2, CDK4, CDK6, cyclin E, and cyclin A were decreased in a time-dependent manner. In addition, HYU-01 up-regulated the expression of the p27, and markedly enhanced the binding of p27 with CDK2, 4, and 6, ultimately resulting in the decrease of their kinase activities. Furthermore, HYU-01 induced the apoptosis through the induction of proapoptotic molecules and reduction of antiapoptotic molecules in association with the activation of caspase-3, -8, and -9. These results suggest that HYU-01 may inhibit the proliferation of HL-60 cells, via apoptosis, as well as G1 block in association with the induction of p27. © 2012 The Authors APMIS © 2012 APMIS.

Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL– and mutant FLT3-expressing cells

PubMed Central

Weisberg, Ellen; Banerji, Lolita; Wright, Renee D.; Barrett, Rosemary; Ray, Arghya; Moreno, Daisy; Catley, Laurence; Jiang, Jingrui; Hall-Meyers, Elizabeth; Sauveur-Michel, Maira; Stone, Richard; Galinsky, Ilene; Fox, Edward; Kung, Andrew L.

2008-01-01

Mediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhi-bitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL–, and induce apoptosis of BCR-ABL–expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL– and mutant FLT3-expressing cells both in vitro and in vivo. PMID:18184863

Chromosome 12p deletions in TEL-AML1 childhood acute lymphoblastic leukemia are associated with retrotransposon elements and occur postnatally

PubMed Central

Hofmann, Jerry; Kang, Michelle; Selzer, Rebecca; Green, Roland; Zhou, Mi; Zhong, Sheng; Zhang, Luoping; Smith, Martyn T.; Marsit, Carmen; Loh, Mignon; Buffler, Patricia; Yeh, Ru-Fang

2008-01-01

TEL-AML1 (ETV6-RUNX1) is the most common translocation in the childhood leukemias, and is a prenatal mutation in most children. This translocation has been detected at a high rate among newborns (∼1%); therefore the rate-limiting event for leukemia appears to be secondary mutations. A frequent such mutation in this subtype is partial deletion of chromosome 12p, trans from the translocation. Nine del(12p) breakpoints within six leukemia cases were sequenced to explore the etiology of this genetic event, and most involved cryptic sterile translocations. Twelve of 18 del(12p) parent sequences involved in these breakpoints were located in repeat regions (8 of these in Long Interspersed Nuclear Elements, or LINEs). This stands in contrast to TEL-AML1, in which only 21 of 110 previously assessed breakpoints (19%) occur in DNA repeats (P = 0.0001). An exploratory assessment of archived neonatal blood cards (ANB cards) revealed significantly more LINE CpG methylation in individuals at birth who were later diagnosed with TEL-AML1 leukemia, compared to individuals who did not contract leukemia (P = 0.01). Nontemplate nucleotides were also more frequent in del(12p) than in TEL-AML1 junctions (P = 0.004) suggesting formation by terminal deoxynucleotidyl transferase. Assessment of six ANB cards indicated that no del(12p) rearrangements backtracked to birth, although two of these patients were previously positive for TEL-AML1 using the same assay with comparable sensitivity. These data are compatible with the a two-stage natural history: TEL-AML1 occurs prenatally, and del(12p) occurs postnatally in more mature cells with a structure that suggests the involvement of retrotransposon instability. PMID:19047175

Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia.

PubMed

Wattjes, M P; Krauter, J; Nagel, S; Heidenreich, O; Ganser, A; Heil, G

2000-02-01

The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leukemia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hematological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel competitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both techniques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages.

Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress

PubMed Central

Doshi, Kshama A.; Trotta, Rossana; Natarajan, Karthika; Rassool, Feyruz V.; Tron, Adriana E.; Huszar, Dennis; Perrotti, Danilo; Baer, Maria R.

2016-01-01

Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD. PMID:27374090

Repression of GSK3 restores NK cell cytotoxicity in AML patients

PubMed Central

Parameswaran, Reshmi; Ramakrishnan, Parameswaran; Moreton, Stephen A.; Xia, Zhiqiang; Hou, Yongchun; Lee, Dean A.; Gupta, Kalpana; deLima, Marcos; Beck, Rose C.; Wald, David N.

2016-01-01

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy. PMID:27040177

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

PubMed Central

Stiefelhagen, Marius; Sellner, Leopold; Kleinschmidt, Jürgen A; Jauch, Anna; Laufs, Stephanie; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan; Veldwijk, Marlon R

2008-01-01

Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. PMID:18789140

miR-137 downregulates c-kit expression in acute myeloid leukemia.

PubMed

Hu, Yanping; Dong, Xiaolong; Chu, Guoming; Lai, Guangrui; Zhang, Bijun; Wang, Leitong; Zhao, Yanyan

2017-06-01

The oncogene c-kit plays a vital role in the pathogenesis of acute myeloid leukemia (AML). However, the mechanism of microRNAs targeting c-kit in AML has not been determined in detail. Moreover, the role miR-137 in tumor cell proliferation remains controversial. The aim of this work was to verify whether miR-137 targets c-kit and to research the biological effects of restoring miR-137 expression in leukemia cells. We found that miR-137 binds specifically to the 3'-UTR of c-kit and suppresses the expression and activities of c-kit. There is a negative correlation between miR-137 and c-kit expression in both patients and cell lines determined by screening large clinical samples. We found that miR-137 can inhibit proliferation, promote apoptosis, and induce differentiation of c-kit+ AML cells. We determined that miR-137 can participate in the leukemogenesis by regulating c-kit, which could be used as a therapeutic target for acute myeloid leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro.

PubMed

Wang, Hai-rong; Xiao, Zhen-yu; Chen, Miao; Wang, Fei-long; Liu, Jia; Zhong, Hua; Zhong, Ji-hua; Ou-Yang, Ren-rong; Shen, Yan-lin; Pan, Shu-ming

2012-06-01

Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus. The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot. We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated. CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.

Jumping translocations in hematological malignancies: a cytogenetic study of five cases.

PubMed

Manola, Kalliopi N; Georgakakos, Vasileios N; Stavropoulou, Chryssa; Spyridonidis, Alexandros; Angelopoulou, Maria K; Vlachadami, Ioanna; Katsigiannis, Andreas; Roussou, Paraskevi; Pantelias, Gabriel E; Sambani, Constantina

2008-12-01

Jumping translocations (JT) are rare cytogenetic aberrations in hematological malignancies that include unbalanced translocations involving a donor chromosome arm or chromosome segment that has fused to two or more different recipient chromosomes in different cell lines. We report five cases associated with different hematologic disorders and JT to contribute to the investigation of the origin, pathogenesis, and clinical significance of JT. These cases involve JT of 1q in a case of acute myeloblastic leukemia (AML)-M1, a case of Burkitt lymphoma, and a case of BCR/ABL-positive acute lymphoblastic leukemia, as well as a JT of 13q in a case of AML-M5, and a JT of 11q segment in a case of undifferentiated leukemia. To our knowledge, with regard to hematologic malignancies, this study presents the first case of JT associated with AML-M1, the first case of JT involving 13q as a donor chromosome, and the first report of JT involving a segment of 11q containing two copies of the MLL gene, jumping on to two recipient chromosomes in each cell line and resulting in six copies of the MLL gene. Our investigation suggests that JT may not contribute to the pathogenesis but rather to the progression of the disease, and it demonstrates that chromosome band 1q10 as a breakpoint of the donor chromosome 1q is also implicated in AML, not only in multiple myeloma as it has been known until now.

Histone deacetylase inhibitors reduce differentiating osteoblast-mediated protection of acute myeloid leukemia cells from cytarabine

PubMed Central

Sterner, Rosalie M.; Kremer, Kimberly N.; Al-Kali, Aref; Patnaik, Mrinal M.; Gangat, Naseema; Litzow, Mark R.; Kaufmann, Scott H.; Westendorf, Jennifer J.; van Wijnen, Andre J.; Hedin, Karen E.

2017-01-01

The bone marrow microenvironment protects acute myeloid leukemia (AML) cells during chemotherapy and is a major factor in relapse. Here, we examined which type(s) of bone marrow cells are responsible for the relapse of AML following treatment with cytarabine (Ara-C), and we identified a means to inhibit this protection. To determine the protective cell type(s), AML cells were treated with Ara-C, and AML cell survival in the presence or absence of osteoblast lineage cells was assessed. Cultured AML cells and patient bone marrow isolates were each significantly protected from Ara-C-induced apoptosis by co-culture with differentiating osteoblasts. Moreover, pretreating differentiating osteoblasts with the histone deacetylase inhibitors (HDACi) vorinostat and panobinostat abrogated the ability of the differentiating osteoblasts to protect AML cells. Together, our results indicate that differentiating osteoblasts have the potential to promote residual AML in the bone marrow following standard chemotherapy and act via a mechanism requiring HDACi-sensitive gene expression. Using HDACi to target the leukemic microenvironment in combination with Ara-C could potentially improve treatment of AML. Moreover, other strategies for manipulating bone marrow osteoblasts may also help eradicate AML cells and reduce relapse. PMID:29212250

Breakdown of the FLT3-ITD/STAT5 axis and synergistic apoptosis induction by the histone deacetylase inhibitor panobinostat and FLT3-specific inhibitors.

PubMed

Pietschmann, Kristin; Bolck, Hella Anna; Buchwald, Marc; Spielberg, Steffi; Polzer, Harald; Spiekermann, Karsten; Bug, Gesine; Heinzel, Thorsten; Böhmer, Frank-Dietmar; Krämer, Oliver H

2012-11-01

Activating mutations of the class III receptor tyrosine kinase FLT3 are the most frequent molecular aberration in acute myeloid leukemia (AML). Mutant FLT3 accelerates proliferation, suppresses apoptosis, and correlates with poor prognosis. Therefore, it is a promising therapeutic target. Here, we show that RNA interference against FLT3 with an internal tandem duplication (FLT3-ITD) potentiates the efficacy of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) against AML cells expressing FLT3-ITD. Similar to RNA interference, tyrosine kinase inhibitors (TKI; AC220/cpd.102/PKC412) in combination with LBH589 exhibit superior activity against AML cells. Median dose-effect analyses of drug-induced apoptosis rates of AML cells (MV4-11 and MOLM-13) revealed combination index (CI) values indicating strong synergism. AC220, the most potent and FLT3-specific TKI, shows highest synergism with LBH589 in the low nanomolar range. A 4-hour exposure to LBH589 + AC220 already generates more than 50% apoptosis after 24 hours. Different cell lines lacking FLT3-ITD as well as normal peripheral blood mononuclear cells are not significantly affected by LBH589 + TKI, showing the specificity of this treatment regimen. Immunoblot analyses show that LBH589 + TKI induce apoptosis via degradation of FLT3-ITD and its prosurvival target STAT5. Previously, we showed the LBH589-induced proteasomal degradation of FLT3-ITD. Here, we show that activated caspase-3 also contributes to the degradation of FLT3-ITD and that STAT5 is a direct target of this protease. Our data strongly emphasize HDACi/TKI drug combinations as promising modality for the treatment of FLT3-ITD-positive AMLs. ©2012 AACR.

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.

PubMed

Rathe, Susan K; Moriarity, Branden S; Stoltenberg, Christopher B; Kurata, Morito; Aumann, Natalie K; Rahrmann, Eric P; Bailey, Natashay J; Melrose, Ellen G; Beckmann, Dominic A; Liska, Chase R; Largaespada, David A

2014-08-13

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC₅₀ of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

Increase in Ara-C cytotoxicity in the presence of valproate, a histone deacetylase inhibitor, is associated with the concurrent expression of cyclin D1 and p27(Kip 1) in acute myeloblastic leukemia cells.

PubMed

Siitonen, Timo; Koistinen, Pirjo; Savolainen, Eeva-Riitta

2005-11-01

The effects of valproate and butyrate were investigated in an acute myeloblastic cell line (OCI/AML-2) on cytotoxicity, cell cycle profile and expression of cell cycle regulating proteins in the presence of cytarabine (Ara-C) and etoposide. As a single agent valproate and butyrate inhibited AML cell growth but did not significantly induce cell death. A dramatic increase in cytotoxicity was observed when combining valproate or butyrate with Ara-C, whereas, co-addition of them with etoposide had much smaller effect on cell death. Valproate induced a clear G1 phase arrest and up-regulated cyclin D1 expression in the presence of Ara-C and etoposide. In addition, valporate was able to block the Ara-C-induced down-regulation of p27(Kip1) expression but not that induced by etoposide.

Therapeutic effect of Northern Labrador tea extracts for acute myeloid leukemia.

PubMed

McGill, Colin M; Tomco, Patrick L; Ondrasik, Regina M; Belknap, Kaitlyn C; Dwyer, Gaelen K; Quinlan, Daniel J; Kircher, Thomas A; Andam, Cheryl P; Brown, Timothy J; Claxton, David F; Barth, Brian M

2018-04-27

Acute myeloid leukemia (AML) is an aggressive hematological malignancy that is one of the more common pediatric malignancies in addition to occurring with high incidence in the aging population. Unfortunately, these patient groups are quite sensitive to toxicity from chemotherapy. Northern Labrador tea, or Rhododendron tomentosum Harmaja (a.k.a. Ledum palustre subsp. decumbens) or "tundra tea," is a noteworthy medicinal plant used by indigenous peoples in Alaska, Canada, and Greenland to treat a diversity of ailments. However, laboratory investigations of Northern Labrador tea, and other Labrador tea family members, as botanical sources for anticancer compounds have been limited. Utilizing an AML cell line in both in vitro and in vivo studies, as well as in vitro studies using primary human AML patient samples, this study demonstrated for the first time that Northern Labrador tea extracts can exert anti-AML activity and that this may be attributed to ursolic acid as a constituent component. Therefore, this medicinal herb holds the potential to serve as a source for further drug discovery efforts to isolate novel anti-AML compounds. Copyright © 2018 John Wiley & Sons, Ltd.

Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells

PubMed Central

Zhang, Xin; Nelson, Erik; Sattler, Martin; Liu, Feiyang; Nicolais, Maria; Zhang, Jianming; Mitsiades, Constantine; Smith, Robert W.; Stone, Richard; Galinsky, Ilene; Nonami, Atsushi; Griffin, James D.; Gray, Nathanael

2013-01-01

Objectives Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells. Methods We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy. Results and Conclusions Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment. PMID:23437141

Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, G.S.; Wang, C.; Minkin, S.

The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. The authors report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C).more » In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) suicide technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR.« less

Association of a murine leukaemia stem cell gene signature based on nucleostemin promoter activity with prognosis of acute myeloid leukaemia in patients.

PubMed

Ali, Mohamed A E; Naka, Kazuhito; Yoshida, Akiyo; Fuse, Kyoko; Kasada, Atsuo; Hoshii, Takayuki; Tadokoro, Yuko; Ueno, Masaya; Ohta, Kumiko; Kobayashi, Masahiko; Takahashi, Chiaki; Hirao, Atsushi

2014-07-18

Acute myeloid leukaemia (AML) is a heterogeneous neoplastic disorder in which a subset of cells function as leukaemia-initiating cells (LICs). In this study, we prospectively evaluated the leukaemia-initiating capacity of AML cells fractionated according to the expression of a nucleolar GTP binding protein, nucleostemin (NS). To monitor NS expression in living AML cells, we generated a mouse AML model in which green fluorescent protein (GFP) is expressed under the control of a region of the NS promoter (NS-GFP). In AML cells, NS-GFP levels were correlated with endogenous NS mRNA. AML cells with the highest expression of NS-GFP were very immature blast-like cells, efficiently formed leukaemia colonies in vitro, and exhibited the highest leukaemia-initiating capacity in vivo. Gene expression profiling analysis revealed that cell cycle regulators and nucleotide metabolism-related genes were highly enriched in a gene set associated with leukaemia-initiating capacity that we termed the 'leukaemia stem cell gene signature'. This gene signature stratified human AML patients into distinct clusters that reflected prognosis, demonstrating that the mouse leukaemia stem cell gene signature is significantly associated with the malignant properties of human AML. Further analyses of gene regulation in leukaemia stem cells could provide novel insights into diagnostic and therapeutic approaches to AML. Copyright © 2014 Elsevier Inc. All rights reserved.

Activity of the hypoxia-activated prodrug, TH-302, in preclinical human acute myeloid leukemia models.

PubMed

Portwood, Scott; Lal, Deepika; Hsu, Yung-Chun; Vargas, Rodrigo; Johnson, Megan K; Wetzler, Meir; Hart, Charles P; Wang, Eunice S

2013-12-01

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm. Recent evidence has shown the bone marrow microenvironment in patients with AML to be intrinsically hypoxic. Adaptive cellular responses by leukemia cells to survive under low oxygenation also confer chemoresistance. We therefore asked whether therapeutic exploitation of marrow hypoxia via the hypoxia-activated nitrogen mustard prodrug, TH-302, could effectively inhibit AML growth. We assessed the effects of hypoxia and TH-302 on human AML cells, primary samples, and systemic xenograft models. We observed that human AML cells and primary AML colonies cultured under chronic hypoxia (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic controls. TH-302 treatment resulted in dose- and hypoxia-dependent apoptosis and cell death in diverse AML cells. TH-302 preferentially decreased proliferation, reduced HIF-1α expression, induced cell-cycle arrest, and enhanced double-stranded DNA breaks in hypoxic AML cells. Hypoxia-induced reactive oxygen species by AML cells were also diminished. In systemic human AML xenografts (HEL, HL60), TH-302 [50 mg/kg intraperitoneally (i.p.) 5 times per week] inhibited disease progression and prolonged overall survival. TH-302 treatment reduced the number of hypoxic cells within leukemic bone marrows and was not associated with hematologic toxicities in nonleukemic or leukemic mice. Later initiation of TH-302 treatment in advanced AML disease was as effective as earlier TH-302 treatment in xenograft models. Our results establish the preclinical activity of TH-302 in AML and provide the rationale for further clinical studies of this and other hypoxia-activated agents for leukemia therapy. ©2013 AACR.

Augmentation of autologous T cell reactivity with acute myeloid leukemia (AML) blasts by Toll-like receptor (TLR) agonists

PubMed Central

Zhong, RuiKun; Li, Hongying; Messer, Karen; Lane, Thomas A.; Zhou, Jiehua; Ball, Edward D.

2016-01-01

This study investigated whether TNF-α, Toll-like receptors (TLRs) 7/8 agonist resiquimod (R848), the TLR4 agonist lipopolysaccharide (LPS) and their combinations can enhance autologous AML-reactive T cell generation in an in vitro culture. AML peripheral blood or bone marrow mononuclear cells were cultured in medium supplemented with GM-CSF/IL-4 to induce dendritic cell (DC) differentiation of AML blasts (AML-DC). The impact of TNF-α, LPS, R848 and their combinations on AML-DC cultures was analyzed. Significantly enhanced CD80, CD40, CD83, CD54, HLADR and CD86 expression of AML cells was observed by addition of TNF-α, LPS, R848 alone or combinations. Induced CD80 expression of AML cells was significantly higher through the combination of TNF-α, LPS and R848 (T + L + R) than that by T alone. CTL induced from T + L + R, T + R, T + L, L + R and R, but not T, L alone stimulated cultures showed significantly higher IFN-γ release than the medium control in response to autologous AML cells. IFN-γ release by T + L + R was significantly higher than T or L alone, and T + R was significantly higher than T alone. CTL generated from T + L + R, T + L, T + R, L + R and L alone exerted significantly higher AML cell killing than medium control. AML cell killing by T + L + R and T + R was significantly higher than T or R alone. These results indicate that the combination of T + L + R induces a significantly enhanced antigen presentation effect of AML-DC. We speculate that the complementary effects of reagent combinations may better address the heterogeneity of responses to any single agent in AML cells from different patients. PMID:25795133

ABCC4 Is a Determinant of Cytarabine‐Induced Cytotoxicity and Myelosuppression

PubMed Central

Drenberg, CD; Hu, S; Li, L; Buelow, DR; Orwick, SJ; Gibson, AA; Schuetz, JD; Sparreboom, A

2016-01-01

Resistance to cytarabine remains a major challenge in the treatment of acute myeloid leukemia (AML). Based on previous studies implicating ABCC4/MRP4 in the transport of nucleosides, we hypothesized that cytarabine is sensitive to ABCC4‐mediated efflux, thereby decreasing its cytotoxic response against AML blasts. The uptake of cytarabine and its monophosphate metabolite was found to be facilitated in ABCC4‐expressing vesicles and intracellular retention was significantly impaired by overexpression of human ABCC4 or mouse Abcc4 (P < 0.05). ABCC4 was expressed highly in AML primary blasts and cell lines, and cytotoxicity of cytarabine in cells was increased in the presence of the ABCC4 inhibitors MK571 or sorafenib, as well as after ABCC4 siRNA. In Abcc4‐null mice, cytarabine‐induced hematological toxicity was enhanced and ex vivo colony‐forming assays showed that Abcc4‐deficiency sensitized myeloid progenitors to cytarabine. Collectively, these studies demonstrate that ABCC4 plays a protective role against cytarabine‐mediated insults in leukemic and host myeloid cells. PMID:26842729

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

PubMed

Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X

1998-01-01

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

Downregulation of miR‑135a predicts poor prognosis in acute myeloid leukemia and regulates leukemia progression via modulating HOXA10 expression.

PubMed

Xu, Hongwei; Wen, Quan

2018-05-23

MicroRNA‑135a (miR‑135a) has been shown to exert important roles in various human cancer types, such as glioblastoma, thyroid carcinoma and renal carcinoma. However, the function of miR‑135a in acute myeloid leukemia (AML) remains largely unknown. In the present study, it was demonstrated that miR‑135a expression was significantly downregulated in AML cells compared with normal control cells. Furthermore, the downregulation of miR‑135a in patients with AML predicted poor prognosis. Through functional experiments, overexpression of miR‑135a was demonstrated to significantly inhibit the proliferation and cell cycle of AML cells, while it promoted cellular apoptosis. miR‑135a directly targeted HOXA10 in AML cells. miR‑135a overexpression significantly suppressed the mRNA and protein levels of HOXA10 in AML cells. Moreover, there was an inverse association between miR‑135a expression and HOXA10 level in AML samples. Additionally, by ectopic expression of HOXA10, restoration of HOXA10 significantly abolished the effects of miR‑135a overexpression on AML cell proliferation, cell cycle and apoptosis. In conclusion, the present study demonstrated that miR‑135a serves as a tumor suppressor in AML by targeting HOXA10, and miR‑135a may be a promising prognostic biomarker for AML patients.

Trib2 expression in granulocyte-monocyte progenitors drives a highly drug resistant acute myeloid leukaemia linked to elevated Bcl2

PubMed Central

O’Connor, Caitriona; Yalla, Krishna; Salomé, Mara; Moka, Hothri Ananyambica; Castañeda, Eduardo Gómez; Eyers, Patrick A.; Keeshan, Karen

2018-01-01

Trib2 pseudokinase has oncogenic and tumour suppressive functions depending on the cellular context. We investigated the ability of Trib2 to transform different haemopoietic stem and progenitor cells (HSPCs). Our study identified the granulocyte-macrophage progenitor (GMP) subpopulation as a potent leukaemia initiating cell of Trib2-driven AML in vivo. Trib2 transformed GMPs generated a fully penetrant and short latency AML. AML cells expressing elevated Trib2 led to a chemoresistant phenotype following chemotherapy treatment. We show that Trib2 overexpression results in an increase in BCL2 expression, and high Trib2 expressing cells are highly sensitive to cell killing by BCL2 inhibition (ABT199). Combined treatment with chemotherapeutic agents and BCL2 inhibition resulted in synergistic killing of Trib2+ AML cells. Trib2 transformed GMP AML cells showed more chemoresistance compared with HSPC derived Trib2 AML cells associated with higher Bcl2 expression. There is significant correlation of high TRIB2 and BCL2 expression in patient derived human AML cells. These data demonstrate that the cell of origin influences the leukaemic profile and chemotherapeutic response of Trib2+ AML. Combined TRIB2 and BCL2 expression in AML cells may have clinical utility relevant for monitoring drug resistance and disease relapse. PMID:29599919

Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells

PubMed Central

Alfano, Daniela; Gorrasi, Anna; Li Santi, Anna; Ricci, Patrizia; Montuori, Nunzia; Selleri, Carmine; Ragno, Pia

2015-01-01

The urokinase-type plasminogen activator (uPA) receptor (uPAR) focuses uPA proteolytic activity on the cell membrane, promoting localized degradation of extracellular matrix (ECM), and binds vitronectin (VN), mediating cell adhesion to the ECM. uPAR-bound uPA and VN induce proteolysis-independent intracellular signalling, regulating cell adhesion, migration, survival and proliferation. uPAR cross-talks with CXCR4, the receptor for the stroma-derived factor 1 chemokine. CXCR4 is crucial in the trafficking of hematopoietic stem cells from/to the bone marrow, which involves also uPAR. Both uPAR and CXCR4 are expressed in acute myeloid leukaemia (AML), with a lower expression in undifferentiated and myeloid subsets, and higher expression in myelomonocytic and promyelocytic subsets. We hypothesized a microRNA (miR)-mediated co-regulation of uPAR and CXCR4 expression, which could allow their cross-talk at the cell surface. We identified three miRs, miR-146a, miR-335 and miR-622, regulating the expression of both uPAR and CXCR4 in AML cell lines. Indeed, these miRs directly target the 3′untranslated region of both uPAR- and CXCR4-mRNAs; accordingly, uPAR/CXCR4 expression is reduced by their overexpression in AML cells and increased by their specific inhibitors. Overexpression of all three miRs impairs migration, invasion and proliferation of myelomonocytic cells. Interestingly, we observed an inverse relationship between uPAR/CXCR4 expression and miR-146a and miR-335 levels in AML blasts, suggesting their possible role in the regulation of uPAR/CXCR4 expression also in vivo. PMID:26082201

All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo.

PubMed

Ma, Hayley S; Greenblatt, Sarah M; Shirley, Courtney M; Duffield, Amy S; Bruner, J Kyle; Li, Li; Nguyen, Bao; Jung, Eric; Aplan, Peter D; Ghiaur, Gabriel; Jones, Richard J; Small, Donald

2016-06-09

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) portends a poor prognosis, and ineffective targeting of the leukemic stem cell (LSC) population remains one of several obstacles in treating this disease. All-trans retinoic acid (ATRA) has been used in several clinical trials for the treatment of nonpromyelocytic AML with limited clinical activity observed. FLT3 tyrosine kinase inhibitors (TKIs) used as monotherapy also achieve limited clinical responses and are thus far unable to affect cure rates in AML patients. We explored the efficacy of combining ATRA and FLT3 TKIs to eliminate FLT3/internal tandem duplication (ITD)(+) LSCs. Our studies reveal highly synergistic drug activity, preferentially inducing apoptosis in FLT3/ITD(+) cell lines and patient samples. Colony-forming unit assays further demonstrate decreased clonogenicity of FLT3/ITD(+) cells upon treatment with ATRA and TKI. Most importantly, the drug combination depletes FLT3/ITD(+) LSCs in a genetic mouse model of AML, and prolongs survival of leukemic mice. Furthermore, engraftment of primary FLT3/ITD(+) patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drug's apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD(+) LSCs and reduce the rate of relapse in AML patients with FLT3 mutations.

The rarity of ALDH(+) cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients.

PubMed

Hoang, Van T; Buss, Eike C; Wang, Wenwen; Hoffmann, Isabel; Raffel, Simon; Zepeda-Moreno, Abraham; Baran, Natalia; Wuchter, Patrick; Eckstein, Volker; Trumpp, Andreas; Jauch, Anna; Ho, Anthony D; Lutz, Christoph

2015-08-01

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH(+) cells; <1.9%; ALDH-rare AML), whereas 24 patients had relatively numerous ALDH(+) cells (≥1.9%; ALDH-numerous AML). In patients with ALDH-rare AML, normal HSC could be separated by their CD34(+) ALDH(+) phenotype, whereas LSC were exclusively detected among CD34(+) ALDH(-) cells. For patients with ALDH-numerous AML, the CD34(+) ALDH(+) subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH(+) cells from ALDH-numerous AML were quiescent, refractory to ARA-C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long-term outcome were also characteristic for patients with ALDH-numerous AML providing an additional risk-stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible. © 2014 UICC.

The novel HSP90 inhibitor NVP-AUY922 shows synergistic anti-leukemic activity with cytarabine in vivo.

PubMed

Wendel, Torunn; Zhen, Yan; Suo, Zenhe; Bruheim, Skjalg; Wiedlocha, Antoni

2016-01-15

HSP90 is a molecular chaperone essential for stability, activity and intracellular sorting of many proteins, including oncoproteins, such as tyrosine kinases, transcription factors and cell cycle regulatory proteins. Therefore, inhibitors of HSP90 are being investigated for their potential as anti-cancer drugs. Here we show that the HSP90 inhibitor NVP-AUY922 induced degradation of the fusion oncoprotein FOP2-FGFR1 in a human acute myeloid leukemia (AML) cell line, KG-1a. Concordantly, downstream signaling cascades, such as STAT1, STAT3 and PLCγ were abrogated. At concentrations that caused FOP2-FGFR1 degradation and signaling abrogation, NVP-AUY922 treatment caused significant cell death and inhibition of proliferation of KG-1a cells in vitro. In an animal model for AML, NVP-AUY922 administrated alone showed no anti-leukemic activity. However, when NVP-AUY922 was administered in combination with cytarabine, the two compounds showed significant synergistic anti-leukemic activity in vivo. Thus NVP-AUY922 and cytarabine combination therapy might be a prospective strategy for AML treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

The biologic properties of recombinant human thrombopoietin in the proliferation and megakaryocytic differentiation of acute myeloblastic leukemia cells.

PubMed

Matsumura, I; Kanakura, Y; Kato, T; Ikeda, H; Horikawa, Y; Ishikawa, J; Kitayama, H; Nishiura, T; Tomiyama, Y; Miyazaki, H; Matsuzawa, Y

1996-10-15

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis. However, the biologic effects of TPO on human acute myeloblastic leukemia (AML) cells are largely unknown. To determine if recombinant human (rh) TPO has proliferation-supporting and differentiation-inducing activities in AML cells, 15 cases of AML cells that were exclusively composed of undifferentiated leukemia cells and showed growth response to rhTPO in a short-term culture (72 hours) were subjected to long-term suspension culture with or without rhTPO. Of 15 cases, rhTPO supported proliferation of AML cells for 2 to 4 weeks in 4 cases whose French-American-British subtypes were M0, M2, M4, and M7, respectively. In addition to the proliferation-supporting activity, rhTPO was found to induce AML cells to progress to some degree of megakaryocytic differentiation at both morphologic and surface-phenotypic level in 2 AML cases with M0 and M7 subtypes. The treatment of AML cells with rhTPO resulted in rapid tyrosine phosphorylation of the TPO-receptor, c-mpl, and STAT3 in all of cases tested. By contrast, the expression of erythroid/megakaryocyte-specific transcription factors (GATA-1, GATA-2, and NF-E2) was markedly induced or enhanced in only 2 AML cases that showed megakaryocytic differentiation in response to rhTPO. These results suggested that, at least in a fraction of AML cases, TPO could not only support the proliferation of AML cells irrespective of AML subtypes, but could also induce megakaryocytic differentiation, possibly through activation of GATA-1, GATA-2, and NF-E2.

Fatty Acid Binding Protein FABP4 Mechanistically Links Obesity with Aggressive AML by Enhancing Aberrant DNA Methylation in AML Cells

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhang, YW; Rao, EY; Bode, AM; Al-Kali, A; Zhang, DE; Litzow, MR; Li, B; Liu, SJ

2016-01-01

Obesity is becoming more prevalent worldwide and is a major risk factor for cancer development. Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a frequently fatal disease. Here, we investigated the molecular mechanisms by which obesity favors AML growth and uncovered the fatty acid binding protein 4 (FABP4) and DNA methyltransferase 1 (DNMT1) regulatory axis that mediates aggressive AML in obesity. We showed that leukemia burden was much higher in high-fat diet-induced obese mice, which had higher levels of FABP4 and IL-6 in sera. Upregulation of environmental and cellular FABP4 accelerated AML cell growth in both a cell-autonomous and cell-non-autonomous manner. Genetic disruption of FABP4 in AML cells or in mice blocked cell proliferation in vitro and induced leukemia regression in vivo. Mechanistic investigations showed that FABP4 upregulation increased IL-6 expression and STAT3 phosphorylation leading to DNMT1 overexpression and further silencing of the p15INK4B tumor suppressor gene in AML cells. Conversely, FABP4 ablation reduced DNMT1-dependent DNA methylation and restored p15INK4B expression, thus conferring substantial protection against AML growth. Our findings reveal the FABP4/DNMT1 axis in the control of AML cell fate in obesity, and suggest that interference with the FABP4/DNMT1 axis might be a new strategy to treat leukemia. PMID:27885273

Fatty acid-binding protein FABP4 mechanistically links obesity with aggressive AML by enhancing aberrant DNA methylation in AML cells.

PubMed

Yan, F; Shen, N; Pang, J X; Zhang, Y W; Rao, E Y; Bode, A M; Al-Kali, A; Zhang, D E; Litzow, M R; Li, B; Liu, S J

2017-06-01

Obesity is becoming more prevalent worldwide and is a major risk factor for cancer development. Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a frequently fatal disease. Here we investigated the molecular mechanisms by which obesity favors AML growth and uncovered the fatty acid-binding protein 4 (FABP4) and DNA methyltransferase 1 (DNMT1) regulatory axis that mediates aggressive AML in obesity. We showed that leukemia burden was much higher in high-fat diet-induced obese mice, which had higher levels of FABP4 and interleukin (IL)-6 in the sera. Upregulation of environmental and cellular FABP4 accelerated AML cell growth in both a cell-autonomous and cell-non-autonomous manner. Genetic disruption of FABP4 in AML cells or in mice blocked cell proliferation in vitro and induced leukemia regression in vivo. Mechanistic investigations showed that FABP4 upregulation increased IL-6 expression and signal transducer and activator of transcription factor 3 phosphorylation leading to DNMT1 overexpression and further silencing of the p15 INK4B tumor-suppressor gene in AML cells. Conversely, FABP4 ablation reduced DNMT1-dependent DNA methylation and restored p15 INK4B expression, thus conferring substantial protection against AML growth. Our findings reveal the FABP4/DNMT1 axis in the control of AML cell fate in obesity and suggest that interference with the FABP4/DNMT1 axis might be a new strategy to treat leukemia.

Effect of thalidomide and arsenic trioxide on the release of tumor necrosis factor-α and vascular endothelial growth factor from the KG-1a human acute myelogenous leukemia cell line.

PubMed

Girgis, Erian H; Mahoney, John P; Khalil, Rafaat H; Soliman, Magdi R

2010-07-01

Studies conducted in our lab have indicated that thalidomide cytotoxicity in the KG-1a human acute myelogenous leukemia (AML) cell line was enhanced by combining it with arsenic trioxide. The current investigation was conducted in order to evaluate the effect of thalidomide either alone or in combination with arsenic trioxide on the release of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) from this cell line in an attempt to clarify its possible cytotoxic mechanism(s). Human AML cell line KG-1a was used in this study. The cells were cultured for 48 h in the presence or absence of thalidomide (5 mg/l), and or arsenic trioxide (4 μM). The levels of TNF-α and VEGF in the supernatant were determined by ELISA. Results obtained indicate that the levels of TNF-α in the supernatant of KG-1a cell cultures incubated with thalidomide, arsenic trioxide, or combination were statistically lower than those observed in the supernatant of control cells (2.89, 5.07, 4.15 and 16.88 pg/ml, respectively). However, the levels of VEGF in the supernatant of thalidomide-treated cells were statistically higher than those in the supernatant of control cells (69.61 vs. 11.48 pg/l). Arsenic trioxide, whether alone or in combination with thalidomide, did not produce any statistically significant difference in the levels of VEGF as compared to the control or thalidomide-treated cell supernatant. These findings indicate that thalidomide and the arsenic trioxide inhibition of TNF-α production by KG-1a cells may play an important role in their cytotoxic effect.

Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells

PubMed Central

George, Joshy; Uyar, Asli; Young, Kira; Kuffler, Lauren; Waldron-Francis, Kaiden; Marquez, Eladio; Ucar, Duygu; Trowbridge, Jennifer J.

2016-01-01

The precise identity of a tumour's cell of origin can influence disease prognosis and outcome. Methods to reliably define tumour cell of origin from primary, bulk tumour cell samples has been a challenge. Here we use a well-defined model of MLL-rearranged acute myeloid leukaemia (AML) to demonstrate that transforming haematopoietic stem cells (HSCs) and multipotent progenitors results in more aggressive AML than transforming committed progenitor cells. Transcriptome profiling reveals a gene expression signature broadly distinguishing stem cell-derived versus progenitor cell-derived AML, including genes involved in immune escape, extravasation and small GTPase signal transduction. However, whole-genome profiling of open chromatin reveals precise and robust biomarkers reflecting each cell of origin tested, from bulk AML tumour cell sampling. We find that bulk AML tumour cells exhibit distinct open chromatin loci that reflect the transformed cell of origin and suggest that open chromatin patterns may be leveraged as prognostic signatures in human AML. PMID:27397025

Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

PubMed Central

Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

2016-01-01

The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

Inhibition of KSP by ARRY-520 Induces Cell Cycle Block and Cell Death via the Mitochondrial Pathway in AML Cells

PubMed Central

Carter, Bing Z.; Mak, Duncan H.; Woessner, Richard; Gross, Stefan; Schober, Wendy D.; Estrov, Zeev; Kantarjian, Hagop; Andreeff, Michael

2013-01-01

Kinesin spindle protein (KSP), a microtubule-associated motor protein essential for cell cycle progression, is overexpressed in many cancers and a potential anti-tumor target. We found that inhibition of KSP by a selective inhibitor, ARRY-520, blocked cell cycle progression, leading to apoptosis in acute myeloid leukemia cell lines which express high levels of KSP. Knockdown of p53, overexpression of XIAP, and mutation in caspase-8 did not significantly affect sensitivity to ARRY-520, suggesting that the response is independent of p53, XIAP, and the extrinsic apoptotic pathway. Although ARRY-520 induced mitotic arrest in both HL-60 and Bcl-2-overexpressing HL-60Bcl-2 cells, cell death was blunted in HL-60Bcl-2 cells, suggesting that the apoptotic program is executed through the mitochondrial pathway. Accordingly, inhibition of Bcl-2 by ABT-737 was synergistic with ARRY-520 in HL-60Bcl-2 cells. Furthermore, ARRY-520 increased Bim protein levels prior to caspase activation in HL-60 cells. ARRY-520 significantly inhibited tumor growth of xenografts in SCID mice and inhibited AML blast but not normal colony formation, supporting a critical role for KSP in proliferation of leukemic progenitor cells. These results demonstrate that ARRY-520 potently induces cell cycle block and subsequent death in leukemic cells via the mitochondrial pathway and has potential to eradicate AML progenitor cells. PMID:19458629

Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia – Impact on enzyme activity and response to cytotoxics

PubMed Central

Morad, Samy A. F.; Tan, Su-Fern; Feith, David J.; Kester, Mark; Claxton, David F.; Loughran, Thomas P.; Barth, Brian M.; Fox, Todd E.; Cabot, Myles C.

2015-01-01

The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N –desmethyltamoxifen (DMT) block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 μM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT, i ) increased the levels of endogenous C16:0- and C24:1 ceramide molecular species, ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, iii ) drastically reduced levels of sphingosine ( to 9% of control), and iv ) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. Co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug resistant setting. PMID:25769964

Pin1 inhibition exerts potent activity against acute myeloid leukemia through blocking multiple cancer-driving pathways.

PubMed

Lian, Xiaolan; Lin, Yu-Min; Kozono, Shingo; Herbert, Megan K; Li, Xin; Yuan, Xiaohong; Guo, Jiangrui; Guo, Yafei; Tang, Min; Lin, Jia; Huang, Yiping; Wang, Bixin; Qiu, Chenxi; Tsai, Cheng-Yu; Xie, Jane; Cao, Ziang Jeff; Wu, Yong; Liu, Hekun; Zhou, Xiaozhen; Lu, Kunping; Chen, Yuanzhong

2018-05-30

The increasing genomic complexity of acute myeloid leukemia (AML), the most common form of acute leukemia, poses a major challenge to its therapy. To identify potent therapeutic targets with the ability to block multiple cancer-driving pathways is thus imperative. The unique peptidyl-prolyl cis-trans isomerase Pin1 has been reported to promote tumorigenesis through upregulation of numerous cancer-driving pathways. Although Pin1 is a key drug target for treating acute promyelocytic leukemia (APL) caused by a fusion oncogene, much less is known about the role of Pin1 in other heterogeneous leukemia. The mRNA and protein levels of Pin1 were detected in samples from de novo leukemia patients and healthy controls using real-time quantitative RT-PCR (qRT-PCR) and western blot. The establishment of the lentiviral stable-expressed short hairpin RNA (shRNA) system and the tetracycline-inducible shRNA system for targeting Pin1 were used to analyze the biological function of Pin1 in AML cells. The expression of cancer-related Pin1 downstream oncoproteins in shPin1 (Pin1 knockdown) and Pin1 inhibitor all-trans retinoic acid (ATRA) treated leukemia cells were examined by western blot, followed by evaluating the effects of genetic and chemical inhibition of Pin1 in leukemia cells on transformed phenotype, including cell proliferation and colony formation ability, using trypan blue, cell counting assay, and colony formation assay in vitro, as well as the tumorigenesis ability using in vivo xenograft mouse models. First, we found that the expression of Pin1 mRNA and protein was significantly increased in both de novo leukemia clinical samples and multiple leukemia cell lines, compared with healthy controls. Furthermore, genetic or chemical inhibition of Pin1 in human multiple leukemia cell lines potently inhibited multiple Pin1 substrate oncoproteins and effectively suppressed leukemia cell proliferation and colony formation ability in cell culture models in vitro. Moreover, tetracycline-inducible Pin1 knockdown and slow-releasing ATRA potently inhibited tumorigenicity of U937 and HL-60 leukemia cells in xenograft mouse models. We demonstrate that Pin1 is highly overexpressed in human AML and is a promising therapeutic target to block multiple cancer-driving pathways in AML.

Survival in acute myeloid leukemia is associated with NKp44 splice variants

PubMed Central

Hadad, Uzi; Teltsh, Omri; Edri, Avishay; Rubin, Eitan; Campbell, Kerry S.; Rosental, Benyamin; Porgador, Angel

2016-01-01

NKp44 is a receptor encoded by the NCR2 gene, which is expressed by cytokine-activated natural killer (NK) cells that are involved in anti-AML immunity. NKp44 has three splice variants corresponding to NKp44ITIM+ (NKp44-1) and NKp44ITIM− (NKp44-2, and NKp44-3) isoforms. RNAseq data of AML patients revealed similar survival of NKp46+NKp44+ and NKp46+NKp44− patients. However, if grouped according to the NKp44 splice variant profile, NKp44-1 expression was significantly associated with poor survival of AML patients. Moreover, activation of PBMC from healthy controls showed co-dominant expression of NKp44-1 and NKp44-3, while primary NK clones show more diverse NKp44 splice variant profiles. Cultured primary NK cells resulted in NKp44-1 dominance and impaired function associated with PCNA over-expression by target cells. This impaired functional phenotype could be rescued by blocking of NKp44 receptor. Human NK cell lines revealed co-dominant expression of NKp44-1 and NKp44-3 and showed a functional phenotype that was not inhibited by PCNA over-expression. Furthermore, transfection-based overexpression of NKp44-1, but not NKp44-2/NKp44-3, reversed the endogenous resistance of NK-92 cells to PCNA-mediated inhibition, and resulted in poor formation of stable lytic immune synapses. This research contributes to the understanding of AML prognosis by shedding new light on the functional implications of differential splicing of NKp44. PMID:27102296

Disruption of Redox Homeostasis in Tumor Necrosis Factor-Induced Apoptosis in a Murine Hepatocyte Cell Line

PubMed Central

Pierce, Robert H.; Campbell, Jean S.; Stephenson, Alyssa B.; Franklin, Christopher C.; Chaisson, Michelle; Poot, Martin; Kavanagh, Terrance J.; Rabinovitch, Peter S.; Fausto, Nelson

2000-01-01

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours ∼50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFκB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant α-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses. PMID:10880392

Discovery of a BTK/MNK dual inhibitor for lymphoma and leukemia.

PubMed

Wu, H; Hu, C; Wang, A; Weisberg, E L; Chen, Y; Yun, C-H; Wang, W; Liu, Y; Liu, X; Tian, B; Wang, J; Zhao, Z; Liang, Y; Li, B; Wang, L; Wang, B; Chen, C; Buhrlage, S J; Qi, Z; Zou, F; Nonami, A; Li, Y; Fernandes, S M; Adamia, S; Stone, R M; Galinsky, I A; Wang, X; Yang, G; Griffin, J D; Brown, J R; Eck, M J; Liu, J; Gray, N S; Liu, Q

2016-01-01

Bruton's tyrosine kinase (BTK) kinase is a member of the TEC kinase family and is a key regulator of the B-cell receptor (BCR)-mediated signaling pathway. It is important for B-cell maturation, proliferation, survival and metastasis. Pharmacological inhibition of BTK is clinically effective against a variety of B-cell malignances, such as mantle cell lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and activated B-cell-diffuse large B-cell lymphoma. MNK kinase is one of the key downstream regulators in the RAF-MEK-ERK signaling pathway and controls protein synthesis via regulating the activity of eIF4E. Inhibition of MNK activity has been observed to moderately inhibit the proliferation of AML cells. Through a structure-based drug-design approach, we have discovered a selective and potent BTK/MNK dual kinase inhibitor (QL-X-138), which exhibits covalent binding to BTK and noncovalent binding to MNK. Compared with the BTK kinase inhibitor (PCI-32765) and the MNK kinase inhibitor (cercosporamide), QL-X-138 enhanced the antiproliferative efficacies in vitro against a variety of B-cell cancer cell lines, as well as AML and CLL primary patient cells, which respond moderately to BTK inhibitor in vitro. The agent can effectively arrest the growth of lymphoma and leukemia cells at the G0-G1 stage and can induce strong apoptotic cell death. These primary results demonstrate that simultaneous inhibition of BTK and MNK kinase activity might be a new therapeutic strategy for B-cell malignances.

Immunogenic apoptosis in human acute myeloid leukemia (AML): primary human AML cells expose calreticulin and release heat shock protein (HSP) 70 and HSP90 during apoptosis.

PubMed

Fredly, Hanne; Ersvær, Elisabeth; Gjertsen, Bjørn-Tore; Bruserud, Oystein

2011-06-01

Several previous studies have demonstrated that both conventional cytotoxic drugs as well as targeted therapeutics can induce apoptosis in primary human acute myelogenous leukemia (AML) cells. However, the apoptotic phenotype of dying AML cells has been less extensively characterized. Even though specific antileukemic immune reactivity is important in AML, especially for allotransplanted patients, it has not been investigated whether dying primary human AML cells show phenotypic characteristics consistent with immunogenic apoptosis [calreticulin exposure, heat shock protein (HSP) release]. We therefore investigated whether in vitro cultured primary human acute myeloid leukemia (AML) cells show calreticulin exposure and HSP70/HSP90 release during spontaneous (stress-induced) apoptosis when cultured in medium alone and when cultured in the presence of antileukemic drugs. Both surface exposure of calreticulin and release of HSP70 and HSP90 was detected but showed a wide variation between patients. This variation was also maintained when the AML cells were cultured in the presence of cytotoxic drugs (cytarabine, daunorubicin, mitomycin), all-trans retinoic acid (ATRA) and valproic acid. Finally, AML cells collected during in vivo ATRA therapy showed increased calreticulin exposure during spontaneous in vitro apoptosis, suggesting that in vivo pharmacotherapy can modulate the apoptotic phenotype. To conclude, apoptotic AML cells can show phenotypic characteristics consistent with immunogenic apoptosis, but there is a wide variation between patients and the level of calreticulin exposure/HSP release seems to depend on individual patient characteristics rather than the apoptosis-inducing agent.

Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia

PubMed Central

Milella, Michele; Kornblau, Steven M.; Estrov, Zeev; Carter, Bing Z.; Lapillonne, Hélène; Harris, David; Konopleva, Marina; Zhao, Shourong; Estey, Elihu; Andreeff, Michael

2001-01-01

The mitogen-activated protein kinase (MAPK) pathway regulates growth and survival of many cell types, and its constitutive activation has been implicated in the pathogenesis of a variety of malignancies. In this study we demonstrate that small-molecule MEK inhibitors (PD98059 and PD184352) profoundly impair cell growth and survival of acute myeloid leukemia (AML) cell lines and primary samples with constitutive MAPK activation. These agents abrogate the clonogenicity of leukemic cells but have minimal effects on normal hematopoietic progenitors. MEK blockade also results in sensitization to spontaneous and drug-induced apoptosis. At a molecular level, these effects correlate with modulation of the expression of cyclin-dependent kinase inhibitors (p27Kip1 and p21Waf1/CIP1) and antiapoptotic proteins of the inhibitor of apoptosis proteins (IAP) and Bcl-2 families. Interruption of constitutive MEK/MAPK signaling therefore represents a promising therapeutic strategy in AML. PMID:11560954

Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation.

PubMed

Bousquet, Marina; Quelen, Cathy; Rosati, Roberto; Mansat-De Mas, Véronique; La Starza, Roberta; Bastard, Christian; Lippert, Eric; Talmant, Pascaline; Lafage-Pochitaloff, Marina; Leroux, Dominique; Gervais, Carine; Viguié, Franck; Lai, Jean-Luc; Terre, Christine; Beverlo, Berna; Sambani, Costantina; Hagemeijer, Anne; Marynen, Peter; Delsol, Georges; Dastugue, Nicole; Mecucci, Cristina; Brousset, Pierre

2008-10-27

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34(+) cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

Gamma-irradiation enhances apoptosis induced by cannabidiol, a non-psychotropic cannabinoid, in cultured HL-60 myeloblastic leukemia cells.

PubMed

Gallily, Ruth; Even-Chena, Tal; Katzavian, Galia; Lehmann, Dan; Dagan, Arie; Mechoulam, Raphael

2003-10-01

Two non-psychotropic cannabinoids, cannabidiol (CBD) and cannabidiol-dimethylheptyl (CBD-DMH), induced apoptosis in a human acute myeloid leukemia (AML) HL-60 cell line. Apoptosis was determined by staining with bisBenzimide and propidium iodide. A dose dependent increase of apoptosis was noted, reaching 61 and 43% with 8 microg/ml CBD and 15 microg/ml CBD-DMH, respectively, after a 24 h treatment. Prior exposure of the cells to gamma-irradiation (800 cGy) markedly enhanced apoptosis, reaching values of 93 and 95%, respectively. Human monocytes from normal individuals were resistant to either cannabinoids or gamma-irradiation. Caspase-3 activation was observed after the cannabinoid treatment, and may represent a mechanism for the apoptosis. Our data suggest a possible new approach to treatment of AML.

Tumor Trp53 status and genotype affect the bone marrow microenvironment in acute myeloid leukemia

PubMed Central

Jacamo, Rodrigo; Davis, R. Eric; Ling, Xiaoyang; Sonnylal, Sonali; Wang, Zhiqiang; Ma, Wencai; Zhang, Min; Ruvolo, Peter; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Lowe, Scott; Zuber, Johannes; Kornblau, Steven M.; Konopleva, Marina; Andreeff, Michael

2017-01-01

The genetic heterogeneity of acute myeloid leukemia (AML) and the variable responses of individual patients to therapy suggest that different AML genotypes may influence the bone marrow (BM) microenvironment in different ways. We performed gene expression profiling of bone marrow mesenchymal stromal cells (BM-MSC) isolated from normal C57BL/6 mice or mice inoculated with syngeneic murine leukemia cells carrying different human AML genotypes, developed in mice with Trp53 wild-type or nullgenetic backgrounds. We identified a set of genes whose expression in BM-MSC was modulated by all four AML genotypes tested. In addition, there were sets of differentially-expressed genes in AML-exposed BM-MSC that were unique to the particular AML genotype or Trp53 status. Our findings support the hypothesis that leukemia cells alter the transcriptome of surrounding BM stromal cells, in both common and genotype-specific ways. These changes are likely to be advantageous to AML cells, affecting disease progression and response to chemotherapy, and suggest opportunities for stroma-targeting therapy, including those based on AML genotype. PMID:29137349

Epigenetically induced ectopic expression of UNCX impairs the proliferation and differentiation of myeloid cells

PubMed Central

Daniele, Giulia; Simonetti, Giorgia; Fusilli, Caterina; Iacobucci, Ilaria; Lonoce, Angelo; Palazzo, Antonio; Lomiento, Mariana; Mammoli, Fabiana; Marsano, Renè Massimiliano; Marasco, Elena; Mantovani, Vilma; Quentmeier, Hilmar; Drexler, Hans G; Ding, Jie; Palumbo, Orazio; Carella, Massimo; Nadarajah, Niroshan; Perricone, Margherita; Ottaviani, Emanuela; Baldazzi, Carmen; Testoni, Nicoletta; Papayannidis, Cristina; Ferrari, Sergio; Mazza, Tommaso; Martinelli, Giovanni; Storlazzi, Clelia Tiziana

2017-01-01

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1. We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML. PMID:28411256

Epigenetically induced ectopic expression of UNCX impairs the proliferation and differentiation of myeloid cells.

PubMed

Daniele, Giulia; Simonetti, Giorgia; Fusilli, Caterina; Iacobucci, Ilaria; Lonoce, Angelo; Palazzo, Antonio; Lomiento, Mariana; Mammoli, Fabiana; Marsano, Renè Massimiliano; Marasco, Elena; Mantovani, Vilma; Quentmeier, Hilmar; Drexler, Hans G; Ding, Jie; Palumbo, Orazio; Carella, Massimo; Nadarajah, Niroshan; Perricone, Margherita; Ottaviani, Emanuela; Baldazzi, Carmen; Testoni, Nicoletta; Papayannidis, Cristina; Ferrari, Sergio; Mazza, Tommaso; Martinelli, Giovanni; Storlazzi, Clelia Tiziana

2017-07-01

We here describe a leukemogenic role of the homeobox gene UNCX , activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX -positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1 , was revealed. Similar results were obtained in UNCX -transduced CD34 + cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1 We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX , associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML. Copyright© 2017 Ferrata Storti Foundation.

RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia

PubMed Central

Zuber, Johannes; Shi, Junwei; Wang, Eric; Rappaport, Amy R.; Herrmann, Harald; Sison, Edward A.; Magoon, Daniel; Qi, Jun; Blatt, Katharina; Wunderlich, Mark; Taylor, Meredith J.; Johns, Christopher; Chicas, Agustin; Mulloy, James C.; Kogan, Scott C.; Brown, Patrick; Valent, Peter; Bradner, James E.; Lowe, Scott W.; Vakoc, Christopher R.

2012-01-01

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention. PMID:21814200

Azelaic Acid Exerts Antileukemic Activity in Acute Myeloid Leukemia

PubMed Central

Pan, Yunbao; Liu, Dong; Wei, Yongchang; Su, Dan; Lu, Chenyang; Hu, Yanchao; Zhou, Fuling

2017-01-01

Acute myeloid leukemia (AML) is an acute leukemia common in most adults; its prevalence intensifies with age. The overall survival of AML is very poor because of therapeutic resistance. Azelaic acid (AZA) is non-toxic, non-teratogenic, and non-mutagenic and its antitumor effect on various tumor cells is well established; Nonetheless, its therapeutic effects in AML cells are largely unknown. In this study, it was shown that AZA significantly inhibits the cell viability and induces apoptosis in AML cells in a dose-dependent manner. Additionally, AZA suppressed the expression of phosphorylated Akt, Jab1 and Trx, and this suppression was enhanced by treatment with Jab1 siRNA. Furthermore, AZA sensitized AML cells to Ara-c chemotherapy. The suppressive effect of AZA on tumor growth was examined in vivo by subcutaneously inoculated AML cells in a tumor model using nude mice. These findings indicate that AZA is useful as an effective ingredient in antineoplastic activity. PMID:28659796

Azelaic Acid Exerts Antileukemic Activity in Acute Myeloid Leukemia.

PubMed

Pan, Yunbao; Liu, Dong; Wei, Yongchang; Su, Dan; Lu, Chenyang; Hu, Yanchao; Zhou, Fuling

2017-01-01

Acute myeloid leukemia (AML) is an acute leukemia common in most adults; its prevalence intensifies with age. The overall survival of AML is very poor because of therapeutic resistance. Azelaic acid (AZA) is non-toxic, non-teratogenic, and non-mutagenic and its antitumor effect on various tumor cells is well established; Nonetheless, its therapeutic effects in AML cells are largely unknown. In this study, it was shown that AZA significantly inhibits the cell viability and induces apoptosis in AML cells in a dose-dependent manner. Additionally, AZA suppressed the expression of phosphorylated Akt, Jab1 and Trx, and this suppression was enhanced by treatment with Jab1 siRNA. Furthermore, AZA sensitized AML cells to Ara-c chemotherapy. The suppressive effect of AZA on tumor growth was examined in vivo by subcutaneously inoculated AML cells in a tumor model using nude mice. These findings indicate that AZA is useful as an effective ingredient in antineoplastic activity.

ABCC4 Is a Determinant of Cytarabine-Induced Cytotoxicity and Myelosuppression.

PubMed

Drenberg, C D; Hu, S; Li, L; Buelow, D R; Orwick, S J; Gibson, A A; Schuetz, J D; Sparreboom, A; Baker, S D

2016-02-01

Resistance to cytarabine remains a major challenge in the treatment of acute myeloid leukemia (AML). Based on previous studies implicating ABCC4/MRP4 in the transport of nucleosides, we hypothesized that cytarabine is sensitive to ABCC4-mediated efflux, thereby decreasing its cytotoxic response against AML blasts. The uptake of cytarabine and its monophosphate metabolite was found to be facilitated in ABCC4-expressing vesicles and intracellular retention was significantly impaired by overexpression of human ABCC4 or mouse Abcc4 (P < 0.05). ABCC4 was expressed highly in AML primary blasts and cell lines, and cytotoxicity of cytarabine in cells was increased in the presence of the ABCC4 inhibitors MK571 or sorafenib, as well as after ABCC4 siRNA. In Abcc4-null mice, cytarabine-induced hematological toxicity was enhanced and ex vivo colony-forming assays showed that Abcc4-deficiency sensitized myeloid progenitors to cytarabine. Collectively, these studies demonstrate that ABCC4 plays a protective role against cytarabine-mediated insults in leukemic and host myeloid cells. © 2016 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Silver nanoparticles exhibit size-dependent differential toxicity and induce expression of syncytin-1 in FA-AML1 and MOLT-4 leukaemia cell lines.

PubMed

Alqahtani, Sultan; Promtong, Pawika; Oliver, Anthony W; He, Xiaotong T; Walker, Thomas D; Povey, Andrew; Hampson, Lynne; Hampson, Ian N

2016-11-01

Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp's being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Peptide microarray profiling identifies phospholipase C gamma 1 (PLC-γ1) as a potential target for t(8;21) AML

PubMed Central

Mahmud, Hasan; Scherpen, Frank J.G.; de Boer, Tiny Meeuwsen; Lourens, Harm-Jan; Schoenherr, Caroline; Eder, Matthias; Scherr, Michaela; Guryev, Victor; De Bont, Eveline S.

2017-01-01

The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) which has a need for improved therapeutic strategies. We found PLC-γ1 as one of the highest phosphorylated peptides in t(8;21) AML samples compared to NBM or CN-AML in our previous peptide microarray. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in t(8;21) AML. In this study, PLC-γ1 expression was significantly higher in t(8;21) AML compared to other karyotypes. The PLC-γ1 protein expression was suppressed in AML1-ETO knock down cells indicating that it might induce kasumi-1 cell death. ShRNA-mediated PLC-γ1 knockdown in kasumi-1 cells significantly blocked cell growth, induced apoptosis and cell cycle arrest which was explained by the increased activation of apoptotic related and cell cycle regulatory protein expressions. Gene expression array analysis showed the up-regulation of apoptotic and DNA damage response genes together with the downregulation of cell growth, proliferation and differentiation genes in the PLC-γ1 suppressed kasumi-1 cells, consistent with the observed phenotypic effects. Importantly, PLC-γ1 suppressed kasumi-1 cells showed higher chemosensitivity to the chemotherapeutic drug treatments and lower cell proliferation upon hypoxic stress. Taken together, these in vitro finding strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cells and identify PLC-γ1 as a potential therapeutic target for t(8;21) AML treatment. PMID:28978037

Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-11-10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

Pharmacological targeting of the PI3K/mTOR pathway alters the release of angioregulatory mediators both from primary human acute myeloid leukemia cells and their neighboring stromal cells.

PubMed

Reikvam, Håkon; Nepstad, Ina; Bruserud, Øystein; Hatfield, Kimberley Joanne

2013-06-01

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects.

Pharmacological targeting of the PI3K/mTOR pathway alters the release of angioregulatory mediators both from primary human acute myeloid leukemia cells and their neighboring stromal cells

PubMed Central

Reikvam, Håkon; Nepstad, Ina; Bruserud, Øystein; Hatfield, Kimberley Joanne

2013-01-01

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects. PMID:23919981

Classification of biological cells using a sound wave based flow cytometer

NASA Astrophysics Data System (ADS)

Strohm, Eric M.; Gnyawali, Vaskar; Van De Vondervoort, Mia; Daghighi, Yasaman; Tsai, Scott S. H.; Kolios, Michael C.

2016-03-01

A flow cytometer that uses sound waves to determine the size of biological cells is presented. In this system, a microfluidic device made of polydimethylsiloxane (PDMS) was developed to hydrodynamically flow focus cells in a single file through a target area. Integrated into the microfluidic device was an ultrasound transducer with a 375 MHz center frequency, aligned opposite the transducer was a pulsed 532 nm laser focused into the device by a 10x objective. Each passing cell was insonfied with a high frequency ultrasound pulse, and irradiated with the laser. The resulting ultrasound and photoacoustic waves from each cell were analyzed using signal processing methods, where features in the power spectra were compared to theoretical models to calculate the cell size. Two cell lines with different size distributions were used to test the system: acute myeloid leukemia cells (AML) and melanoma cells. Over 200 cells were measured using this system. The average calculated diameter of the AML cells was 10.4 +/- 2.5 μm using ultrasound, and 11.4 +/- 2.3 μm using photoacoustics. The average diameter of the melanoma cells was 16.2 +/- 2.9 μm using ultrasound, and 18.9 +/- 3.5 μm using photoacoustics. The cell sizes calculated using ultrasound and photoacoustic methods agreed with measurements using a Coulter Counter, where the AML cells were 9.8 +/- 1.8 μm and the melanoma cells were 16.0 +/- 2.5 μm. These results demonstrate a high speed method of assessing cell size using sound waves, which is an alternative method to traditional flow cytometry techniques.

MicroRNA-29b mediates altered innate immune development in acute leukemia

PubMed Central

Mundy-Bosse, Bethany L.; Scoville, Steven D.; Chen, Li; McConnell, Kathleen; Mao, Hsiaoyin C.; Ahmed, Elshafa H.; Zorko, Nicholas; Harvey, Sophia; Cole, Jordan; Zhang, Xiaoli; Costinean, Stefan; Croce, Carlo M.; Larkin, Karilyn; Byrd, John C.; Vasu, Sumithira; Blum, William; Yu, Jianhua; Freud, Aharon G.; Caligiuri, Michael A.

2016-01-01

Natural killer (NK) cells can have potent antileukemic activity following haplo-mismatched, T cell–depleted stem cell transplantations for the treatment of acute myeloid leukemia (AML), but they are not successful in eradicating de novo AML. Here, we have used a mouse model of de novo AML to elucidate the mechanisms by which AML evades NK cell surveillance. NK cells in leukemic mice displayed a marked reduction in the cytolytic granules perforin and granzyme B. Further, as AML progressed, we noted the selective loss of an immature subset of NK cells in leukemic mice and in AML patients. This absence was not due to elimination by cell death or selective reduction in proliferation, but rather to the result of a block in NK cell differentiation. Indeed, NK cells from leukemic mice and humans with AML showed lower levels of TBET and EOMES, transcription factors that are critical for terminal NK cell differentiation. Further, the microRNA miR-29b, a regulator of T-bet and EOMES, was elevated in leukemic NK cells. Finally, deletion of miR-29b in NK cells reversed the depletion of this NK cell subset in leukemic mice. These results indicate that leukemic evasion of NK cell surveillance occurs through miR-mediated dysregulation of lymphocyte development, representing an additional mechanism of immune escape in cancer. PMID:27775550

HBV life cycle is restricted in mouse hepatocytes expressing human NTCP.

PubMed

Li, Hanjie; Zhuang, Qiuyu; Wang, Yuze; Zhang, Tianying; Zhao, Jinghua; Zhang, Yali; Zhang, Junfang; Lin, Yi; Yuan, Quan; Xia, Ningshao; Han, Jiahuai

2014-03-01

Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1-6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.

Rheb1 promotes tumor progression through mTORC1 in MLL-AF9-initiated murine acute myeloid leukemia.

PubMed

Gao, Yanan; Gao, Juan; Li, Minghao; Zheng, Yawei; Wang, Yajie; Zhang, Hongyan; Wang, Weili; Chu, Yajing; Wang, Xiaomin; Xu, Mingjiang; Cheng, Tao; Ju, Zhenyu; Yuan, Weiping

2016-04-12

The constitutive hyper-activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways has frequently been associated with acute myeloid leukemia (AML). While many inhibitors targeting these pathways have been developed, the anti-leukemic effect was not as robust as expected. As part of the molecular link between PI3K/Akt and mTOR kinase, the role of Rheb1 in AML remains unexplored. Our study aims to explore the role of Rheb1 in AML and estimate whether Rheb1 could be a potential target of AML treatment. The expressions of Rheb1 and other indicated genes were analyzed using real-time PCR. AML mouse model was established by retrovirus transduction. Leukemia cell properties and related signaling pathways were dissected by in vitro and in vivo studies. The transcriptional changes were analyzed via gene chip analysis. Molecular reagents including mTOR inhibitor and mTOR activator were used to evaluate the function of related signaling pathway in the mouse model. We observed that Rheb1 is overexpressed in AML patients and the change of Rheb1 level in AML patients is associated with their median survival. Using a Rheb1-deficient MLL-AF9 murine AML model, we revealed that Rheb1 deletion prolonged the survival of AML mice by weakening LSC function. In addition, Rheb1 deletion arrested cell cycle progression and enhanced apoptosis of AML cells. Furthermore, while Rheb1 deletion reduced mTORC1 activity in AML cells, additional rapamycin treatment further decreased mTORC1 activity and increased the apoptosis of Rheb1 (Δ/Δ) AML cells. The mTOR activator 3BDO partially rescued mTORC1 signaling and inhibited apoptosis in Rheb1 (Δ/Δ) AML cells. Our data suggest that Rheb1 promotes AML progression through mTORC1 signaling pathway and combinational drug treatments targeting Rheb1 and mTOR might have a better therapeutic effect on leukemia.

Anti-apoptotic ARC protein confers chemoresistance by controlling leukemia-microenvironment interactions through a NFκB/IL1β signaling network

PubMed Central

Carter, Bing Z.; Mak, Po Yee; Chen, Ye; Mak, Duncan H.; Mu, Hong; Jacamo, Rodrigo; Ruvolo, Vivian; Arold, Stefan T.; Ladbury, John E.; Burks, Jared K.; Kornblau, Steven; Andreeff, Michael

2016-01-01

To better understand how the apoptosis repressor with caspase recruitment domain (ARC) protein confers drug resistance in acute myeloid leukemia (AML), we investigated the role of ARC in regulating leukemia-mesenchymal stromal cell (MSC) interactions. In addition to the previously reported effect on AML apoptosis, we have demonstrated that ARC enhances migration and adhesion of leukemia cells to MSCs both in vitro and in a novel human extramedullary bone/bone marrow mouse model. Mechanistic studies revealed that ARC induces IL1β expression in AML cells and increases CCL2, CCL4, and CXCL12 expression in MSCs, both through ARC-mediated activation of NFκB. Expression of these chemokines in MSCs increased by AML cells in an ARC/IL1β-dependent manner; likewise, IL1β expression was elevated when leukemia cells were co-cultured with MSCs. Further, cells from AML patients expressed the receptors for and migrated toward CCL2, CCL4, and CXCL12. Inhibition of IL1β suppressed AML cell migration and sensitized the cells co-cultured with MSCs to chemotherapy. Our results suggest the existence of a complex ARC-regulated circuit that maintains intimate connection of AML with the tumor microenvironment through NFκB/IL1β-regulated chemokine receptor/ligand axes and reciprocal crosstalk resulting in cytoprotection. The data implicate ARC as a promising drug target to potentially sensitize AML cells to chemotherapy. PMID:26956049

Histone Deacetylase Inhibitors Target the Leukemic Microenvironment by Enhancing a Nherf1-Protein Phosphatase 1α-TAZ Signaling Pathway in Osteoblasts*

PubMed Central

Kremer, Kimberly N.; Dudakovic, Amel; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Kaufmann, Scott H.; Westendorf, Jennifer J.; van Wijnen, Andre J.; Hedin, Karen E.

2015-01-01

Disrupting the protective signals provided by the bone marrow microenvironment will be critical for more effective combination drug therapies for acute myeloid leukemia (AML). Cells of the osteoblast lineage that reside in the endosteal niche have been implicated in promoting survival of AML cells. Here, we investigated how to prevent this protective interaction. We previously showed that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis of AML cells, unless the leukemic cells receive protective signals provided by differentiating osteoblasts (8, 10). We now identify a novel signaling pathway in differentiating osteoblasts that can be manipulated to disrupt the osteoblast-mediated protection of AML cells. Treating differentiating osteoblasts with histone deacetylase inhibitors (HDACi) abrogated their ability to protect co-cultured AML cells from SDF-1-induced apoptosis. HDACi prominently up-regulated expression of the Nherf1 scaffold protein, which played a major role in preventing osteoblast-mediated protection of AML cells. Protein phosphatase-1α (PP1α) was identified as a novel Nherf1 interacting protein that acts as the downstream mediator of this response by promoting nuclear localization of the TAZ transcriptional modulator. Moreover, independent activation of either PP1α or TAZ was sufficient to prevent osteoblast-mediated protection of AML cells even in the absence of HDACi. Together, these results indicate that HDACi target the AML microenvironment by enhancing activation of the Nherf1-PP1α-TAZ pathway in osteoblasts. Selective drug targeting of this osteoblast signaling pathway may improve treatments of AML by rendering leukemic cells in the bone marrow more susceptible to apoptosis. PMID:26491017

KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activity in preclinical models of T-cell acute lymphoblastic leukaemia and acute myeloid leukaemia.

PubMed

Etchin, Julia; Sanda, Takaomi; Mansour, Marc R; Kentsis, Alex; Montero, Joan; Le, Bonnie T; Christie, Amanda L; McCauley, Dilara; Rodig, Scott J; Kauffman, Michael; Shacham, Sharon; Stone, Richard; Letai, Anthony; Kung, Andrew L; Thomas Look, A

2013-04-01

This study explored the anti-leukaemic efficacy of novel irreversible inhibitors of the major nuclear export receptor, chromosome region maintenance 1 (CRM1, also termed XPO1). We found that these novel CRM1 antagonists, termed SINE (Selective Inhibitors of Nuclear Export), induced rapid apoptosis at low nanomolar concentrations in a panel of 14 human T-cell acute lymphoblastic leukaemia (T-ALL) cell lines representing different molecular subtypes of the disease. To assess in vivo anti-leukaemia cell activity, we engrafted immunodeficient mice intravenously with the human T-ALL MOLT-4 cells, which harbour activating mutations of NOTCH1 and NRAS as well as loss of function of the CDKN2A, PTEN and TP53 tumour suppressors and express a high level of oncogenic transcription factor TAL1. Importantly, we examined the in vivo anti-leukaemic efficacy of the clinical SINE compound KPT-330 against T-ALL and acute myeloid leukaemia (AML) cells. These studies demonstrated striking in vivo activity of KPT-330 against T-ALL and AML cells, with little toxicity to normal murine haematopoietic cells. Taken together, our results show that SINE CRM1 antagonists represent promising 'first-in-class' drugs with a novel mechanism of action and wide therapeutic index, and imply that drugs of this class show promise for the targeted therapy of T-ALL and AML. © 2013 Blackwell Publishing Ltd.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

PubMed Central

Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

The targeted histone deacetylase inhibitor tefinostat (CHR-2845) shows selective in vitro efficacy in monocytoid-lineage leukaemias

PubMed Central

Zabkiewicz, Joanna; Gilmour, Marie; Hills, Robert; Vyas, Pares; Bone, Elizabeth; Davidson, Alan; Burnett, Alan; Knapper, Steven

2016-01-01

Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase (HDAC) inhibitor which is cleaved into its active acid by the intracellular esterase human carboxylesterase-1 (hCE-1). The in vitro efficacy of tefinostat was characterised in cell lines and in a cohort of 73 primary AML and CMML samples. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in myelomonocytic (M4), monocytic/monoblastic (M5) and CMML samples in comparison to non-monocytoid AML sub-types (p = 0.007). Importantly, no growth inhibitory effects were seen in normal bone marrow CD34+ cells exposed to AML-toxic doses of tefinostat in clonogenic assays. Expression of hCE-1 was measured by intracellular flow cytometry and immunoblotting across the cohort, with highest levels seen in M5 AML patients. hCE-1 levels correlated with significantly increased tefinostat sensitivity (low EC50) as measured by growth inhibition assays (p = 0.001) and concomitant elevation of the mature monocytoid marker CD14+. Strong induction of intracellular histone protein acetylation was observed in tefinostat-responsive samples, as were high levels of the DNA damage sensor γ-H2A.X, highlighting potential biomarkers of patient responsiveness. Synergistic interaction between tefinostat and the current standard treatment cytarabine was demonstrated in dose response and clonogenic assays using simultaneous drug addition in primary samples (median Combination Index value = 0.51). These data provide a strong rationale for the further clinical evaluation of tefinostat in monocytoid-lineage haematological neoplasms including CMML and monocyte-lineage AMLs. PMID:26934551

Potent Activity of Ponatinib (AP24534) in Models of FLT3-Driven Acute Myeloid Leukemia and Other Hematologic Malignancies

PubMed Central

Gozgit, Joseph M.; Wong, Matthew J.; Wardwell, Scott; Tyner, Jeffrey W.; Loriaux, Marc M.; Mohemmad, Qurish K.; Narasimhan, Narayana I.; Shakespeare, William C.; Wang, Frank; Druker, Brian J.; Clackson, Tim; Rivera, Victor M.

2011-01-01

Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, fibroblast growth factor receptor 1 (FGFR1), and platelet derived growth factor receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to 20 nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD+/+) but not RS4;11 (FLT3-ITD−/−) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD–driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα. PMID:21482694

CD8+T cells expressing both PD-1 and TIGIT but not CD226 are dysfunctional in acute myeloid leukemia (AML) patients.

PubMed

Wang, Mengjie; Bu, Jin; Zhou, Maohua; Sido, Jessica; Lin, Yu; Liu, Guanfang; Lin, Qiwen; Xu, Xiuzhang; Leavenworth, Jianmei W; Shen, Erxia

2018-05-01

Acute myeloid leukemia (AML) is one of the most common types of leukemia among adults with an overall poor prognosis and very limited treatment management. Immune checkpoint blockade of PD-1 alone or combined with other immune checkpoint blockade has gained impressive results in murine AML models by improving anti-leukemia CD8 + T cell function, which has greatly promoted the strategy to utilize combined immune checkpoint inhibitors to treat AML patients. However, the expression profiles of these immune checkpoint receptors, such as co-inhibitory receptors PD-1 and TIGIT and co-stimulatory receptor CD226, in T cells from AML patients have not been clearly defined. Here we have defined subsets of CD8 + and CD4 + T cells in the peripheral blood (PB) from newly diagnosed AML patients and healthy controls (HCs). We have observed increased frequencies of PD-1- and TIGIT- expressing CD8 + T cells but decreased occurrence of CD226-expressing CD8 + T cells in AML patients. Further analysis of these CD8 + T cells revealed a unique CD8 + T cell subset that expressed PD-1 and TIGIT but displayed lower levels of CD226 was associated with failure to achieve remission after induction chemotherapy and FLT3-ITD mutations which predict poor clinical prognosis in AML patients. Importantly, these PD-1 + TIGIT + CD226 - CD8 + T cells are dysfunctional with lower expression of intracellular IFN-γ and TNF-α than their counterparts in HCs. Therefore, our studies revealed that an increased frequency of a unique CD8 + T cell subset, PD-1 + TIGIT + CD226 - CD8 + T cells, is associated with CD8 + T cell dysfunction and poor clinical prognosis of AML patients, which may reveal critical diagnostic or prognostic biomarkers and direct more efficient therapeutic strategies. Copyright © 2017. Published by Elsevier Inc.

A randomized assessment of adding the kinase inhibitor lestaurtinib to first-line chemotherapy for FLT3-mutated AML.

PubMed

Knapper, Steven; Russell, Nigel; Gilkes, Amanda; Hills, Robert K; Gale, Rosemary E; Cavenagh, James D; Jones, Gail; Kjeldsen, Lars; Grunwald, Michael R; Thomas, Ian; Konig, Heiko; Levis, Mark J; Burnett, Alan K

2017-03-02

The clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had FLT3 -internal tandem duplication mutations, 23% FLT3 -tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; P = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; P = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed FLT3 -mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside front-line AML treatment. The UK AML15 and AML17 trials are registered at www.isrctn.com/ISRCTN17161961 and www.isrctn.com/ISRCTN55675535 respectively. © 2017 by The American Society of Hematology.

Cannabidiol stimulates Aml-1a-dependent glial differentiation and inhibits glioma stem-like cells proliferation by inducing autophagy in a TRPV2-dependent manner.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Amantini, Consuelo; Liberati, Sonia; Santoni, Matteo; Ricci-Vitiani, Lucia; Pallini, Roberto; Santoni, Giorgio

2015-10-15

Glioma stem-like cells (GSCs) correspond to a tumor cell subpopulation, involved in glioblastoma multiforme (GBM) tumor initiation and acquired chemoresistance. Currently, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population. Recently, the effect of cannabinoids (CBs) in promoting glial differentiation and inhibiting gliomagenesis has been evidenced. Herein, we demonstrated that cannabidiol (CBD) by activating transient receptor potential vanilloid-2 (TRPV2) triggers GSCs differentiation activating the autophagic process and inhibits GSCs proliferation and clonogenic capability. Above all, CBD and carmustine (BCNU) in combination overcome the high resistance of GSCs to BCNU treatment, by inducing apoptotic cell death. Acute myeloid leukemia (Aml-1) transcription factors play a pivotal role in GBM proliferation and differentiation and it is known that Aml-1 control the expression of several nociceptive receptors. So, we evaluated the expression levels of Aml-1 spliced variants (Aml-1a, b and c) in GSCs and during their differentiation. We found that Aml-1a is upregulated during GSCs differentiation, and its downregulation restores a stem cell phenotype in differentiated GSCs. Since it was demonstrated that CBD induces also TRPV2 expression and that TRPV2 is involved in GSCs differentiation, we evaluated if Aml-1a interacted directly with TRPV2 promoters. Herein, we found that Aml-1a binds TRPV2 promoters and that Aml-1a expression is upregulated by CBD treatment, in a TRPV2 and PI3K/AKT dependent manner. Altogether, these results support a novel mechanism by which CBD inducing TRPV2-dependent autophagic process stimulates Aml-1a-dependent GSCs differentiation, abrogating the BCNU chemoresistance in GSCs. © 2015 UICC.

A potential therapeutic target for FLT3-ITD AML: PIM1 Kinase

PubMed Central

Fathi, Amir T.; Arowojolu, Omotayo; Swinnen, Ian; Sato, Takashi; Rajkhowa, Trivikram; Small, Donald; Marmsater, Fredrik; Robinson, John E.; Gross, Stefan David; Martinson, Matthew; Allen, Shelley; Kallan, Nicholas C.; Levis, Mark

2011-01-01

Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) mutation have a poor prognosis, and FLT3 inhibitors are now under clinical investigation. PIM1, a serine/threonine kinase, is up-regulated in FLT3-ITD AML and may be involved in FLT3-mediated leukemogenesis. We employed a PIM1 inhibitor, AR00459339 (Array Biopharma Inc.), to investigate the effect of PIM1 inhibition in FLT3-mutant AML. Like FLT3 inhibitors, AR00459339 was preferentially cytotoxic to FLT3-ITD cells, as demonstrated in the MV4-11, Molm-14, and TF/ITD cell lines, as well as 12 FLT3-ITD primary samples. Unlike FLT3 inhibitors, AR00459339 did not suppress phosphorylation of FLT3, but did promote the de-phosphorylation of downstream FLT3 targets, STAT5, AKT, and BAD. Combining AR00459339 with a FLT3 inhibitor resulted in additive to mildly synergistic cytotoxic effects. AR00459339 was cytotoxic to FLT3-ITD samples from patients with secondary resistance to FLT3 inhibitors, suggesting a novel benefit to combining these agents. We conclude that PIM1 appears to be closely associated with FLT3 signaling, and that inhibition of PIM1 may hold therapeutic promise, either as monotherapy, or by overcoming resistance to FLT3 inhibitors. PMID:21802138

Prognostic significance of huntingtin interacting protein 1 expression on patients with acute myeloid leukemia.

PubMed

Wang, Jinghan; Yu, Mengxia; Guo, Qi; Ma, Qiuling; Hu, Chao; Ma, Zhixin; Yin, Xiufeng; Li, Xia; Wang, Yungui; Pan, Hanzhang; Wang, Dongmei; Huang, Jiansong; Meng, Haitao; Tong, Hongyan; Qian, Wenbin; Jin, Jie

2017-04-28

Huntingtin interacting protein 1 (HIP1) is an endocytic protein which is overexpressed in a variety of human cancers and involved in cancer-causing translocation in leukemia. However, the prognostic impact of HIP1 expression on AML remains unclear. In this study, quantification of HIP1 transcript by real-time quantitative PCR in bone marrow blasts was performed in 270 AML patients. As a result, high HIP1 expression was seen more frequently in older patients, M4/M5 morphology and genes of NPM1 and DNMT3A mutations, and underrepresented in favorable karyotype subgroups and CEBPA double allele mutations in our AML patients. We also found high HIP1 expressers showed lower levels of hemoglobin. In addition, overexpression of HIP1 was associated with an inferior overall survival. The prognostic value of HIP1 expression was validated in patients from an independent TCGA cohort. Notably, up-regulation of miR-16, miR-15a, miR-28 and miR-660 were seen in high HIP1 expressers from the two independent cohorts. In vitro, interfereing of HIP1 expression by siRNA suppressed the proliferation of leukemic cells, and downregulation of these miRNAs were seen in THP-1 and Kasumi cell lines after silencing HIP1 expression. In conclusion, the HIP1 gene expression might serve as a reliable predictor for overall survival in AML patients.

Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

PubMed

Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

2018-05-01

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

mTORC1 is essential for leukemia propagation but not stem cell self-renewal

PubMed Central

Hoshii, Takayuki; Tadokoro, Yuko; Naka, Kazuhito; Ooshio, Takako; Muraguchi, Teruyuki; Sugiyama, Naoyuki; Soga, Tomoyoshi; Araki, Kimi; Yamamura, Ken-ichi; Hirao, Atsushi

2012-01-01

Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence. PMID:22622041

ent-Jungermannenone C Triggers Reactive Oxygen Species-Dependent Cell Differentiation in Leukemia Cells.

PubMed

Yue, Zongwei; Xiao, Xinhua; Wu, Jinbao; Zhou, Xiaozhou; Liu, Weilong; Liu, Yaxi; Li, Houhua; Chen, Guoqiang; Wu, Yingli; Lei, Xiaoguang

2018-02-23

Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal proliferation of myeloid blasts. Despite the progress that has been made in the treatment of various malignant hematopoietic diseases, the effective treatment of AML remains very challenging. Differentiation therapy has emerged as a promising approach for leukemia treatment, and new and effective chemical agents to trigger the differentiation of AML cells, especially drug-resistant cells, are urgently required. Herein, the natural product jungermannenone C, a tetracyclic diterpenoid isolated from liverworts, is reported to induce cell differentiation in AML cells. Interestingly, the unnatural enantiomer of jungermannenone C (1) was found to be more potent than jungermannenone C in inducing cell differentiation. Furthermore, compound 1 targets peroxiredoxins I and II by selectively binding to the conserved cysteine residues and leads to cellular reactive oxygen species accumulation. Accordingly, ent-jungermannenone C (1) shows potential for further investigation as an effective differentiation therapy against AML.

Overexpression of microRNA-125b inhibits human acute myeloid leukemia cells invasion, proliferation and promotes cells apoptosis by targeting NF-κB signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yan; Tang, Ping; Chen, Yanli

microRNA-125b has been reported to play an novel biological function in the progression and development of several kinds of leukemia. However, the detail role of miR-125b in acute myeloid leukemia (AML) is remains largely unknown. The present study aimed to investigate the biological role of miR-125b in AML and the potential molecular mechanism involved in this process. Our results showed that overexpression of miR-125b suppressed AML cells proliferation, invasion and promotes cells apoptosis in a dose-dependent manner, while the miR-NC did not show the same effect. In addition, miR-125b induced AML cells G2/M cell cycle arrest in vitro. Overexpression of miR-125bmore » resulted in a significant decrease of the expression of p-IκB-α and inhibition of IκB-α degradation, and the nuclear translocation of NF-κB subunit p65 was abrogated by miR-125b simutaneously. To further verify that miR-125b targeted NF-κB signaling pathway, the NF-κB-regulated downstream genes that were associated with cell cycle arrest and apoptosis was also determined. The results showed that, miR-125b also affect NF-κB-regulated genes expression involved in cell cycle arrest and apoptosis. In conclusion, the present work certificates that miR-125b can significantly inhibit human AML cells invasion, proliferation and promotes cells apoptosis by targeting the NF-κB signaling pathway, and thus it can be viewed as an promising therapeutic target for AML. - Highlights: • Overexpression of miR-125b suppressed AML cells proliferation, invasion and promotes cells apoptosis. • miR-125b induced AML cells G2/M cell cycle arrest in vitro. • miR-125b suppressed AML cells tumorigenicity and promoted cells apoptosis by targeting NF-κB pathway.« less

Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia

PubMed Central

Kamga, Paul Takam; Bassi, Giulio; Cassaro, Adriana; Midolo, Martina; Di Trapani, Mariano; Gatti, Alessandro; Carusone, Roberta; Resci, Federica; Perbellini, Omar; Gottardi, Michele; Bonifacio, Massimiliano; Kamdje, Armel Hervé Nwabo; Ambrosetti, Achille; Krampera, Mauro

2016-01-01

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB. These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML. PMID:26967055

Cell markers in the recognition of acute myeloblastic leukaemia subtypes.

PubMed

Andoljsek, Dusan; Preloznik Zupan, Irena; Zontar, Darja; Cernelc, Peter; Mlakar, Uros; Modic, Mojca; Pretnar, Joze; Zver, Samo

2002-01-01

The diagnosis of acute myeloblastic leukaemia (AML) is based on cell morphology, cytogenetic and molecular changes, cell markers and clinical data. Our aim was to establish whether morphology and cell markers are comparable in the evaluation of AML. Bone marrow smears were analysed, and flow cytometry and monoclonal antibodies were used to determine cell type and maturity. Morphology and cell markers correlated differently in different AML subtypes.

Inhibition of poly(ADP-ribose) polymerase 1 protects against acute myeloid leukemia by suppressing the myeloproliferative leukemia virus oncogene

PubMed Central

Wang, Lingbo; Cai, Weili; Zhang, Wei; Chen, Xueying; Dong, Wenqian; Tang, Dongqi; Zhang, Yun; Ji, Chunyan; Zhang, Mingxiang

2015-01-01

An abnormal expression of poly(ADP-ribose) polymerase 1 (PARP-1) has been described in many tumors. PARP-1 promotes tumorigenesis and cancer progression by acting on different molecular pathways. PARP-1 inhibitors can be used with radiotherapy or chemotherapy to enhance the susceptibility of tumor cells to the treatment. However, the specific mechanism of PARP-1 in acute myeloid leukemia (AML) remains unknown. Our study showed that expression of PARP-1 was upregulated in AML patients. PARP-1 inhibition slowed AML cell proliferation, arrested the cell cycle, induced apoptosis in vitro and improved AML prognosis in vivo. Mechanistically, microarray assay of AML cells with loss of PARP-1 function revealed that the myeloproliferative leukemia virus oncogene (MPL) was significantly downregulated. In human AML samples, MPL expression was increased, and gain-of-function and loss-of-function analysis demonstrated that MPL promoted cell growth. Moreover, PARP-1 and MPL expression were positively correlated in AML samples, and their overexpression was associated with an unfavorable prognosis. Furthermore, PARP-1 and MPL consistently acted on Akt and ERK1/2 pathways, and the anti-proliferative and pro-apoptotic function observed with PARP-1 inhibition were reversed in part via MPL activation upon thrombopoietin stimulation or gene overexpression. These data highlight the important function of PARP-1 in the progression of AML, which suggest PARP-1 as a potential target for AML treatment. PMID:26314963

Autophagy is dispensable for Kmt2a/Mll-Mllt3/Af9 AML maintenance and anti-leukemic effect of chloroquine.

PubMed

Chen, Xiaoyi; Clark, Jason; Wunderlich, Mark; Fan, Cuiqing; Davis, Ashley; Chen, Song; Guan, Jun-Lin; Mulloy, James C; Kumar, Ashish; Zheng, Yi

2017-05-04

Recently, macroautophagy/autophagy has emerged as a promising target in various types of solid tumor treatment. However, the impact of autophagy on acute myeloid leukemia (AML) maintenance and the validity of autophagy as a viable target in AML therapy remain unclear. Here we show that Kmt2a/Mll-Mllt3/Af9 AML (MA9-AML) cells have high autophagy flux compared with normal bone marrow cells, but autophagy-specific targeting, either through Rb1cc1-disruption to abolish autophagy initiation, or via Atg5-disruption to prevent phagophore (the autophagosome precursor) membrane elongation, does not affect the growth or survival of MA9-AML cells, either in vitro or in vivo. Mechanistically, neither Atg5 nor Rb1cc1 disruption impairs endolysosome formation or survival signaling pathways. The autophagy inhibitor chloroquine shows autophagy-independent anti-leukemic effects in vitro but has no efficacy in vivo likely due to limited achievable drug efficacy in blood. Further, vesicular exocytosis appears to mediate chloroquine resistance in AML cells, and exocytotic inhibition significantly enhances the anti-leukemic effect of chloroquine. Thus, chloroquine can induce leukemia cell death in vitro in an autophagy-independent manner but with inadequate efficacy in vivo, and vesicular exocytosis is a possible mechanism of chloroquine resistance in MA9-AML. This study also reveals that autophagy-specific targeting is unlikely to benefit MA9-AML therapy.

Knockdown of SALL4 Protein Enhances All-trans Retinoic Acid-induced Cellular Differentiation in Acute Myeloid Leukemia Cells*

PubMed Central

Liu, Li; Liu, Liang; Leung, Lai-Han; Cooney, Austin J.; Chen, Changyi; Rosengart, Todd K.; Ma, Yupo; Yang, Jianchang

2015-01-01

All-trans retinoic acid (ATRA) is a differentiation agent that revolutionized the treatment of acute promyelocytic leukemia. However, it has not been useful for other types of acute myeloid leukemia (AML). Here we explored the effect of SALL4, a stem cell factor, on ATRA-induced AML differentiation in both ATRA-sensitive and ATRA-resistant AML cells. Aberrant SALL4 expression has been found in nearly all human AML cases, whereas, in normal bone marrow and peripheral blood cells, its expression is only restricted to hematopoietic stem/progenitor cells. We reason that, in AMLs, SALL4 activation may prevent cell differentiation and/or protect self-renewal that is seen in normal hematopoietic stem/progenitor cells. Indeed, our studies show that ATRA-mediated myeloid differentiation can be largely blocked by exogenous expression of SALL4, whereas ATRA plus SALL4 knockdown causes significantly increased AML differentiation and cell death. Mechanistic studies indicate that SALL4 directly associates with retinoic acid receptor α and modulates ATRA target gene expression. SALL4 is shown to recruit lysine-specific histone demethylase 1 (LSD1) to target genes and alter the histone methylation status. Furthermore, coinhibition of LSD1 and SALL4 plus ATRA treatment exhibited the strongest anti-AML effect. These findings suggest that SALL4 plays an unfavorable role in ATRA-based regimes, highlighting an important aspect of leukemia therapy. PMID:25737450

Myeloid cell differentiation arrest by miR-125b-1 in myelodysplasic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation

PubMed Central

Bousquet, Marina; Quelen, Cathy; Rosati, Roberto; Mansat-De Mas, Véronique; La Starza, Roberta; Bastard, Christian; Lippert, Eric; Talmant, Pascaline; Lafage-Pochitaloff, Marina; Leroux, Dominique; Gervais, Carine; Viguié, Franck; Lai, Jean-Luc; Terre, Christine; Beverlo, Berna; Sambani, Costantina; Hagemeijer, Anne; Marynen, Peter; Delsol, Georges; Dastugue, Nicole; Mecucci, Cristina; Brousset, Pierre

2008-01-01

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34+ cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity. PMID:18936236

Gene expression profiling of acute myeloid leukemia samples from adult patients with AML-M1 and -M2 through boutique microarrays, real-time PCR and droplet digital PCR.

PubMed

Handschuh, Luiza; Kaźmierczak, Maciej; Milewski, Marek C; Góralski, Michał; Łuczak, Magdalena; Wojtaszewska, Marzena; Uszczyńska-Ratajczak, Barbara; Lewandowski, Krzysztof; Komarnicki, Mieczysław; Figlerowicz, Marek

2018-03-01

Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American‑British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1 gene expression levels correlated with the response to therapy. CAT expression was highest in patients who remained longer under complete remission, whereas WT1 expression increased with treatment resistance. On the whole, this study demonstrates that the AML-array can potentially serve as a first-line screening tool, and may be helpful for the diagnosis of AML, whereas the differentiation between AML subgroups can be more successfully performed with PCR-based analysis of a few marker genes.

Homoharringtonine combined with aclarubicin and cytarabine synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein.

PubMed

Cao, Jiang; Feng, Hao; Ding, Ning-Ning; Wu, Qing-Yun; Chen, Chong; Niu, Ming-Shan; Chen, Wei; Qiu, Ting-Ting; Zhu, Hong-Hu; Xu, Kai-Lin

2016-11-01

Homoharringtonine combined with aclarubicin and cytarabine (HAA) is a highly effective treatment for acute myeloid leukemia (AML), especially for t(8;21) AML. However, the underlying mechanisms by which HAA kills t(8;21) AML cells remain unclear. In this study, SKNO-1 and Kasumi-1 cells with t(8;21) were used. Compared with individual or pairwise administration of homoharringtonine, aclarubicin, or cytarabine, HAA showed the strongest inhibition of growth and induction of apoptosis in SKNO-1 and Kasumi-1 cells. HAA caused cleavage of the AML1-ETO (AE) oncoprotein to form truncated AE (ΔAE). Pretreatment with the caspase-3 inhibitor caspase-3 inhibitor Q-DEVD-OPh (QDO) not only suppressed HAA-induced apoptosis but also abrogated the cleavage of AE and generation of ΔAE. These results suggest that HAA synergistically induces apoptosis in t(8;21) leukemia cells and triggers caspase-3-mediated cleavage of the AML1-ETO oncoprotein, thus providing direct evidence for the strong activity of HAA toward t(8;21) AML. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis*

PubMed Central

Wojtuszkiewicz, Anna; Schuurhuis, Gerrit J.; Kessler, Floortje L.; Piersma, Sander R.; Knol, Jaco C.; Pham, Thang V.; Jansen, Gerrit; Musters, René J. P.; van Meerloo, Johan; Assaraf, Yehuda G.; Kaspers, Gertjan J. L.; Zweegman, Sonja; Cloos, Jacqueline; Jimenez, Connie R.

2016-01-01

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance. PMID:26801919

The clinical implications of mixed lymphocyte reaction with leukemic cells.

PubMed

Kim, Hee-Je; Kim, Tai-Gyu; Cho, Hyun Il; Han, Hoon; Min, Woo-Sung; Kim, Chun-Choo

2002-11-01

To evaluate the clinical implications of a mixed lymphocyte reaction between leukemic cells and lymphocytes from HLA-matched sibling donors, we attempted to generate donor-derived, graft-versus-leukemia-effective cells and to define their characteristics. We studied 8 patients with chronic myelogenous leukemia (CML), including 5 patients in the chronic phase (CP), 3 patients in the accelerated phase (AP), and 2 patients with acute myelogenous leukemia (AML) in their first complete remission. Cells from these patients were used as stimulators in a mixed lymphocyte reaction.The effects of natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) were separated by observing tests for cytotoxicity to target cells, including K562 cells, the patient's leukemic cells, and phytohemagglutinin (PHA) blasts. Donor-derived antileukemic CTLs againstthe patient's own leukemic cells are productive in vitro. The efficacy of generating CTLs against leukemic target cells was (in decreasing order) AML, CML-CP, and CML-AP. Cytotoxic activity against leukemic targets was prominent in 4 cases--2 CML-CP and the 2 AML cases. On the contrary, the 3 cases of CML-AP showed low CTL activity. In cases showing 1 positive result among 3 targets (K562 cells, the patient's leukemic cells, and PHA blasts), the relapse rate was significantly lower (P = .022) on follow-up (median, 33 months; 7-40 months) after hematopoietic stem cell transplantation. By a combined analysis of the cytotoxicity effects for all 3 target cells, we were able to demonstrate a correlation between leukemic relapse and the variable degree of the cytotoxicity test results. Although the total sample numbers for this study were low, we speculate that these results may come from differences in the individual characteristics of the leukemic cells that are in line with their clinical disease status.

CD34+CD38-CD123+ Cells Are Present in Virtually All Acute Myeloid Leukaemia Blasts: A Promising Single Unique Phenotype for Minimal Residual Disease Detection.

PubMed

Al-Mawali, Adhra; Pinto, Avinash Daniel; Al-Zadjali, Shoaib

In CD34-positive acute myeloid leukaemia (AML), the leukaemia-initiating event likely takes place in the CD34+CD38- cell compartment. CD123 has been shown to be a unique marker of leukaemic stem cells within the CD34+CD38- compartment. The aim of this study was to identify the percentage of CD34+CD38-CD123+ cells in AML blasts, AML CD34+CD38- stem cells, and normal and regenerating bone marrow CD34+CD38- stem cells from non-myeloid malignancies. Thirty-eight adult de novo AML patients with intention to treat were enrolled after the application of inclusion criteria from February 2012 to February 2017. The percentage of the CD34+CD38-CD123+ phenotype in the blast population at diagnosis was determined using a CD45-gating strategy and CD34+ backgating by flow cytometry. We studied the CD34+CD38-CD123+ fraction in AML blasts at diagnosis, and its utility as a unique phenotype for minimal residual disease (MRD) of AML patients. CD123+ cells were present in 97% of AML blasts in patients at diagnosis (median 90%; range 21-99%). CD123+ cells were also present in 97% of the CD34+CD38- compartment (median 0.8164%, range 0.0262-39.7%). Interestingly, CD123 was not present in normal and regenerating CD34+CD38- bone marrow stem cells (range 0.002- 0.067 and 0.004-0.086, respectively). The CD34+CD38-CD123+ phenotype is present in virtually all AML blasts and it may be used as a unique single phenotype for MRD detection in AML patients. © 2017 The Author(s) Published by S. Karger AG, Basel.

[Expression of c-MPL in leukemic stem cells from acute myeloid leukemia patients].

PubMed

Yu, Pei; Qiu, Shao-Wei; Rao, Qing; Lin, Dong; Xing, Hai-Yan; Tang, Ke-Jing; Tian, Zheng; Wang, Min; Wang, Jian-Xiang

2012-10-01

This study was aimed to investigate the expression of c-MPL in acute myeloid leukemia (AML) and the correlation of the c-MPL expression with CD34 and CD38, so as to define the expression of c-MPL in leukemic stem cells. The expression levels of CD34, CD38 and c-MPL were detected by flow cytometry in bone marrow cells from 29 newly diagnosed AML patients. The relationship of c-MPL positive cell ratio with clinical parameters and correlation of c-MPL with CD34 and CD38 expression in AML patients were analyzed. The results showed that expression level of c-MPL in AML patients was significantly higher than that of normal controls (P < 0.05), and the expression level of c-MPL did not correlate with age, sex, white blood cell count, AML1-ETO fusion gene and remission after chemotherapy, but the expression of c-MPL in M2 and M5 patients was higher than that of normal control (P < 0.05). Expression of c-MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P < 0.01). c-MPL was significantly higher expressed in CD34(+) cells than that in CD34(-) cells (P < 0.001), while c-MPL expression was not significantly different between CD34(+)CD38(-) and CD34(+)CD38(-) cell groups. Positive correlation between c-MPL and CD34 expression was observed (r = 0.380, P = 0.042). It is concluded that expression of c-MPL is higher in AML patients, and positively correlates with the expression level of CD34. The c-MPL expresses in leukemic stem cells.

Concurrent Inhibition of Pim and FLT3 Kinases Enhances Apoptosis of FLT3-ITD Acute Myeloid Leukemia Cells through Increased Mcl-1 Proteasomal Degradation.

PubMed

Kapoor, Shivani; Natarajan, Karthika; Baldwin, Patrick R; Doshi, Kshama A; Lapidus, Rena G; Mathias, Trevor J; Scarpa, Mario; Trotta, Rossana; Davila, Eduardo; Kraus, Manfred; Huszar, Dennis; Tron, Adriana E; Perrotti, Danilo; Baer, Maria R

2018-01-01

Purpose: fms -like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor-induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G 1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234-47. ©2017 AACR . ©2017 American Association for Cancer Research.

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

PubMed

Shlush, Liran I; Zandi, Sasan; Mitchell, Amanda; Chen, Weihsu Claire; Brandwein, Joseph M; Gupta, Vikas; Kennedy, James A; Schimmer, Aaron D; Schuh, Andre C; Yee, Karen W; McLeod, Jessica L; Doedens, Monica; Medeiros, Jessie J F; Marke, Rene; Kim, Hyeoung Joon; Lee, Kwon; McPherson, John D; Hudson, Thomas J; Brown, Andrew M K; Yousif, Fouad; Trinh, Quang M; Stein, Lincoln D; Minden, Mark D; Wang, Jean C Y; Dick, John E

2014-02-20

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

PubMed

Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

2016-01-29

Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis.

PubMed

Dietrich, Philipp A; Yang, Chen; Leung, Halina H L; Lynch, Jennifer R; Gonzales, Estrella; Liu, Bing; Haber, Michelle; Norris, Murray D; Wang, Jianlong; Wang, Jenny Yingzi

2014-11-20

β-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML. © 2014 by The American Society of Hematology.

A novel glycogen synthase kinase-3 inhibitor optimized for acute myeloid leukemia differentiation activity

PubMed Central

Stetson, Lindsay; Ignatz-Hoover, James; Moreton, Stephen; Chakrabarti, Amit; Xia, Zhiqiang; Karan, Goutam; de Lima, Marcos; Agrawal, Mukesh K; Wald, David N

2016-01-01

Standard therapies used for the treatment of Acute Myeloid Leukemia (AML) are cytotoxic agents that target rapidly proliferating cells. Unfortunately, this therapeutic approach has limited efficacy and significant toxicity and the majority of AML patients still die of their disease. In contrast to the poor prognosis of most AML patients, most individuals with a rare subtype of AML, Acute Promyelocytic Leukemia (APL), can be cured by differentiation therapy using regimens containing all-trans retinoic acid. GSK3 has previously been identified as a therapeutic target in AML where its inhibition can lead to the differentiation and growth arrest of leukemic cells. Unfortunately, existing GSK3 inhibitors lead to suboptimal differentiation activity making them less useful as clinical AML differentiation agents. Here we describe the discovery of a novel GSK3 inhibitor, GS87. GS87 was discovered in efforts to optimize GSK3 inhibition for AML differentiation activity. Despite GS87's dramatic ability to induce AML differentiation, kinase profiling reveals its high specificity in targeting GSK3 as compared to other kinases. GS87 demonstrates high efficacy in a mouse AML model system and unlike current AML therapeutics, exhibits little effect on normal bone marrow cells. GS87 induces potent differentiation by more effectively activating GSK3-dependent signaling components including MAPK signaling as compared to other GSK3 inhibitors. GS87 is a novel GSK3 inhibitor with therapeutic potential as a differentiation agent for non-promyelocytic AML. PMID:27196775

Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia.

PubMed

Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

2015-11-25

The challenge for glycogen synthase kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML), may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy.

CpG methylation patterns and decitabine treatment response in acute myeloid leukemia cells and normal hematopoietic precursors

PubMed Central

Negrotto, Soledad; Ng, Kwok Peng; Jankowska, Ania M.; Bodo, Juraj; Gopalan, Banu; Guinta, Kathryn; Mulloy, James C.; Hsi, Eric; Maciejewski, Jaroslaw; Saunthararajah, Yogen

2011-01-01

The DNA hypomethylating drug decitabine maintains normal hematopoietic stem cell (HSC) self-renewal but induces terminal differentiation in acute myeloid leukemia (AML) cells. The basis for these contrasting cell-fates, and for selective CpG hypomethylation by decitabine, is poorly understood. Promoter CpGs, with methylation measured by microarray, were classified by the direction of methylation change with normal myeloid maturation. In AML cells, the methylation pattern at maturation-responsive CpG suggested at least partial maturation. Consistent with partial maturation, in gene expression analyses, AML cells expressed high levels of the key lineage-specifying factor CEBPA, but relatively low levels of the key late-differentiation driver CEBPE. In methylation analysis by mass-spectrometry, CEBPE promoter CpG that are usually hypomethylated during granulocyte maturation were significantly hypermethylated in AML cells. Decitabine treatment induced cellular differentiation of AML cells, and the largest methylation decreases were at CpG that are hypomethylated with myeloid maturation, including CEBPE promoter CpG. In contrast, decitabine-treated normal HSC retained immature morphology, and methylation significantly decreased at CpG that are less methylated in immature cells. High expression of lineage-specifying factor and aberrant epigenetic repression of some key late-differentiation genes distinguishes AML cells from normal HSC and could explain the contrasting differentiation and methylation responses to decitabine. PMID:21836612

Enhancers of Polycomb EPC1 and EPC2 sustain the oncogenic potential of MLL leukemia stem cells

PubMed Central

Huang, Xu; Spencer, Gary J; Lynch, James T; Ciceri, Filippo; Somerville, Tim D D; Somervaille, Tim C P

2013-01-01

Through a targeted knockdown (KD) screen of chromatin regulatory genes we identified the EP400 complex components EPC1 and EPC2 as critical oncogenic co-factors in acute myeloid leukemia (AML). EPC1 and EPC2 were required for the clonogenic potential of human AML cells of multiple molecular subtypes. Focusing on MLL-mutated AML as an exemplar, Epc1 or Epc2 KD induced apoptosis of murine MLL-AF9 AML cells and abolished leukemia stem cell potential. By contrast, normal hematopoietic stem and progenitor cells (HSPC) were spared. Similar selectivity was observed for human primary AML cells versus normal CD34+ HSPC. In keeping with these distinct functional consequences, Epc1 or Epc2 KD induced divergent transcriptional consequences in murine MLL-AF9 granulocyte-macrophage progenitor-like (GMP) cells versus normal GMP, with a signature of increased MYC activity in leukemic but not normal cells. This was caused by accumulation of MYC protein and was also observed following KD of other EP400 complex genes. Pharmacological inhibition of MYC:MAX dimerization, or concomitant MYC KD, reduced apoptosis following EPC1 KD, linking the accumulation of MYC to cell death. Therefore EPC1 and EPC2 are components of a complex which directly or indirectly serves to prevent MYC accumulation and AML cell apoptosis, thus sustaining oncogenic potential. PMID:24166297

Oncogenic deregulation of NKL homeobox gene MSX1 in mantle cell lymphoma.

PubMed

Nagel, Stefan; Ehrentraut, Stefan; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

2014-08-01

NKL homeobox gene MSX1 is physiologically expressed during embryonic hematopoiesis. Here, we detected MSX1 overexpression in three examples of mantle cell lymphoma (MCL) and one of acute myeloid leukemia (AML) by screening 96 leukemia/lymphoma cell lines via microarray profiling. Moreover, in silico analysis identified significant overexpression of MSX1 in 3% each of patients with MCL and AML, confirming aberrant activity in subsets of both types of malignancies. Comparative expression profiling analysis and subsequent functional studies demonstrated overexpression of histone acetyltransferase PHF16 together with transcription factors FOXC1 and HLXB9 as activators of MSX1 transcription. Additionally, we identified regulation of cyclin D1/CCND1 by MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization in MCL cells showed that t(11;14)(q13;q32) results in detachment of CCND1 from its corresponding repressive MSX1 binding site. Taken together, we uncovered regulators and targets of homeobox gene MSX1 in leukemia/lymphoma cells, supporting the view of a recurrent genetic network that is reactivated in malignant transformation.

Prognostic significance of huntingtin interacting protein 1 expression on patients with acute myeloid leukemia

PubMed Central

Wang, Jinghan; Yu, Mengxia; Guo, Qi; Ma, Qiuling; Hu, Chao; Ma, Zhixin; Yin, Xiufeng; Li, Xia; Wang, Yungui; Pan, Hanzhang; Wang, Dongmei; Huang, Jiansong; Meng, Haitao; Tong, Hongyan; Qian, Wenbin; Jin, Jie

2017-01-01

Huntingtin interacting protein 1 (HIP1) is an endocytic protein which is overexpressed in a variety of human cancers and involved in cancer-causing translocation in leukemia. However, the prognostic impact of HIP1 expression on AML remains unclear. In this study, quantification of HIP1 transcript by real-time quantitative PCR in bone marrow blasts was performed in 270 AML patients. As a result, high HIP1 expression was seen more frequently in older patients, M4/M5 morphology and genes of NPM1 and DNMT3A mutations, and underrepresented in favorable karyotype subgroups and CEBPA double allele mutations in our AML patients. We also found high HIP1 expressers showed lower levels of hemoglobin. In addition, overexpression of HIP1 was associated with an inferior overall survival. The prognostic value of HIP1 expression was validated in patients from an independent TCGA cohort. Notably, up-regulation of miR-16, miR-15a, miR-28 and miR-660 were seen in high HIP1 expressers from the two independent cohorts. In vitro, interfereing of HIP1 expression by siRNA suppressed the proliferation of leukemic cells, and downregulation of these miRNAs were seen in THP-1 and Kasumi cell lines after silencing HIP1 expression. In conclusion, the HIP1 gene expression might serve as a reliable predictor for overall survival in AML patients. PMID:28452374

IDH1/2 Mutations Sensitize Acute Myeloid Leukemia to PARP Inhibition and This Is Reversed by IDH1/2-Mutant Inhibitors.

PubMed

Molenaar, Remco J; Radivoyevitch, Tomas; Nagata, Yasunobu; Khurshed, Mohammed; Przychodzen, Bartolomiej; Makishima, Hideki; Xu, Mingjiang; Bleeker, Fonnet E; Wilmink, Johanna W; Carraway, Hetty E; Mukherjee, Sudipto; Sekeres, Mikkael A; van Noorden, Cornelis J F; Maciejewski, Jaroslaw P

2018-04-01

Purpose: Somatic mutations in IDH1/2 occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2 MUT enzymes produce D -2-hydroxyglutarate ( D 2HG), which associates with increased DNA damage and improved responses to chemo/radiotherapy and PARP inhibitors in solid tumor cells. Whether this also holds true for IDH1/2 MUT AML is not known. Experimental Design: Well-characterized primary IDH1 MUT , IDH2 MUT , and IDH1/2 WT AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors. Results: IDH1/2 MUT caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2 MUT inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of IDH1/2 MUT AML cells. We provide evidence that the therapy sensitivity of IDH1/2 MUT cells was caused by D 2HG-mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2 MUT cells. Conclusions: IDH1/2 MUT AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA-damaging agent, such as daunorubicin, whereas concomitant administration of IDH1/2 MUT inhibitors during cytotoxic therapy decrease the efficacy of both agents in IDH1/2 MUT AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in IDH1/2 MUT AML. Clin Cancer Res; 24(7); 1705-15. ©2018 AACR . ©2018 American Association for Cancer Research.

Novel mechanism of regulation of fibrosis in kidney tumor with tuberous sclerosis

PubMed Central

2013-01-01

Background Deficiency in tuberin results in activation the mTOR pathway and leads to accumulation of cell matrix proteins. The mechanisms by which tuberin regulates fibrosis in kidney angiomyolipomas (AMLs) of tuberous sclerosis patients are not fully known. Method In the present study, we investigated the potential role of tuberin/mTOR pathway in the regulation of cell fibrosis in AML cells and kidney tumor tissue from tuberous sclerosis complex (TSC) patients. Results AML cells treated with rapamycin shows a significant decrease in mRNA and protein expression as well as in promoter transcriptional activity of alpha-smooth muscle actin (α-SMA) compared to untreated cells. In addition, cells treated with rapamycin significantly decreased the protein expression of the transcription factor YY1. Rapamycin treatment also results in the redistribution of YY1 from the nucleus to cytoplasm in AML cells. Moreover, cells treated with rapamycin resulted in a significant reduce of binding of YY1 to the αSMA promoter element in nuclear extracts of AML cells. Kidney angiomyolipoma tissues from TSC patients showed lower levels of tuberin and higher levels of phospho-p70S6K that resulted in higher levels of mRNA and protein of αSMA expression compared to control kidney tissues. In addition, most of the α-SMA staining was identified in the smooth muscle cells of AML tissues. YY1 was also significantly increased in tumor tissue of AMLs compared to control kidney tissue suggesting that YY1 plays a major role in the regulation of αSMA. Conclusions These data comprise the first report to provide one mechanism whereby rapamycin might inhibit the cell fibrosis in kidney tumor of TSC patients. PMID:23705901

Discovery of agents that eradicate leukemia stem cells using an in silico screen of public gene expression data

PubMed Central

Hassane, Duane C.; Guzman, Monica L.; Corbett, Cheryl; Li, Xiaojie; Abboud, Ramzi; Young, Fay; Liesveld, Jane L.; Carroll, Martin

2008-01-01

Increasing evidence indicates that malignant stem cells are important for the pathogenesis of acute myelogenous leukemia (AML) and represent a reservoir of cells that drive the development of AML and relapse. Therefore, new treatment regimens are necessary to prevent relapse and improve therapeutic outcomes. Previous studies have shown that the sesquiterpene lactone, parthenolide (PTL), ablates bulk, progenitor, and stem AML cells while causing no appreciable toxicity to normal hematopoietic cells. Thus, PTL must evoke cellular responses capable of mediating AML selective cell death. Given recent advances in chemical genomics such as gene expression-based high-throughput screening (GE-HTS) and the Connectivity Map, we hypothesized that the gene expression signature resulting from treatment of primary AML with PTL could be used to search for similar signatures in publicly available gene expression profiles deposited into the Gene Expression Omnibus (GEO). We therefore devised a broad in silico screen of the GEO database using the PTL gene expression signature as a template and discovered 2 new agents, celastrol and 4-hydroxy-2-nonenal, that effectively eradicate AML at the bulk, progenitor, and stem cell level. These findings suggest the use of multicenter collections of high-throughput data to facilitate discovery of leukemia drugs and drug targets. PMID:18305216

mTOR up-regulation of PFKFB3 is essential for acute myeloid leukemia cell survival

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Yonghuai; Institute of Hematology, Peking University, Beijing; Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing

Although mTOR (mammalian target of rapamycin) activation is frequently observed in acute myeloid leukemia (AML) patients, the precise function and the downstream targets of mTOR are poorly understood. Here we revealed that PFKFB3, but not PFKFB1, PFKFB2 nor PFKFB4 was a novel downstream substrate of mTOR signaling pathway as PFKFB3 level was augmented after knocking down TSC2 in THP1 and OCI-AML3 cells. Importantly, PFKFB3 silencing suppressed glycolysis and cell proliferation of TSC2 silencing OCI-AML3 cells and activated apoptosis pathway. These results suggested that mTOR up-regulation of PFKFB3 was essential for AML cells survival. Mechanistically, Rapamycin treatment or Raptor knockdown reducedmore » the expression of PFKFB3 in TSC2 knockdown cells, while Rictor silencing did not have such effect. Furthermore, we also revealed that mTORC1 up-regulated PFKFB3 was dependent on hypoxia-inducible factor 1α (HIF1α), a positive regulator of glycolysis. Moreover, PFKFB3 inhibitor PFK15 and rapamycin synergistically blunted the AML cell proliferation. Taken together, PFKFB3 was a promising drug target in AML patients harboring mTOR hyper-activation.« less

Future prospects of therapeutic clinical trials in acute myeloid leukemia

PubMed Central

Khan, Maliha; Mansoor, Armaghan-e-Rehman; Kadia, Tapan M

2017-01-01

Acute myeloid leukemia (AML) is a markedly heterogeneous hematological malignancy that is most commonly seen in elderly adults. The response to current therapies to AML is quite variable, and very few new drugs have been recently approved for use in AML. This review aims to discuss the issues with current trial design for AML therapies, including trial end points, patient enrollment, cost of drug discovery and patient heterogeneity. We also discuss the future directions in AML therapeutics, including intensification of conventional therapy and new drug delivery mechanisms; targeted agents, including epigenetic therapies, cell cycle regulators, hypomethylating agents and chimeric antigen receptor T-cell therapy; and detail of the possible agents that may be incorporated into the treatment of AML in the future. PMID:27771959

Brief Report: Human Acute Myeloid Leukemia Reprogramming to Pluripotency Is a Rare Event and Selects for Patient Hematopoietic Cells Devoid of Leukemic Mutations.

PubMed

Lee, Jong-Hee; Salci, Kyle R; Reid, Jennifer C; Orlando, Luca; Tanasijevic, Borko; Shapovalova, Zoya; Bhatia, Mickie

2017-09-01

Induced pluripotent stem cell reprogramming has provided critical insights into disease processes by modeling the genetics and related clinical pathophysiology. Human cancer represents highly diverse genetics, as well as inter- and intra-patient heterogeneity, where cellular model systems capable of capturing this disease complexity would be invaluable. Acute myeloid leukemia (AML) represents one of most heterogeneous cancers and has been divided into genetic subtypes correlated with unique risk stratification over the decades. Here, we report our efforts to induce pluripotency from the heterogeneous population of human patients that represents this disease in the clinic. Using robust optimized reprogramming methods, we demonstrate that reprogramming of AML cells harboring leukemic genomic aberrations is a rare event with the exception of those with de novo mixed-lineage leukemia (MLL) mutations that can be reprogrammed and model drug responses in vitro. Our findings indicate that unlike hematopoietic cells devoid of genomic aberrations, AML cells harboring driver mutations are refractory to reprogramming. Expression of MLL fusion proteins in AML cells did not contribute to induced reprogramming success, which continued to select for patient derived cells devoid of AML patient-specific aberrations. Our study reveals that unanticipated blockades to achieving pluripotency reside within the majority of transformed AML patient cells. Stem Cells 2017;35:2095-2102. © 2017 AlphaMed Press.

Impaired B cell immunity in acute myeloid leukemia patients after chemotherapy.

PubMed

Goswami, Meghali; Prince, Gabrielle; Biancotto, Angelique; Moir, Susan; Kardava, Lela; Santich, Brian H; Cheung, Foo; Kotliarov, Yuri; Chen, Jinguo; Shi, Rongye; Zhou, Huizhi; Golding, Hana; Manischewitz, Jody; King, Lisa; Kunz, Lauren M; Noonan, Kimberly; Borrello, Ivan M; Smith, B Douglas; Hourigan, Christopher S

2017-07-10

Changes in adaptive immune cells after chemotherapy in adult acute myeloid leukemia (AML) may have implications for the success of immunotherapy. This study was designed to determine the functional capacity of the immune system in adult patients with AML who have completed chemotherapy and are potential candidates for immunotherapy. We used the response to seasonal influenza vaccination as a surrogate for the robustness of the immune system in 10 AML patients in a complete remission post-chemotherapy and performed genetic, phenotypic, and functional characterization of adaptive immune cell subsets. Only 2 patients generated protective titers in response to vaccination, and a majority of patients had abnormal frequencies of transitional and memory B-cells. B-cell receptor sequencing showed a B-cell repertoire with little evidence of somatic hypermutation in most patients. Conversely, frequencies of T-cell populations were similar to those seen in healthy controls, and cytotoxic T-cells demonstrated antigen-specific activity after vaccination. Effector T-cells had increased PD-1 expression in AML patients least removed from chemotherapy. Our results suggest that while some aspects of cellular immunity recover quickly, humoral immunity is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. The observed B-cell abnormalities may explain the poor response to vaccination often seen in AML patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immunity and increased PD-1 expression shortly after chemotherapy might have implications for the success of several modalities of immunotherapy.

HZE Radiation Leukemogenesis in Mice

NASA Astrophysics Data System (ADS)

Peng, Yuanlin

Radiation exposure is a risk factor for acute myeloid leukemia (AML). The Leukemogenesis NSCOR was developed to compare this risk for low LET vs HZE radiations as a means to better assess the leukemia risk to astronauts posed by space radiation. Individual projects within the NSCOR explore HZE radiation leukemogenesis in murine model systems and extend the findings to AML in humans. AML sensitive CBA/CaJ mice have been irradiated with 1 GeV 56 Fe particles at NSRL and with 137 Cs gamma-rays at Colorado State University and followed to 800 days of age for the development of AML. Molecular and cytogenetic analyses of HZE- and gamma-induced AML, including assays for chromosomal aberrations, PU.1 deletion, gene expression, array CGH and microsatellite instability are ongoing. Preliminary data indicate that 56 Fe particles are no more effective in inducing AML or shortening lifespan than gamma-rays. Studies designed to address the individual molecular steps in leukemogenesis and determine the effects of radiation and genetic background on each step have been initiated using knockout mice. Deletion of the PU.1 gene on mouse chromosome 2 is a critical step in this murine model of radiation leukemogenesis. Two of the three HZE-induced AMLs that could be assayed and thirteen of fourteen γ-induced AMLs had PU.1 loss as determined by Fluorescence in Situ Hybridization (FISH). We have found that AML sensitive CBA/CaJ mice have a higher incidence of Chr. 2 deletion in bone marrow cells following 56 Fe irradiation than AML resistant C57BL/6 mice. This study is being extended to proton irradiated mice. Our preliminary results indicate that microsatellite instability may be common in HZE irradiated progenitor cells. To determine if these cytogenetic changes can be induced in human myeloid progenitor cells by gamma, proton or HZE irradiation we are generating NOD/SCID mice that have been "humanized" by being transplanted with human hematopoietic stem cells. We are currently irradiating the humanized NOD/SCID mice with gamma-rays and then harvesting human cells from their bone marrow. These cells will be assayed for specific cytogenetic and molecular changes consistent with AML. In addition to screening the cells for chromosomal aberrations and specific deletions and translocations, we will also screen them for microsatellite instability by small pool PCR.(Funded by NASA Grant NAG9 1569)

Inhibition of mTOR pathway sensitizes acute myeloid leukemia cells to aurora inhibitors by suppression of glycolytic metabolism.

PubMed

Liu, Ling-Ling; Long, Zi-Jie; Wang, Le-Xun; Zheng, Fei-Meng; Fang, Zhi-Gang; Yan, Min; Xu, Dong-Fan; Chen, Jia-Jie; Wang, Shao-Wu; Lin, Dong-Jun; Liu, Quentin

2013-11-01

Aurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) induced polyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantly increased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggesting that polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway by mTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in the polyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptake and lactate production in polyploidy cells as well as the expression of p62/SQSTM1. Moreover, knockdown of p62/SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy. Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidy cells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatment in AML. ©2013 AACR.

Mechanisms of apoptosis induction by simultaneous inhibition of PI3K and FLT3-ITD in AML cells in the hypoxic bone marrow microenvironment

PubMed Central

Jin, Linhua; Tabe, Yoko; Lu, Hongbo; Borthakur, Gautam; Miida, Takashi; Kantarjian, Hagop; Andreeff, Michael; Konopleva, Marina

2013-01-01

We investigated the antileukemia effects and molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in AML cells grown under hypoxia in co-cultures with bone marrow stromal cells. Combined treatment with selective class I PI3K inhibitor GDC-0941 and sorafenib reversed the protective effects of bone marrow stromal cells on FLT3-mutant AML cells in hypoxia, which was associated with downregulation of Pim-1 and Mcl-1 expression levels. These findings suggest that combined inhibition of PI3K and FLT3-ITD may constitute a targeted approach to eradicating chemoresistant AML cells sequestered in hypoxic bone marrow niches. PMID:23036488

Mechanisms of apoptosis induction by simultaneous inhibition of PI3K and FLT3-ITD in AML cells in the hypoxic bone marrow microenvironment.

PubMed

Jin, Linhua; Tabe, Yoko; Lu, Hongbo; Borthakur, Gautam; Miida, Takashi; Kantarjian, Hagop; Andreeff, Michael; Konopleva, Marina

2013-02-01

We investigated the antileukemia effects and molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in AML cells grown under hypoxia in co-cultures with bone marrow stromal cells. Combined treatment with selective class I PI3K inhibitor GDC-0941 and sorafenib reversed the protective effects of bone marrow stromal cells on FLT3-mutant AML cells in hypoxia, which was associated with downregulation of Pim-1 and Mcl-1 expression levels. These findings suggest that combined inhibition of PI3K and FLT3-ITD may constitute a targeted approach to eradicating chemoresistant AML cells sequestered in hypoxic bone marrow niches. Copyright © 2012 Elsevier Ltd. All rights reserved.

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment.

PubMed

Jin, Linhua; Tabe, Yoko; Kojima, Kensuke; Shikami, Masato; Benito, Julina; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Ciurea, Stefan O; Miida, Takashi; Andreeff, Michael; Konopleva, Marina

2013-12-01

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here, we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737-induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. Combined blockade of PI3K and Bcl-2 pathways down-regulates anti-apoptotic Mcl-1 expression PI3K and Bcl-2 induced Mcl-1 down-regulation activates BAX PI3K and Bcl-2 blockage induces apoptosis in AML under hypoxic BM microenvironment.

Resveratrol Downregulates Interleukin-6-Stimulated Sonic Hedgehog Signaling in Human Acute Myeloid Leukemia

PubMed Central

Su, Yu-Chieh; Li, Szu-Chin; Wu, Yin-Chi; Wang, Li-Min; Chao, K. S. Clifford; Liao, Hui-Fen

2013-01-01

IL-6 and sonic hedgehog (Shh) signaling molecules are considered to maintain the growth of cancer stem cells (CSCs). Resveratrol, an important integrant in traditional Chinese medicine, possesses certain antitumor effects. However, the mechanisms on regulating acute myeloid leukemia (AML) are unclear. This study first used human subjects to demonstrate that the plasma levels of IL-6 and IL-1β in AML patients were higher and lower, respectively, than healthy donors. The expression of Shh preproproteins, and C- and N-terminal Shh peptides increased in bone marrow and peripheral blood mononuclear cells isolated from AML patients, and the plasma N-Shh secretion was greater. To further clarify the effect of IL-6 and resveratrol in Shh signaling, human AML HL-60 cells were tested. IL-6 upregulated Shh and Gli-1 expression and was accompanied by an increase of cell viability. Resveratrol significantly decreased CSC-related Shh expression, Gli-1 nuclear translocation, and cell viability in IL-6-treated HL-60 cells and had synergistic effect with Shh inhibitor cyclopamine on inhibiting cell growth. Conclusions. IL-6 stimulated the growth of AML cells through Shh signaling, and this effect might be blocked by resveratrol. Further investigations of Shh as a prognostic marker and resveratrol as a therapeutic drug target to CSCs in AML are surely warranted. PMID:23533494

Comprehensive genetic analysis of cytarabine sensitivity in a cell-based model identifies polymorphisms associated with outcome in AML patients

PubMed Central

Gamazon, Eric R.; Lamba, Jatinder K.; Pounds, Stanley; Stark, Amy L.; Wheeler, Heather E.; Cao, Xueyuan; Im, Hae K.; Mitra, Amit K.; Rubnitz, Jeffrey E.; Ribeiro, Raul C.; Raimondi, Susana; Campana, Dario; Crews, Kristine R.; Wong, Shan S.; Welsh, Marleen; Hulur, Imge; Gorsic, Lidija; Hartford, Christine M.; Zhang, Wei; Cox, Nancy J.; Dolan, M. Eileen

2013-01-01

A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10−5) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10−6) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084. PMID:23538338

SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia.

PubMed

Schneider, Constanze; Oellerich, Thomas; Baldauf, Hanna-Mari; Schwarz, Sarah-Marie; Thomas, Dominique; Flick, Robert; Bohnenberger, Hanibal; Kaderali, Lars; Stegmann, Lena; Cremer, Anjali; Martin, Margarethe; Lohmeyer, Julian; Michaelis, Martin; Hornung, Veit; Schliemann, Christoph; Berdel, Wolfgang E; Hartmann, Wolfgang; Wardelmann, Eva; Comoglio, Federico; Hansmann, Martin-Leo; Yakunin, Alexander F; Geisslinger, Gerd; Ströbel, Philipp; Ferreirós, Nerea; Serve, Hubert; Keppler, Oliver T; Cinatl, Jindrich

2017-02-01

The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.

Circulating endothelial cells and their progenitors in acute myeloid leukemia

PubMed Central

Zahran, Asmaa Mohammed; Aly, Sanaa Shaker; Altayeb, Hanan Ahmed; Ali, Arwa Mohammed

2016-01-01

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by the accumulation of immature myeloid progenitor cells in the bone marrow. Studies are required to investigate the prognostic and predictive value of surrogate biomarkers. Given the importance of angiogenesis in oncology in terms of pathogenesis as well as being a target for treatment, circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) are promising candidates to serve as such markers. The aim of the present study was to quantify CECs and EPCs in patients with AML at initial diagnosis and following induction chemotherapy, and to correlate these findings with the response to treatment in AML patients. The present study included 40 patients with de novo AML and 20 age- and gender-matched healthy controls. CECs and EPCs were evaluated by flow cytometry at initial diagnosis and after induction chemotherapy (3+7 protocol for AML other than M3 and all-trans-retinoic acid plus anthracycline for M3 disease). CECs and EPCs were significantly higher in AML patients at diagnosis and after induction chemotherapy than in controls. After induction chemotherapy, CECs and EPCs were significantly decreased compared with the levels at initial diagnosis. Patients who achieved complete response (n=28) had lower initial CEC and EPC levels compared with patients who did not respond to treatment. These results suggest that CEC levels are higher in AML patients and may correlate with disease status and treatment response. Further investigations are required to better determine the predictive value and implication of these cells in AML management. PMID:27602121

p53 independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid leukemia

PubMed Central

Ng, Kwok Peng; Ebrahem, Quteba; Negrotto, Soledad; Mahfouz, Reda Z.; Link, Kevin A.; Hu, Zhenbo; Gu, Xiaorong; Advani, Anjali; Kalaycio, Matt; Sobecks, Ronald; Sekeres, Mikkael; Copelan, Edward; Radivoyevitch, Tomas; Maciejewski, Jaroslaw; Mulloy, James C.; Saunthararajah, Yogen

2013-01-01

Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the deoxycytidine analogue decitabine that deplete the chromatin modifying enzyme DNA methyl-transferase 1 (DNMT1) without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine up regulated the key late differentiation factors CEBPε and p27/CDKN1B, induced cellular differentiation, and terminated AML cell-cycle, even in cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xeno-transplanted AML cells was abrogated but normal hematopoietic stem cell (HSC) engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S-phase specific decitabine therapy. In xeno-transplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared to conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy. PMID:21701495

Inhibiting glutamine uptake represents an attractive new strategy for treating acute myeloid leukemia

PubMed Central

Willems, Lise; Jacque, Nathalie; Jacquel, Arnaud; Neveux, Nathalie; Trovati Maciel, Thiago; Lambert, Mireille; Schmitt, Alain; Poulain, Laury; Green, Alexa S.; Uzunov, Madalina; Kosmider, Olivier; Radford-Weiss, Isabelle; Moura, Ivan Cruz; Auberger, Patrick; Ifrah, Norbert; Bardet, Valérie; Chapuis, Nicolas; Lacombe, Catherine; Mayeux, Patrick; Tamburini, Jérôme

2013-01-01

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase–induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML. PMID:24014241

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL*

PubMed Central

Bansal, Hima; Seifert, Theresea; Bachier, Carlos; Rao, Manjeet; Tomlinson, Gail; Iyer, Swaminathan Padmanabhan; Bansal, Sanjay

2012-01-01

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias. PMID:22898820

The transcription factor Wilms tumor 1 confers resistance in myeloid leukemia cells against the proapoptotic therapeutic agent TRAIL (tumor necrosis factor α-related apoptosis-inducing ligand) by regulating the antiapoptotic protein Bcl-xL.

PubMed

Bansal, Hima; Seifert, Theresea; Bachier, Carlos; Rao, Manjeet; Tomlinson, Gail; Iyer, Swaminathan Padmanabhan; Bansal, Sanjay

2012-09-21

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.

Immunomodulatory action of SGI-110, a hypomethylating agent, in acute myeloid leukemia cells

PubMed Central

Srivastava, Pragya; Paluch, Benjamin E.; Matsuzaki, Junko; James, Smitha R.; Collamat-Lai, Golda; Karbach, Julia; Nemeth, Michael J.; Taverna, Pietro; Karpf, Adam R.; Griffiths, Elizabeth A.

2017-01-01

The mechanism of clinical action for the FDA approved hypomethylating drugs azacitidine and decitabine remains unresolved and in this context the potential immunomodulatory effect of these agents on leukemic cells is an area of active investigation. Induced expression of methylated Cancer Testis Antigen (CTA) genes has been demonstrated in leukemic cell lines following exposure to hypomethylating drugs in vitro. SGI-110 is a novel hypomethylating dinucleotide with prolonged in vivo exposure and clinical activity in patients with MDS and AML. We demonstrate that this agent, like decitabine, produces robust re-expression of the CTAs NY-ESO-1 and MAGE-A, both in vitro and in leukemia-bearing AML xenografts. Upregulation of these genes in vitro was sufficient to induce cytotoxicity by HLA-compatible CD8+ T-cells specific for NY-ESO-1, a well-recognized and immunogenic CTA. Additionally, exposure to SGI-110 enhances MHC class I and co-stimulatory molecule expression, potentially contributing to recognition of CTAs. SGI-110, like the parent compound decitabine, induces expression of CTAs and might modulate immune recognition of myeloid malignancy. PMID:25260825

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.

PubMed

van Rhenen, Anna; van Dongen, Guus A M S; Kelder, Angèle; Rombouts, Elwin J; Feller, Nicole; Moshaver, Bijan; Stigter-van Walsum, Marijke; Zweegman, Sonja; Ossenkoppele, Gert J; Jan Schuurhuis, Gerrit

2007-10-01

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.

An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

PubMed Central

Vick, Binje; Rothenberg, Maja; Sandhöfer, Nadine; Carlet, Michela; Finkenzeller, Cornelia; Krupka, Christina; Grunert, Michaela; Trumpp, Andreas; Corbacioglu, Selim; Ebinger, Martin; André, Maya C.; Hiddemann, Wolfgang; Schneider, Stephanie; Subklewe, Marion; Metzeler, Klaus H.; Spiekermann, Karsten; Jeremias, Irmela

2015-01-01

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups. PMID:25793878

Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huai, Lei; Wang, Cuicui; Zhang, Cuiping

2012-06-08

Highlights: Black-Right-Pointing-Pointer Metformin induces differentiation in NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces activation of the MEK/ERK signaling pathway in APL cells. Black-Right-Pointing-Pointer Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces the relocalization and degradation of the PML-RAR{alpha} fusion protein. Black-Right-Pointing-Pointer The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acutemore » promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RAR{alpha} and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.« less

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment

PubMed Central

Kojima, Kensuke; Shikami, Masato; Benito, Julina; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Ciurea, Stefan O.; Miida, Takashi; Andreeff, Michael; Konopleva, Marina

2013-01-01

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737–induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. PMID:23955073

[Acute myeloid leukemia possibly originating from the same clone of testicular germ cell tumor].

PubMed

Suyama, Takuya; Obara, Naoshi; Kawai, Koji; Yamada, Kenji; Kusakabe, Manabu; Kurita, Naoki; Nishikii, Hidekazu; Yokoyama, Yasuhisa; Suzukawa, Kazumi; Hasegawa, Yuichi; Noguchi, Masayuki; Chiba, Shigeru

2013-08-01

This report describes a 30-year-old man with a testicular germ cell tumor, which later developed into acute myeloid leukemia (AML) with a common chromosomal abnormality. Testicular germ cell tumors had developed at the age of 26. He was successfully treated with surgery followed by chemotherapy.Four years after the onset of the germ cell tumor, he developed pancytopenia with elevated serum LDH. More than 95% of the bone marrow was occupied by blastic cells. These cells were CD13+, CD34+ but CD45- and MPO-. Amplification of the short arm of chromosome 12 was recognized by fluorescence in situ hybridization using the blastic cells in the bone marrow and the previous testicular tumor specimen. Because testicular germ cell tumor recurrence and other malignant tumors could be ruled out pathologically, he was diagnosed as having AML.Allogeneic stem cell transplantation from a HLA-matched sibling donor was performed after chemotherapy. As of 19 months after the transplantation, recurrence of neither AML nor testicular tumors has been observed. Because the same genetic abnormality was observed in the testicular germ cell tumor and AML in this case, the possibility of AML having a common origin with the testicular germ cell tumor is indicated.

MicroRNA-155 expression and function in AML: An evolving paradigm.

PubMed

Narayan, Nisha; Bracken, Cameron P; Ekert, Paul G

2018-06-01

Acute myeloid leukemia (AML) arises when immature myeloid blast cells acquire multiple, recurrent genetic and epigenetic changes that result in dysregulated proliferation. Acute leukemia is the most common form of pediatric cancer, with AML accounting for ~20% of all leukemias in children. The genomic aberrations that drive AML inhibit myeloid differentiation and activate signal transduction pathways that drive proliferation. MicroRNAs, a class of small (~22 nucleotide) noncoding RNAs that posttranscriptionally suppress the expression of specifically targeted transcripts, are also frequently dysregulated in AML, which may prove useful for the purposes of disease classification, prognosis, and future therapeutic approaches. MicroRNA expression profiles are associated with patient prognosis and responses to standard chemotherapy, including predicting therapy resistance in AML. miR-155 is the primary focus of this review because it has been repeatedly associated with poorer survival across multiple cohorts of adult and pediatric AML. We discuss some novel features of miR-155 expression in AML, in particular how the levels of expression can critically influence function. Understanding the role of microRNAs in AML and the ways in which microRNA expression influences AML biology is one means to develop novel and more targeted therapies. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Minimal PU.1 reduction induces a preleukemic state and promotes development of acute myeloid leukemia

PubMed Central

Will, Britta; Vogler, Thomas O.; Narayanagari, Swathi; Bartholdy, Boris; Todorova, Tihomira I.; da Silva Ferreira, Mariana; Chen, Jiahao; Yu, Yiting; Mayer, Jillian; Barreyro, Laura; Carvajal, Luis; Ben Neriah, Daniela; Roth, Michael; van Oers, Johanna; Schaetzlein, Sonja; McMahon, Christine; Edelmann, Winfried; Verma, Amit; Steidl, Ulrich

2016-01-01

Modest transcriptional changes caused by genetic or epigenetic mechanisms are frequent in human cancer. Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces acute myeloid leukemia (AML) in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet moderate PU.1 inhibition is common in AML patients. We assessed functional consequences of modest reduction of PU.1 expression on leukemia development in mice harboring DNA lesions resembling those acquired during human stem cell aging. Heterozygous deletion of an enhancer of PU.1, which resulted in 35% reduction of PU.1 expression, was sufficient to induce myeloid biased preleukemic stem cells and subsequent transformation to AML in a DNA mismatch repair-deficient background. AML progression was mediated by inhibition of expression of a PU.1 cooperating transcription factor, Irf8. Strikingly, we found significant molecular similarities with human myelodysplastic syndrome and AML. This study demonstrates that minimal reduction of a key lineage-specific transcription factor that commonly occurs in human disease is sufficient to initiate cancer development and provides mechanistic insight into the formation and progression of preleukemic stem cells in AML. PMID:26343801

A new ETV6-NTRK3 cell line model reveals MALAT1 as a novel therapeutic target - a short report.

PubMed

Chen, Suning; Nagel, Stefan; Schneider, Bjoern; Dai, Haiping; Geffers, Robert; Kaufmann, Maren; Meyer, Corinna; Pommerenke, Claudia; Thress, Kenneth S; Li, Jiao; Quentmeier, Hilmar; Drexler, Hans G; MacLeod, Roderick A F

2018-02-01

Previously, the chromosomal translocation t(12;15)(p13;q25) has been found to recurrently occur in both solid tumors and leukemias. This translocation leads to ETV6-NTRK3 (EN) gene fusions resulting in ectopic expression of the NTRK3 neurotropic tyrosine receptor kinase moiety as well as oligomerization through the donated ETV6-sterile alpha motif domain. As yet, no in vitro cell line model carrying this anomaly is available. Here we genetically characterized the acute promyelocytic leukemia (APL) cell line AP-1060 and, by doing so, revealed the presence of a t(12;15)(p13;q25). Subsequently, we evaluated its suitability as a model for this important clinical entity. Spectral karyotyping, fluorescence in situ hybridization (FISH), and genomic and transcriptomic microarray-based profiling were used to screen for the presence of EN fusions. qRT-PCR was used for quantitative expression analyses. Responses to AZ-23 (NTRK) and wortmannin (PI3K) inhibitors, as well as to arsenic trioxide (ATO), were assessed using colorimetric assays. An AZ-23 microarray screen was used to define the EN targetome, which was parsed bioinformatically. MAPK1 and MALAT1 activation were assayed using Western blotting and RNA-FISH, respectively, whereas an AML patient cohort was used to assess the clinical occurrence of MALAT1 activation. An EN fusion was detected in AP1060 cells which, accordingly, turned out to be hypersensitive to AZ-23. We also found that AZ-23 can potentiate the effect of ATO and inhibit the phosphorylation of its canonical target MAPK1. The AZ-23 microarray screen highlighted a novel EN target, MALAT1, which also proved sensitive to wortmannin. Finally, we found that MALAT1 was massively up-regulated in a subset of AML patients. From our data we conclude that AP-1060 may serve as a first publicly available preclinical model for EN. In addition, we conclude that these EN-positive cells are sensitive to the NTRK inhibitor AZ-23 and that this inhibitor may potentiate the therapeutic efficacy of ATO. Our data also highlight a novel AML EN target, MALAT1, which was so far only conspicuous in solid tumors.

[Acute myeloid leukemia].

PubMed

Tabuchi, Ken

2007-02-01

The annual incident rate of pediatric acute myeloid leukemia (AML) is now 10 per million in Japan, against 5 to 9 per million in the USA and Europe. Overall long-term survival has now been achieved for more than 50% of pediatric patients with AML in the USA and in Europe. The prognostic factors of pediatric AML were analyzed,and patients with AML were classified according to prognostic factors. The t(15;17), inv(16) and t(8;21) have emerged as predictors of good prognosis in children with AML. Monosomy 7, monosomy 5 and del (5 q) abnormalities showed a poor prognosis. In addition to chromosomal deletions, FLT 3/ITD identifies pediatric patients with a particularly poor prognosis. Clinical trials of AML feature intensive chemotherapy with or without subsequent stem cell transplantation. Risk group stratification is becoming increasingly important in planning AML therapy. APL can be distinguished from other subtypes of AML by virtue of its excellent response and overall outcome as a result of differentiation therapy with ATRA. Children with Down syndrome and AML have been shown to have a superior prognosis to AML therapy compared to other children with AML. The results of the Japan Cooperative Study Group protocol ANLL 91 was one of the best previously reported in the literature. With the consideration of quality of life (QOL), risk-adapted therapy was introduced in the AML 99 trial conducted by the Japanese Childhood AML Cooperative Study Group. A high survival rate of 79% at 3 years was achieved for childhood de novo AML in the AML 99 trial. To evaluate the efficacy and safety of the treatment strategy according to risk stratification based on leukemia cell biology and response to the initial induction therapy in children with AML, the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) has organized multi-center phase II trials in children with newly diagnosed AML.

Scrutinizing the Expression and Blockade of Inhibitory Molecules Expressed on T Cells from Acute Myeloid Leukemia Patients.

PubMed

Abdolmaleki, Mohsen; Mojtabavi, Nazanin; Zavvar, Mahdi; Vaezi, Mohammad; Noorbakhsh, Farshid; Nicknam, Mohammad Hossein

2018-06-01

T cell exhaustion is an immunosuppressive mechanism which occurs in chronic viral infections, solid tumors and hematologic malignancies. Exhausted T cell has increased the expression of inhibitory receptors, and functional impairment. In this study, we investigated the expression from some of those inhibitory receptors being Programmed death 1 (PD-1), T cell immunoglobulin and mucin domain containing molecules 3 (TIM-3) and CD244 on T cells from Iranian acute myeloid leukemia (AML) patients. Peripheral blood samples were collected from Iranian newly diagnosed AML patients and flow cytometric analysis was accomplished for cell surface expression of PD-1, TIM-3, and CD244 on T lymphocytes. Functionality and proliferation assay were done in the presence of anti-PD-1 and anti-CD244 blocking antibodies. Immunophenotyping of T cells showed a significant increase of PD-1 and CD244 expression on CD4+ and CD8+ T cells of AML patients. Whereas blockade of PD1 and CD244 increased the proliferation of CD4+ and CD8+ T lymphocytes of AML patients but IFN-γ production was not significantly increased. In conclusion, our data indicate that CD4+ and CD8+ T cells from AML patients appeared to be exhausted and blockade of some immune checkpoints can improve the proliferation of those cells.

Drug targeting of NR4A nuclear receptors for treatment of acute myeloid leukemia.

PubMed

Boudreaux, Seth P; Duren, Ryan P; Call, Steven G; Nguyen, Loc; Freire, Pablo R; Narayanan, Padmini; Redell, Michele S; Conneely, Orla M

2018-06-08

NR4As are AML tumor suppressors that are frequently silenced in human acute myeloid leukemia (AML). Despite their potential as novel targets for therapeutic intervention, mechanisms of NR4A silencing and strategies for their reactivation remain poorly defined. Here we show that NR4A silencing in AML occurs through blockade of transcriptional elongation rather than epigenetic promoter silencing. By intersection of NR4A-regulated gene signatures captured upon acute, exogenous expression of NR4As in human AML cells with in silico chemical genomics screening, we identify several FDA-approved drugs including dihydroergotamine (DHE) that reactivate NR4A expression and regulate NR4A-dependent gene signatures. We show that DHE induces NR4A expression via recruitment of the super elongation complex to enable elongation of NR4A promoter paused RNA polymerase II. Finally, DHE exhibits AML selective NR4A-dependent anti-leukemic activity in cytogenetically distinct human AML cells in vitro and delays AML progression in mice revealing its potential as a novel therapeutic agent in AML.

Ibrutinib inhibits SDF1/CXCR4 mediated migration in AML

PubMed Central

Zaitseva, Lyubov; Murray, Megan Y.; Shafat, Manar S.; Lawes, Matthew J.; MacEwan, David J.; Bowles, Kristian M.; Rushworth, Stuart A.

2014-01-01

Pharmacological targeting of BTK using ibrutinib has recently shown encouraging clinical activity in a range of lymphoid malignancies. Recently we reported that ibrutinib inhibits human acute myeloid leukemia (AML) blast proliferation and leukemic cell adhesion to the surrounding bone marrow stroma cells. Here we report that in human AML ibrutinib, in addition, functions to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. It has previously been shown that SDF1/CXCR4-induced migration is dependent on activation of downstream BTK in chronic lymphocytic leukaemia (CLL) and multiple myeloma. Here we show that SDF-1 induces BTK phosphorylation and downstream MAPK signalling in primary AML blast. Furthermore, we show that ibrutinib can inhibit SDF1-induced AKT and MAPK activation. These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. PMID:25294819

Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer?

PubMed Central

Grove, Carolyn S.; Vassiliou, George S.

2014-01-01

Acute myeloid leukaemia (AML) is an uncontrolled clonal proliferation of abnormal myeloid progenitor cells in the bone marrow and blood. Advances in cancer genomics have revealed the spectrum of somatic mutations that give rise to human AML and drawn our attention to its molecular evolution and clonal architecture. It is now evident that most AML genomes harbour small numbers of mutations, which are acquired in a stepwise manner. This characteristic, combined with our ability to identify mutations in individual leukaemic cells and our detailed understanding of normal human and murine haematopoiesis, makes AML an excellent model for understanding the principles of cancer evolution. Furthermore, a better understanding of how AML evolves can help us devise strategies to improve the therapy and prognosis of AML patients. Here, we draw from recent advances in genomics, clinical studies and experimental models to describe the current knowledge of the clonal evolution of AML and its implications for the biology and treatment of leukaemias and other cancers. PMID:25056697

Raman spectroscopy for the assessment of acute myeloid leukemia: a proof of concept study

NASA Astrophysics Data System (ADS)

Vanna, R.; Tresoldi, C.; Ronchi, P.; Lenferink, A. T. M.; Morasso, C.; Mehn, D.; Bedoni, M.; Terstappen, L. W. M. M.; Ciceri, F.; Otto, C.; Gramatica, F.

2014-03-01

Acute myeloid leukemia (AML) is a proliferative neoplasm, that if not properly treated can rapidly cause a fatal outcome. The diagnosis of AML is challenging and the first diagnostic step is the count of the percentage of blasts (immature cells) in bone marrow and blood sample, and their morphological characterization. This evaluation is still performed manually with a bright field light microscope. Here we report results of a study applying Raman spectroscopy for analysis of samples from two patients affected by two AML subtypes characterized by a different maturation stage in the neutrophilic lineage. Ten representative cells per sample were selected and analyzed with high-resolution confocal Raman microscopy by scanning 64x64 (4096) points in a confocal layer through the volume of the whole cell. The average spectrum of each cell was then used to obtain a highly reproducible mean fingerprint of the two different AML subtypes. We demonstrate that Raman spectroscopy efficiently distinguishes these different AML subtypes. The molecular interpretation of the substantial differences between the subtypes is related to granulocytic enzymes (e.g. myeloperoxidase and cytochrome b558), in agreement with different stages of maturation of the two considered AML subtypes . These results are promising for the development of a new, objective, automated and label-free Raman based methods for the diagnosis and first assessment of AML.

Chidamide in FLT3-ITD positive acute myeloid leukemia and the synergistic effect in combination with cytarabine.

PubMed

Li, Xia; Yan, Xiao; Guo, Wenjian; Huang, Xin; Huang, Jiansong; Yu, Mengxia; Ma, Zhixin; Xu, Yu; Huang, ShuJuan; Li, Chenying; Zhou, Yile; Jin, Jie

2017-06-01

Chidamide, a novel histone deacetylase inhibitor (HDACi), has been approved for treatment of T-cell lymphomas in multiple clinical trials. It has been demonstrated that chidamide can inhibit cell cycle, promote apoptosis and induce differentiation in leukemia cells, whereas its effect on acute myeloid leukemia (AML) patients with FLT3-ITD mutation has not been clarified. In this study, we found that chidamide specifically induced G0/G1 arrest and apoptosis in FLT3-ITD positive AML cells in a concentration and time-dependent manner. We also found chidamide had the cytotoxicity effect on FLT3-ITD positive and negative AML cells. Moreover, with respect to relapsed/refractory patients, chidamide showed the same effectiveness as that in de novo AML patients. Notably, chidamide synergistically enhanced apoptosis caused by cytarabine. Our results support chidamide alone or combine with cytarabine may be used as an alternative therapeutic choice for AML patients especially those with FLT3-ITD mutation or relapsed/refractory ones. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Late ovarian relapse of TEL/AML1 positive ALL confirming that TEL deletion is a secondary event in leukemogenesis.

PubMed

Ly-Sunnaram, Beatrice; Henry, Catherine; Gandemer, Virginie; Mee, Franseza Le; Burtin, Florence; Blayau, Martine; Cayuela, Jean-Michel; Oster, Magalie; Clech, Philippe; Rambeau, Marc; Marie, Celine; Pampin, Cecilia; Edan, Christine; Gall, Edouard Le; Goasguen, Jean E

2005-09-01

We describe here a late extramedullary ovarian relapse in an 18-year-old female who was diagnosed with hypotetraploid cell acute lymphoblastic leukaemia (cALL) at the age of 6. At both occurrences of the disease cells were analyzed by morphology, immunophenotyping, cytogenetics and molecular methods. TEL/AML1 was detected by RT-PCR and FISH analysis in both events. We demonstrated, using detection of IGH/TCR rearrangements and TEL/AML1 breakpoints sequencing that the cells were clonally related. Moreover, interphasic FISH using TEL and AML1 probes showed the loss of a second TEL at the time of relapse. This observation confirms that TEL/AML1 alone is not sufficient to trigger ALL and that TEL deletion is a secondary event in leukemogenesis. To our knowledge, it is the first complete description of extramedullary ALL relapse combining all methodologies.

A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.

PubMed

Adamia, Sophia; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bar-Natan, Michal; Pevzner, Samuel; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Burke, John; Galinsky, Ilene; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Stone, Richard; Griffin, James D

2014-03-01

Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease. ©2013 AACR

Acute erythremic myelosis (true erythroleukaemia): a variant of AML FAB-M6.

PubMed

Hasserjian, R P; Howard, J; Wood, A; Henry, K; Bain, B

2001-03-01

Classic erythroleukaemia (acute myeloid leukaemia M6, or M6 AML) is defined as an excess of myeloblasts in an erythroid predominant background. Leukaemia variants in which the primitive blast cells are demonstrably erythroid are extremely rare and poorly characterised. Variably referred to as "true erythroleukaemia" or "acute erythremic myelosis", they are often included within the M6 AML category even though they do not meet strict criteria for this type of AML. Two cases of acute erythroid neoplasia are presented with clinical, morphological, immunophenotypic, and cytogenetic analysis. Both patients presented with profound anaemia, one in a setting of long standing myelodysplasia. Bone marrow examination revealed a predominant population of highly dysplastic erythroid cells in both cases. In one case, the liver was infiltrated by neoplastic erythroid cells. Both patients died within four months of diagnosis. This report illustrates that cases of acute leukaemia occur in which the dominant neoplastic cell is a primitive erythroid cell without an accompanying increase in myeloblasts. This does not preclude the neoplastic clone originating in a multipotent haemopoietic stem cell, as suggested by cases arising in patients with myelodysplasia. Acute erythremic myelosis should be recognised as a distinct variant of M6 AML.

Successful treatment of congenital acute myeloid leukemia (AML-M6) in a premature infant.

PubMed

van Dongen, Joyce C A; Dalinghaus, Michiel; Kroon, Andre A; de Vries, Andrica C H; van den Heuvel-Eibrink, Marry M

2009-11-01

Congenital acute myeloid leukemia (AML), and especially AML-M6 is a rare disease with a poor prognosis. Moreover, reports of treatment outcome of congenital AML-M6 in premature infants are not available. We report the first treated case of congenital AML-M6 in a premature girl, who received a full AML protocol. She presented with blueberry-muffin spots, anemia, high white blood cell count, and serious cardiopulmonary distress. Peripheral blood smears showed AML-M6 blasts. After treatment with a sequential low-dose cytarabine after birth and full-dose AML treatment according to the MRC-12 protocol at the age of 2 months, she now is in continuous complete remission for 4 years.

Global Cell Proteome Profiling, Phospho-signaling and Quantitative 
Proteomics for Identification of New Biomarkers in Acute Myeloid 
Leukemia Patients

PubMed Central

Aasebø, Elise; Forthun, Rakel B.; Berven, Frode; Selheim, Frode; Hernandez-Valladares, Maria

2016-01-01

The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging. PMID:26306748

p53−/− synergizes with enhanced NrasG12D signaling to transform megakaryocyte-erythroid progenitors in acute myeloid leukemia

PubMed Central

Kong, Guangyao; Rajagopalan, Adhithi; Lu, Li; Song, Jingming; Hussaini, Mohamed; Zhang, Xinmin; Ranheim, Erik A.; Liu, Yangang; Wang, Jinyong; Gao, Xin; Chang, Yuan-I; Johnson, Kirby D.; Zhou, Yun; Yang, David; Bhatnagar, Bhavana; Lucas, David M.; Bresnick, Emery H.; Zhong, Xuehua; Padron, Eric

2017-01-01

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53−/− mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53−/− bone marrow cells rapidly develop a highly penetrant AML. We find that p53−/− cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53−/− MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53−/− synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML. PMID:27815262

Minimally differentiated erythroleukaemia (AML M6 'variant'): a rare subset of AML distinct from AML M6. Groupe Français d'Hématologie Cellulaire.

PubMed

Garand, R; Duchayne, E; Blanchard, D; Robillard, N; Kuhlein, E; Fenneteau, O; Salomon-Nguyen, F; Grange, M J; Rousselot, P; Demur, C

1995-08-01

We describe eight cases of erythroleukaemia distinct from FAB-AML M6, which demonstrate minimal erythroid differentiation not associated with a myeloblastic component. Three infants (including a Down's syndrome) and two adults presented with a de novo leukaemia. One case was preceded by an untreated refractory anaemia with excess of blasts and one by polycythaemia vera. One case presented with an inaugural blast crisis of chronic myeloid leukaemia. In four patients the leukaemic cells showed a proerythroblast-like morphology. The four other were initially classified as undifferentiated AL (two cases) or AML MO (two cases) because of the immature aspect of the cells, their lack of myeloperoxidase activity and the absence of B, T lymphoid and myeloid (My) marker expressions apart from the CD33 antigen. Immunophenotyping in three cases showed an immature erythroblast profile (glycophorins A and B+, spectrin+). In the five others the erythroid nature was recognized by the expression of ABH blood group system on fresh cells (four cases) and glycophorin A on cells after 3 d in vitro culture with erythropoietin (EPO) + IL3 (two cases). Moreover, an erythroid colony growth of leukaemic origin was observed in three patients. In conclusion, the study of erythroid marker expression is of particular importance when immunophenotyping leukaemic cells with a proerythroblast-like morphology or an undifferentiated aspect and a HLA DR-, CD36++, B-, T-, My- (CD33 +/-) phenotype. We propose the term AML M6 'variant' for this rare type of AML.

DOT1L inhibitor EPZ-5676 displays synergistic antiproliferative activity in combination with standard of care drugs and hypomethylating agents in MLL-rearranged leukemia cells.

PubMed

Klaus, Christine R; Iwanowicz, Dorothy; Johnston, Danielle; Campbell, Carly A; Smith, Jesse J; Moyer, Mikel P; Copeland, Robert A; Olhava, Edward J; Scott, Margaret Porter; Pollock, Roy M; Daigle, Scott R; Raimondi, Alejandra

2014-09-01

EPZ-5676 [(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol], a small-molecule inhibitor of the protein methyltransferase DOT1L, is currently under clinical investigation for acute leukemias bearing MLL-rearrangements (MLL-r). In this study, we evaluated EPZ-5676 in combination with standard of care (SOC) agents for acute leukemias as well as other chromatin-modifying drugs in cellular assays with three human acute leukemia cell lines: MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), and SKM-1 (non-MLL-r). Studies were performed to evaluate the antiproliferative effects of EPZ-5676 combinations in a cotreatment model in which the second agent was added simultaneously with EPZ-5676 at the beginning of the assay, or in a pretreatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. EPZ-5676 was found to act synergistically with the acute myeloid leukemia (AML) SOC agents cytarabine or daunorubicin in MOLM-13 and MV4-11 MLL-r cell lines. EPZ-5676 is selective for MLL-r cell lines as demonstrated by its lack of effect either alone or in combination in the nonrearranged SKM-1 cell line. In MLL-r cells, the combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the chemotherapeutic agents, suggesting that EPZ-5676 sets up a durable, altered chromatin state that enhances the chemotherapeutic effects. Our evaluation of EPZ-5676 in conjunction with other chromatin-modifying drugs also revealed a consistent combination benefit, including synergy with DNA hypomethylating agents. These results indicate that EPZ-5676 is highly efficacious as a single agent and synergistically acts with other chemotherapeutics, including AML SOC drugs and DNA hypomethylating agents in MLL-r cells. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Biologico-clinical significance of DNMT3A variants expression in acute myeloid leukemia.

PubMed

Lin, Na; Fu, Wei; Zhao, Chen; Li, Bixin; Yan, Xiaojing; Li, Yan

2017-12-09

DNA methyltransferase 3A (DNMT3A) catalyzes de novo DNA methylation and plays important roles in the pathogenesis of acute myeloid leukemia. However, the expression status of DNMT3A variants in acute myeloid leukemia remains obscure. This study aimed to assess the expression levels of alternative splicing of DNMT3A variants and explore their roles in acute myeloid leukemia (AML). DNMT3A variants gene expression were assessed, measuring their effects on cell proliferation. In addition, the expression of DNMT3A variants were evaluated in acute myeloid leukemia patients. Four DNMT3A variants were identified, with DNMT3A1 and DNMT3A2V found to be dominant in acute myeloid leukemia cell lines. Moreover, DNMT3A2V overexpression delayed cell proliferation; while, DNMT3A2V R882H mutation promoted cell proliferation. Further, DNMT3A1 and DNMT3A2V were detected in newly diagnosed acute myeloid leukemia (AML) patients and controls with non-malignant hematological disease, with DNMT3A2V significantly up-regulated in AML patients. The main transcript switched from DNMT3A1 to DNMT3A2V in some patients, especially the low risk group based on the NCCN 2016 guidelines. These findings suggest that DNMT3A1 and DNMT3A2V are the main variants in acute myeloid leukemia with different clinical association, and might play important roles in the pathophysiology of acute myeloid leukemia. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis

PubMed Central

Valerio, Daria G.; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E.; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J.; Huang, Chun-Hao; Lujambio, Amaia; Zheng, George; Zuber, Johannes; Pandita, Tej K.; Lowe, Scott W.; Armstrong, Scott A.

2017-01-01

Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore Mof inactivation suppressed leukemia development in a NUP98-HOXA9 driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. PMID:28202522

Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA-Induced Leukemogenesis

PubMed Central

Masuya, Masahiro; Ishii, Satomi; Katayama, Naoyuki

2017-01-01

ABSTRACT PLZF is a transcription factor that confers aberrant self-renewal in leukemogenesis, and the PLZF-RARA fusion gene causes acute promyelocytic leukemia (APL) through differentiation block. However, the molecular mechanisms of aberrant self-renewal underlying PLZF-mediated leukemogenesis are poorly understood. To investigate these mechanisms, comprehensive expression profiling of mouse hematopoietic stem/progenitor cells transduced with Plzf was performed, which revealed the involvement of a key transcriptional coactivator, Eya2, a target molecule shared by Plzf and PLZF-RARA, in the aberrant self-renewal. Indeed, PLZF-RARA as well as Plzf rendered those cells immortalized through upregulation of Eya2. Eya2 also led to immortalization without differentiation block, while depletion of Eya2 suppressed clonogenicity in cells immortalized by PLZF-RARA without influence on differentiation and apoptosis. Interestingly, cancer outlier profile analysis of human samples of acute myeloid leukemia (AML) in The Cancer Genome Atlas (TCGA) revealed a subtype of AML that strongly expressed EYA2. In addition, gene set enrichment analysis of human AML samples, including TCGA data, showed that this subtype of AML was more closely associated with the properties of leukemic stem cells in its gene expression signature than other AMLs. Therefore, EYA2 may be a target for molecular therapy in this subtype of AML, including PLZF-RARA APL. PMID:28416638

RSK2 is a new Pim2 target with pro-survival functions in FLT3-ITD-positive acute myeloid leukemia.

PubMed

Hospital, M-A; Jacquel, A; Mazed, F; Saland, E; Larrue, C; Mondesir, J; Birsen, R; Green, A S; Lambert, M; Sujobert, P; Gautier, E-F; Salnot, V; Le Gall, M; Decroocq, J; Poulain, L; Jacque, N; Fontenay, M; Kosmider, O; Récher, C; Auberger, P; Mayeux, P; Bouscary, D; Sarry, J-E; Tamburini, J

2018-03-01

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

Proliferative status of primitive hematopoietic progenitors from patients with acute myelogenous leukemia (AML).

PubMed

Guan, Y; Hogge, D E

2000-12-01

One possible explanation for the competitive advantage that malignant cells in patients with acute myelogenous leukemia (AML) appear to have over normal hematopoietic elements is that leukemic progenitors proliferate more rapidly than their normal progenitor cell counterparts. To test this hypothesis, an overnight 3H-thymidine (3H-Tdr) suicide assay was used to analyze the proliferative status of malignant progenitors detected in both colony-forming cell (CFC) and long-term culture initiating cell (LTC-IC) assays from the peripheral blood of nine patients with newly diagnosed AML. Culture of AML cells in serum-free medium with 100 ng/ml Steel factor (SF), 20 ng/ml interleukin 3 (IL-3) and 20 ng/ml granulocyte colony-stimulating factor (G-CSF) for 16-24 h maintained the number of AML-CFC and LTC-IC at near input values (mean % input +/- s.d. for CFC and LTC-IC were 78 +/- 33 and 126 +/- 53, respectively). The addition of 20 muCi/ml high specific activity 3H-Tdr to these cultures reduced the numbers of both progenitor cell types from most of the patient samples substantially: mean % kill +/- s.d. for AML-CFC and LTC-IC were 64 +/- 27 and 82 +/- 16, respectively, indicating that a large proportion of both progenitor populations were actively cycling. FISH analysis of colonies from CFC and LTC-IC assays confirmed that most cytogenetically abnormal CFC and LTC-IC were actively cycling (mean % kill +/- s.d.: 68 +/- 26 and 85 +/- 13, respectively). Interestingly, in six patient samples where a significant number of cytogenetically normal LTC-ICs were detected, the % kill of these cells (74 +/- 20) was similar to that of the abnormal progenitors. These data contrast with the predominantly quiescent cell cycle status of CFC and LTC-IC previously observed in steady-state peripheral blood from normal individuals but also provide evidence that a significant proportion of primitive malignant progenitors from AML patients are quiescent and therefore may be resistant to standard chemotherapeutic regimens.

AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo.

PubMed

Yang, Jing; Ikezoe, Takayuki; Nishioka, Chie; Tasaka, Taizo; Taniguchi, Ayuko; Kuwayama, Yoshio; Komatsu, Naoki; Bandobashi, Kentaro; Togitani, Kazuto; Koeffler, H Phillip; Taguchi, Hirokuni; Yokoyama, Akihito

2007-09-15

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation.

Targeting B7x and B7-H3 as New Immunotherapies for Prostate Cancer

DTIC Science & Technology

2017-11-01

treat rheumatoid arthritis and prevent acute kidney transplant rejection (Fiocco et al., 2008; Vincenti et al., 2011). The past decade has witnessed a...cell lines. The first Phase I trial with pidilizumab recruited patients with hematologic malignancies, including acute myeloid leukemia (AML), chronic... radiation , chemotherapy, other coinhibi- tory antibodies, or vaccines can improve the response rate in cancers. Predictive biomarkers need to be developed

Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells.

PubMed

Saenz, D T; Fiskus, W; Qian, Y; Manshouri, T; Rajapakshe, K; Raina, K; Coleman, K G; Crew, A P; Shen, A; Mill, C P; Sun, B; Qiu, P; Kadia, T M; Pemmaraju, N; DiNardo, C; Kim, M-S; Nowak, A J; Coarfa, C; Crews, C M; Verstovsek, S; Bhalla, K N

2017-09-01

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry 'CyTOF' analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

Biology and relevance of human acute myeloid leukemia stem cells.

PubMed

Thomas, Daniel; Majeti, Ravindra

2017-03-23

Evidence of human acute myeloid leukemia stem cells (AML LSCs) was first reported nearly 2 decades ago through the identification of rare subpopulations of engrafting cells in xenotransplantation assays. These AML LSCs were shown to reside at the apex of a cellular hierarchy that initiates and maintains the disease, exhibiting properties of self-renewal, cell cycle quiescence, and chemoresistance. This cancer stem cell model offers an explanation for chemotherapy resistance and disease relapse and implies that approaches to treatment must eradicate LSCs for cure. More recently, a number of studies have both refined and expanded our understanding of LSCs and intrapatient heterogeneity in AML using improved xenotransplant models, genome-scale analyses, and experimental manipulation of primary patient cells. Here, we review these studies with a focus on the immunophenotype, biological properties, epigenetics, genetics, and clinical associations of human AML LSCs and discuss critical questions that need to be addressed in future research. © 2017 by The American Society of Hematology.

Single-cell whole exome and targeted sequencing in NPM1/FLT3 positive pediatric acute myeloid leukemia.

PubMed

Walter, Christiane; Pozzorini, Christian; Reinhardt, Katarina; Geffers, Robert; Xu, Zhenyu; Reinhardt, Dirk; von Neuhoff, Nils; Hanenberg, Helmut

2018-02-01

The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC-enriched blasts from pediatric patients. For three AMLs with mutations in nucleophosmin 1 and/or fms-like tyrosine kinase 3, we performed whole genome amplification on sorted single-cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies. Analysis revealed that read coverage statistics were comparable between single-cell and bulk WES data, indicating high-quality whole genome amplification. From 102 single-cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50-72× failed to detect rare AML subclones in the bulk DNAs. In summary, our study demonstrated that single-cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs. © 2017 Wiley Periodicals, Inc.

Allogeneic stem cell transplantation for acute myeloid leukemia with del(7q) following untreated chronic lymphocytic leukemia.

PubMed

DeFilipp, Zachariah; Huynh, Donny V; Fazal, Salman; Sahovic, Entezam

2012-01-01

The development of hematologic malignancy in the presence of chronic lymphocytic leukemia (CLL) is rare. We present a case of acute myeloid leukemia (AML) with del(7q) occurring in a patient with a 4-year history of untreated CLL. Application of flow cytometry and immunohistochemistry allowed for characterization of two distinct coexisting malignant cell populations. After undergoing induction and consolidation chemotherapy, the patient achieved complete remission of AML with the persistence of CLL. Allogeneic transplantation was pursued given his unfavorable cytogenetics. Subsequent matched unrelated donor allogeneic stem cell transplantation resulted in full engraftment and complete remission, with no evidence of AML or CLL. Due to a scarcity of reported cases, insight into treatment and prognosis in cases of concurrent AML and CLL is limited. However, prognosis seems dependent on the chemosensitivity of AML. CLL did not have a detrimental effect on treatment or transplant outcome in our case. This is the first reported case of concomitant de novo AML and CLL to undergo allogeneic transplantation. The patient remained in complete hematologic and cytogenetic remission of both malignancies over a year after transplantation.

Universal monitoring of minimal residual disease in acute myeloid leukemia.

PubMed

Coustan-Smith, Elaine; Song, Guangchun; Shurtleff, Sheila; Yeoh, Allen Eng-Juh; Chng, Wee Joo; Chen, Siew Peng; Rubnitz, Jeffrey E; Pui, Ching-Hon; Downing, James R; Campana, Dario

2018-05-03

Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to identify distinctive features of AML cells for universal MRD monitoring. We compared genome-wide gene expression of AML cells from 157 patients with that of normal myeloblasts. Markers encoded by aberrantly expressed genes, including some previously associated with leukemia stem cells, were studied by flow cytometry in 240 patients with AML and in nonleukemic myeloblasts from 63 bone marrow samples. Twenty-two (CD9, CD18, CD25, CD32, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a/c, CD366, CD371, and CX3CR1) markers were aberrantly expressed in AML. Leukemia-associated profiles defined by these markers extended to immature CD34+CD38- AML cells; expression remained stable during treatment. The markers yielded MRD measurements matching those of standard methods in 208 samples from 52 patients undergoing chemotherapy and revealed otherwise undetectable MRD. They allowed MRD monitoring in 129 consecutive patients, yielding prognostically significant results. Using a machine-learning algorithm to reduce high-dimensional data sets to 2-dimensional data, the markers allowed a clear visualization of MRD and could detect 1 leukemic cell among more than 100,000 normal cells. The markers uncovered in this study allow universal and sensitive monitoring of MRD in AML. In combination with contemporary analytical tools, the markers improve the discrimination between leukemic and normal cells, thus facilitating data interpretation and, hence, the reliability of MRD results. National Cancer Institute (CA60419 and CA21765); American Lebanese Syrian Associated Charities; National Medical Research Council of Singapore (1299/2011); Viva Foundation for Children with Cancer, Children's Cancer Foundation, Tote Board & Turf Club, and Lee Foundation of Singapore.

Crenolanib is a type I tyrosine kinase inhibitor that inhibits mutant KIT D816 isoforms prevalent in systemic mastocytosis and core binding factor leukemia.

PubMed

Kampa-Schittenhelm, Kerstin Maria; Frey, Julia; Haeusser, Lara A; Illing, Barbara; Pavlovsky, Ashly A; Blumenstock, Gunnar; Schittenhelm, Marcus Matthias

2017-10-10

Activating D816 mutations of the class III receptor tyrosine kinase KIT are associated with the majority of patients with systemic mastocytosis (SM), but also core binding factor (CBF) AML, making KIT mutations attractive therapeutic targets for the treatment of these cancers. Crenolanib is a potent and selective inhibitor of wild-type as well as mutant isoforms of the class III receptor tyrosine kinases FLT3 and PDGFRα/β. Notably, crenolanib inhibits constitutively active mutant-FLT3 isoforms resulting from amino acid substitutions of aspartic acid at codon 835, which is homologous to codon 816 in the KIT gene - suggesting sensitivity against mutant-KIT D816 isoforms as well. Here we demonstrate that crenolanib targets KIT D816 in SM and CBF AML models: crenolanib inhibits cellular proliferation and initiates apoptosis of mastocytosis cell lines expressing these mutations. Target-specificity was confirmed using an isogenic cell model. In addition, we demonstrate that KIT D816 mutations are targetable with clinically achievable doses of crenolanib. Further, a rationale to combine cladribine (2-CDA), the therapeutic standard in SM, with crenolanib is provided. In conclusion, we demonstrate that crenolanib is an inhibitor of mutant-KIT D816 isoforms at clinically achievable concentrations, and thus may be a potential treatment for SM and CBF AML as a monotherapy or in combination approaches.

Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia

PubMed Central

Kenderian, Saad S.; Shen, Feng; Ruella, Marco; Shestova, Olga; Kozlowski, Miroslaw; Li, Yong; Schrank-Hacker, April; Morrissette, Jennifer J. D.; Carroll, Martin; June, Carl H.; Grupp, Stephan A.; Gill, Saar

2017-01-01

We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA–electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti–CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML. PMID:28246194

Functionally distinct roles for different miR-155 expression levels through contrasting effects on gene expression, in acute myeloid leukaemia.

PubMed

Narayan, N; Morenos, L; Phipson, B; Willis, S N; Brumatti, G; Eggers, S; Lalaoui, N; Brown, L M; Kosasih, H J; Bartolo, R C; Zhou, L; Catchpoole, D; Saffery, R; Oshlack, A; Goodall, G J; Ekert, P G

2017-04-01

Enforced expression of microRNA-155 (miR-155) in myeloid cells has been shown to have both oncogenic or tumour-suppressor functions in acute myeloid leukaemia (AML). We sought to resolve these contrasting effects of miR-155 overexpression using murine models of AML and human paediatric AML data sets. We show that the highest miR-155 expression levels inhibited proliferation in murine AML models. Over time, enforced miR-155 expression in AML in vitro and in vivo, however, favours selection of intermediate miR-155 expression levels that results in increased tumour burden in mice, without accelerating the onset of disease. Strikingly, we show that intermediate and high miR-155 expression also regulate very different subsets of miR-155 targets and have contrasting downstream effects on the transcriptional environments of AML cells, including genes involved in haematopoiesis and leukaemia. Furthermore, we show that elevated miR-155 expression detected in paediatric AML correlates with intermediate and not high miR-155 expression identified in our experimental models. These findings collectively describe a novel dose-dependent role for miR-155 in the regulation of AML, which may have important therapeutic implications.

Chimeric antigen receptor T-cell therapy in AML: How close are we?

PubMed Central

Gill, Saar

2016-01-01

The majority of patients presenting with acute myeloid leukemia (AML) initially respond to chemotherapy but post-remission therapy is required to consolidate this response and achieve long-term disease-free survival. The most effective form of post-remission therapy relies on T-cell immunotherapy in the form of allogeneic hematopoietic cell transplantation (HCT). However, patients with active disease cannot usually expect to be cured with HCT. This inherent dichotomy implies that traditional T cell-based immunotherapy in the form of allogeneic HCT stops being efficacious somewhere between the measurable residual disease (MRD) and the morphologically obvious range. This is in part because the full power of T cells must be restrained in order to avoid lethal graft-versus-host disease (GVHD) and partly because only a sub-population of donor T cells are expected to be able to recognize AML cells via their T cell receptor. Chimeric antigen receptor (CAR) T cell therapy, most advanced in the treatment of patients with B-cell malignancies, may circumvent some of these limitations. However, major challenges remain to be overcome before CAR T cell therapy can be safely applied to AML. PMID:27890255

RNA-Seq analysis identifies aberrant RNA splicing of TRIP12 in acute myeloid leukemia patients at remission.

PubMed

Gao, Panke; Jin, Zhen; Cheng, Yingying; Cao, Xiangshan

2014-10-01

Aberrant splicing events play important roles in the pathogenesis of acute myeloid leukemia (AML). To investigate the aberrant splicing events in AML during treatment, we carried out RNA sequencing in peripheral mononuclear cell samples from a patient with complete remission. In addition to the sequencing samples, selected splicing events were confirmed and validated with real-time quantitative RT-PCR in another seven pairs of samples. A total of 4.05 and 3.39 GB clean data of the AML and remission sample were generated, respectively, and 2,223 differentially expressed genes (DEGs) were identified. Integrated with gene expression profiling on T cells from AML patients compared with healthy donors, 82 DEGs were also differentially expressed in AML CD4 T cells and CD8 T cells. Twenty-three alternative splicing events were considered to be confidential, and they were involved in many biological processes, such as RNA processing, cellular macromolecule catabolic process, and DNA binding process. An exon3-skipping event in TRIP12 was detected in patients at remission and further validated in another three independent samples. TRIP12 is an ubiquitin ligase of ARF, which suppresses aberrant cell growth by activating p53 responses. The exon3-skipping isoform of TRIP12 increased significantly after treatment. Our results may provide new understanding of AML, and the confirmed alternative splicing event of TRIP12 may be used as potential target for future investigations.

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients.

PubMed

Siegler, Uwe; Meyer-Monard, Sandrine; Jörger, Simon; Stern, Martin; Tichelli, André; Gratwohl, Alois; Wodnar-Filipowicz, Aleksandra; Kalberer, Christian P

2010-10-01

Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia.

PubMed

Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2015-10-20

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

Monocarboxylate transporter 1 (MCT1), a tool to stratify acute myeloid leukemia (AML) patients and a vehicle to kill cancer cells

PubMed Central

Lopes-Coelho, Filipa; Nunes, Carolina; Gouveia-Fernandes, Sofia; Rosas, Rita; Silva, Fernanda; Gameiro, Paula; Carvalho, Tânia; Gomes da Silva, Maria; Cabeçadas, José; Dias, Sérgio; Gonçalves, Luís G.; Serpa, Jacinta

2017-01-01

Dysregulation of glucose/lactate dynamics plays a role in cancer progression, and MCTs are key elements in metabolic remodeling. VEGF is a relevant growth factor in the maintenance of bone marrow microenvironment and it is also important in hematological diseases. Our aim was to investigate the role of VEGF in the metabolic adaptation of Acute myeloid leukemia (AML) cells by evaluating the metabolic profiles and cell features according to the AML lineage and testing lactate as a metabolic coin. Our in vitro results showed that AML promyelocytic (HL60) and monocytic (THP1) (but not erythroid- HEL) lineages are well adapted to VEGF and lactate rich environment. Their metabolic adaptation relies on high rates of glycolysis to generate intermediates for PPP to support cell proliferation, and on the consumption of glycolysis-generated lactate to supply biomass and energy production. VEGF orchestrates this metabolic network by regulating MCT1 expression. Bromopyruvic acid (BPA) was proven to be an effective cytotoxic in AML, possibly transported by MCT1. Our study reinforces that targeting metabolism can be a good strategy to fight cancer. MCT1 expression at the time of diagnosis can assist on the identification of AML patients that will benefit from BPA therapy. Additionally, MCT1 can be used in targeted delivery of conventional cytotoxic drugs. PMID:29137304

Monocarboxylate transporter 1 (MCT1), a tool to stratify acute myeloid leukemia (AML) patients and a vehicle to kill cancer cells.

PubMed

Lopes-Coelho, Filipa; Nunes, Carolina; Gouveia-Fernandes, Sofia; Rosas, Rita; Silva, Fernanda; Gameiro, Paula; Carvalho, Tânia; Gomes da Silva, Maria; Cabeçadas, José; Dias, Sérgio; Gonçalves, Luís G; Serpa, Jacinta

2017-10-10

Dysregulation of glucose/lactate dynamics plays a role in cancer progression, and MCTs are key elements in metabolic remodeling. VEGF is a relevant growth factor in the maintenance of bone marrow microenvironment and it is also important in hematological diseases. Our aim was to investigate the role of VEGF in the metabolic adaptation of Acute myeloid leukemia (AML) cells by evaluating the metabolic profiles and cell features according to the AML lineage and testing lactate as a metabolic coin. Our in vitro results showed that AML promyelocytic (HL60) and monocytic (THP1) (but not erythroid- HEL) lineages are well adapted to VEGF and lactate rich environment. Their metabolic adaptation relies on high rates of glycolysis to generate intermediates for PPP to support cell proliferation, and on the consumption of glycolysis-generated lactate to supply biomass and energy production. VEGF orchestrates this metabolic network by regulating MCT1 expression. Bromopyruvic acid (BPA) was proven to be an effective cytotoxic in AML, possibly transported by MCT1. Our study reinforces that targeting metabolism can be a good strategy to fight cancer. MCT1 expression at the time of diagnosis can assist on the identification of AML patients that will benefit from BPA therapy. Additionally, MCT1 can be used in targeted delivery of conventional cytotoxic drugs.

Correlation between CD34 expression and chromosomal abnormalities but not clinical outcome in acute myeloid leukemia.

PubMed

Fruchart, C; Lenormand, B; Bastard, C; Boulet, D; Lesesve, J F; Callat, M P; Stamatoullas, A; Monconduit, M; Tilly, H

1996-11-01

The hemopoietic stem cell marker CD34 has been reported to be a useful predictor of treatment outcome in acute myeloid leukemia (AML). Previous data suggested that CD34 expression may be associated with other poor prognosis factors in AML such as undifferentiated leukemia, secondary AML (SAML), and clonal abnormalities involving chromosome 5 and 7. In order to analyze the correlations between the clinicopathologic features, cytogenetic and CD34 expression in AML, we retrospectively investigated 99 patients with newly diagnosed AML: 85 with de novo disease and 14 with secondary AML (SAML). Eighty-six patients who received the same induction chemotherapy were available for clinical outcome. Defining a case as positive when > or = 20% of bone marrow cells collected at diagnosis expressed the CD34 antigen, forty-five patients were included in the CD34 positive group. Ninety patients had adequate cytogenetic analysis. Thirty-two patients (72%) with CD34 positive AML exhibited an abnormal karyotype whereas 15 patients (28%) with CD34 negative AML had abnormal metaphases (P < 0.01). Monosomy 7/7q- or monosomy 5/5q- occurred in 10 patients and 8 of them expressed the CD34 antigen (P < 0.05). All patients with t(8;21) which is considered as a favorable factor in AML had levels of CD34 >/= 20% (P < 0.05). We did not find any association between CD34 expression and attainment of complete remission, overall survival, or disease-free survival. In conclusion, the variations of CD34 expression in AML are correlated with cytogenetic abnormalities associated both with poor and favorable outcome. The evaluation of the correlations between CD34 antigen and clinical outcome in AML should take into account the results of pretreatment karyotype.

HLA-E upregulation on IFN-gamma-activated AML blasts impairs CD94/NKG2A-dependent NK cytolysis after haplo-mismatched hematopoietic SCT.

PubMed

Nguyen, S; Beziat, V; Dhedin, N; Kuentz, M; Vernant, J P; Debre, P; Vieillard, V

2009-05-01

Natural killer (NK) cells generated after haploidentical hematopoietic SCT in patients with AML are characterized by specific phenotypic features and impaired functioning that may affect transplantation outcome. We show that IFN-gamma produced by immature CD56(bright) NK cells upregulates cell surface expression of HLA-E on AML blasts and that this upregulation protects leukemic cells from NK-mediated cell lysis through the mediation of CD94/NKG2A, an inhibitory receptor overexpressed on NK cells after haploidentical SCT. Two years after transplantation, however, maturing NK cells were functionally active, as evidenced by high cytotoxicity and poor IFN-gamma production. This implies that maturation of NK cells is the key to improved immune responses and transplantation outcome.

Renal Sinus Fat Invasion and Tumoral Thrombosis of the Inferior Vena Cava-Renal Vein: Only Confined to Renal Cell Carcinoma

PubMed Central

Harman, Mustafa; Guneyli, Serkan; Sen, Sait; Elmas, Nevra

2014-01-01

Epithelioid angiomyolipoma (E-AML), accounting for 8% of renal angiomyolipoma, is usually associated with tuberous sclerosis (TS) and demonstrates aggressive behavior. E-AML is macroscopically seen as a large infiltrative necrotic tumor with occasional extension into renal vein and/or inferior vena cava. However, without history of TS, renal sinus and venous invasion E-AML would be a challenging diagnosis, which may lead radiologists to misinterpret it as a renal cell carcinoma (RCC). In this case presentation, we aimed to report cross-sectional imaging findings of two cases diagnosed as E-AML and pathological correlation of these aforementioned masses mimicking RCC. PMID:25506021

Renal sinus fat invasion and tumoral thrombosis of the inferior vena cava-renal vein: only confined to renal cell carcinoma.

PubMed

Acar, Turker; Harman, Mustafa; Guneyli, Serkan; Sen, Sait; Elmas, Nevra

2014-01-01

Epithelioid angiomyolipoma (E-AML), accounting for 8% of renal angiomyolipoma, is usually associated with tuberous sclerosis (TS) and demonstrates aggressive behavior. E-AML is macroscopically seen as a large infiltrative necrotic tumor with occasional extension into renal vein and/or inferior vena cava. However, without history of TS, renal sinus and venous invasion E-AML would be a challenging diagnosis, which may lead radiologists to misinterpret it as a renal cell carcinoma (RCC). In this case presentation, we aimed to report cross-sectional imaging findings of two cases diagnosed as E-AML and pathological correlation of these aforementioned masses mimicking RCC.

Leukemogenesis Induced by an Activating β-catenin mutation in Osteoblasts

PubMed Central

Kode, Aruna; Manavalan, John S.; Mosialou, Ioanna; Bhagat, Govind; Rathinam, Chozha V.; Luo, Na; Khiabanian, Hossein; Lee, Albert; Vundavalli, Murty; Friedman, Richard; Brum, Andrea; Park, David; Galili, Naomi; Mukherjee, Siddhartha; Teruya-Feldstein, Julie; Raza, Azra; Rabadan, Raul; Berman, Ellin; Kousteni, Stavroula

2014-01-01

Summary Cells of the osteoblast lineage affect homing, 1, 2 number of long term repopulating hematopoietic stem cells (HSCs) 3, 4, HSC mobilization and lineage determination and B lymphopoiesis 5-8. More recently osteoblasts were implicated in pre-leukemic conditions in mice 9, 10. Yet, it has not been shown that a single genetic event taking place in osteoblasts can induce leukemogenesis. We show here that in mice, an activating mutation of β-catenin in osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukemia (AML) with common chromosomal aberrations and cell autonomous progression. Activated β-catenin stimulates expression of the Notch ligand Jagged-1 in osteoblasts. Subsequent activation of Notch signaling in HSC progenitors induces the malignant changes. Demonstrating the pathogenetic role of the Notch pathway, genetic or pharmacological inhibition of Notch signaling ameliorates AML. Nuclear accumulation and increased β-catenin signaling in osteoblasts was also identified in 38% of patients with MDS/AML. These patients showed increased Notch signaling in hematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce AML, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to AML. PMID:24429522

GSK-3 Inhibition Sensitizes Acute Myeloid Leukemia Cells to 1,25D-Mediated Differentiation

PubMed Central

Gupta, Kalpana; Stefan, Tammy; Ignatz-Hoover, James; Moreton, Stephen; Parizher, Gary; Saunthararajah, Yogen; Wald, David N.

2017-01-01

1,25-dihydroxyvitamin D3 (1,25D), the biologically active form of vitamin D, is widely considered a promising therapy for acute myeloid leukemia (AML) based on its ability to drive differentiation of leukemic cells. However, clinical trials have been disappointing in part to dose-limiting hypercalcemia. Here we show how inhibiting glycogen synthase kinase 3 (GSK3) can improve the differentiation response of AML cells to 1,25D-mediated differentiation. GSK3 inhibition in AML cells enhanced the differentiating effects of low concentrations of 1,25D. In addition, GSK3 inhibition augmented the ability of 1,25D to induce irreversible growth inhibition and slow the progression of AML in mouse models. Mechanistic studies revealed that GSK3 inhibition led to the hyperphosphorylation of the vitamin D receptor (VDR), enabling an interaction between VDR and the coactivator, SRC-3 (NCOA3), thereby increasing transcriptional activity. We also found that activation of JNK-mediated pathways in response to GSK3 inhibition contributed to the potentiation of 1,25D-induced differentiation. Taken together, our findings offer a preclinical rationale to explore the repositioning of GSK3 inhibitors to enhance differentiation-based therapy for AML treatment. PMID:26964622

[Proliferation and IFN-gamma secretion of autologous T lymphocytes stimulated by myeloid leukemia cells induced with rhGM-CSF and rhIL-4].

PubMed

Xie, Yan-Hui; Chen, Qin-Fen; Xie, Yi; Xie, Hong

2002-12-01

To observe the proliferation of T lymphocytes stimulated by CML and AML cells which were induced by rhGM-CSF and rhIL-4, and the secretion of IFN-gamma from proliferated T lymphocytes, the expression of CD80, CD86 and HLA-DR on CML and AML cells induced by GM-CSF and IL-4 was assayed by flow cytometry in vitro. Then one-way mixed lymphocyte reaction was carried out, with induced leukemia cells as stimulating cells and auto-T lymphocytes as reactive cells. The secretion of IFN-gamma from T lymphocytes was determined by double antibody sandwich ELISA. The results showed that GM-CSF and IL-4 significantly upregulated the expression of CD80, CD86 and HLA-DR on CML cells and CD80 and CD86 on AML cells, which could stimulate the T lymphocyte proliferation and high secretion of IFN-gamma (in CML group) of autologous T lymphocytes. It is concluded that the CML and AML cells induced by GM-CSF and IL-4 have the ability to present tumor specific antigen to auto-T lymphocyte.

A novel isoflavone, ME-344, targets the cytoskeleton in acute myeloid leukemia

PubMed Central

Jeyaraju, Danny V.; Hurren, Rose; Wang, Xiaoming; MacLean, Neil; Gronda, Marcela; Shamas-Din, Aisha; Minden, Mark D.; Giaever, Guri; Schimmer, Aaron D.

2016-01-01

The isoflavone ME-344 is a potent anti-cancer agent with preclinical and clinical efficacy in solid tumors. Yet, the mechanism of action of ME-344 has not been fully defined and the preclinical efficacy in leukemia has not been established. Therefore, we investigated the anti-leukemic properties and mechanism of action of ME-344. In a panel of 7 leukemia cell lines, ME-344 was cytotoxic with an IC50 in the range of 70–260 nM. In addition, ME-344 was cytotoxic to primary AML patient samples over normal hematopoietic cells. In an OCI-AML2 xenograft model, ME-344 reduced tumor growth by up to 95% of control without evidence of toxicity. Mechanistically, ME-344 increased mitochondrial ROS generation in leukemic cells. However, antioxidant treatment did not rescue cell death, suggesting that ME-344 had additional targets beyond the mitochondria. We demonstrated that ME-344 inhibited tubulin polymerization by interacting with tubulin near the colchicine-binding site. Furthermore, inhibition of tubulin polymerization was functionally important for ME-344 induced death. Finally, we showed that ME-344 synergizes with vinblastine in leukemia cells. Thus, our study demonstrates that ME-344 displays preclinical efficacy in leukemia through a mechanism at least partly related to targeting tubulin polymerization. PMID:27391350

A novel isoflavone, ME-344, targets the cytoskeleton in acute myeloid leukemia.

PubMed

Jeyaraju, Danny V; Hurren, Rose; Wang, Xiaoming; MacLean, Neil; Gronda, Marcela; Shamas-Din, Aisha; Minden, Mark D; Giaever, Guri; Schimmer, Aaron D

2016-08-02

The isoflavone ME-344 is a potent anti-cancer agent with preclinical and clinical efficacy in solid tumors. Yet, the mechanism of action of ME-344 has not been fully defined and the preclinical efficacy in leukemia has not been established. Therefore, we investigated the anti-leukemic properties and mechanism of action of ME-344. In a panel of 7 leukemia cell lines, ME-344 was cytotoxic with an IC50 in the range of 70-260 nM. In addition, ME-344 was cytotoxic to primary AML patient samples over normal hematopoietic cells. In an OCI-AML2 xenograft model, ME-344 reduced tumor growth by up to 95% of control without evidence of toxicity. Mechanistically, ME-344 increased mitochondrial ROS generation in leukemic cells. However, antioxidant treatment did not rescue cell death, suggesting that ME-344 had additional targets beyond the mitochondria. We demonstrated that ME-344 inhibited tubulin polymerization by interacting with tubulin near the colchicine-binding site. Furthermore, inhibition of tubulin polymerization was functionally important for ME-344 induced death. Finally, we showed that ME-344 synergizes with vinblastine in leukemia cells. Thus, our study demonstrates that ME-344 displays preclinical efficacy in leukemia through a mechanism at least partly related to targeting tubulin polymerization.

Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis.

PubMed

Valerio, Daria G; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J; Huang, Chun-Hao; Lujambio, Amaia; Zheng, YuJun George; Zuber, Johannes; Pandita, Tej K; Lowe, Scott W; Armstrong, Scott A

2017-04-01

Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9 -driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof -deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9 -driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL -rearranged leukemias. Cancer Res; 77(7); 1753-62. ©2017 AACR . ©2017 American Association for Cancer Research.

Immunotherapy of elderly acute myeloid leukemia: light at the end of a long tunnel?

PubMed

Rafelson, William M; Reagan, John L; Fast, Loren D; Lim, Seah H

2017-11-01

Although it is possible to induce remission in the majority of the patients with acute myeloid leukemia (AML), many patients still die due to disease relapse. Immunotherapy is an attractive option. It is more specific. The memory T cells induced by immunotherapy may also provide the long-term tumor immunosurveillance to prevent disease relapse. Although immunotherapy of AML started in the early 1970s, its clinical impact has been disappointing. Recent advances in tumor immunology and immunotherapeutic agents have rekindled interest. Here, we provide a review of the history of AML immunotherapy, discuss why AML is well suited for immunotherapeutic approaches and present the biological obstacles that affect the success of immunotherapy. Finally, we put forward a new paradigm of AML immunotherapy that utilizes a combination of immunotherapeutic agents sequentially to enhance the in vivo tumor immunogenicity and effective priming and propagation of tumor-specific cytotoxic T cells.

Phase 2 trial of CPX-351, a fixed 5:1 molar ratio of cytarabine/daunorubicin, vs cytarabine/daunorubicin in older adults with untreated AML

PubMed Central

Cortes, Jorge E.; Hogge, Donna E.; Tallman, Martin S.; Kovacsovics, Tibor J.; Damon, Lloyd E.; Komrokji, Rami; Solomon, Scott R.; Kolitz, Jonathan E.; Cooper, Maureen; Yeager, Andrew M.; Louie, Arthur C.; Feldman, Eric J.

2014-01-01

CPX-351 is a liposomal formulation of cytarabine:daunorubicin designed to deliver synergistic drug ratios to leukemia cells. In this phase 2 study, newly diagnosed older acute myeloid leukemia (AML) patients were randomized 2:1 to first-line CPX-351 or 7+3 treatment. The goal was to determine efficacy and identify patient subgroups that may benefit from CPX-351 treatment. Response rate (complete remission + incomplete remission) was the primary end point, with event-free survival (EFS) and overall survival (OS) as secondary end points. The 126 patients entered were balanced for disease and patient-specific risk factors. Overall, CPX-351 produced higher response rates (66.7% vs 51.2%, P = .07), meeting predefined criteria for success (P < .1). Differences in EFS and OS were not statistically significant. A planned analysis of the secondary AML subgroup demonstrated an improved response rate (57.6% vs 31.6%, P = .06), and prolongation of EFS (hazard ratio [HR] = 0.59, P = .08) and OS (HR = 0.46, P = .01). Recovery from cytopenias was slower after CPX-351 (median days to absolute neutrophil count ≥1000: 36 vs 32; platelets >100 000: 37 vs 28) with more grade 3-4 infections but without increase in infection-related deaths (3.5% vs 7.3%) or 60-day mortality (4.7% vs 14.6%), indicating acceptable safety. These results suggest a clinical benefit with CPX-351, particularly among patients with secondary AML, and provide the rationale for a phase 3 trial currently underway in newly diagnosed secondary AML patients. This study is registered at Clinicaltrials.gov as #NCT00788892. PMID:24687088

AML associated with previous cytotoxic therapy, MDS or myeloproliferative disorders: results from the MRC's 9th AML trial.

PubMed

Hoyle, C F; de Bastos, M; Wheatley, K; Sherrington, P D; Fischer, P J; Rees, J K; Gray, R; Hayhoe, F G

1989-05-01

The outcome of treatment with standard first line therapy of 66 patients with acute myeloid leukaemia (AML) secondary to preceding chemotherapy (Group 1), a myelodysplastic state (Group 2) or a myeloproliferative disorder (Group 3) was analysed in relation to the preceding disorder, the cytogenetic pattern where available, and the cytology and cytochemistry of blood and bone marrow. The complete remission (CR) rate for the secondary AMLs was 36% (24/66), with 24% (16/66) dying in the induction period and 39% (26/66) having resistant disease. The CR rate was 25% (5/20) for Group 1, 42% (15/36) for Group 2, and 40% (4/10) for Group 3. Even after allowance for the generally older age of the secondary AML patients, they still had a significantly poorer CR rate than the de novo AMLs (P = 0.0004). The lower CR rate was chiefly due to resistant disease. Despite this, overall survival was not significantly worse for the secondary AML patients (P = 0.15). For the 36% that achieved remission, remission duration appeared similar to that of de novo cases. Of 62 cases with adequate cytology, 38 (61%) had evidence of erythroid and/or megakaryocytic dysplasia with a CR rate of 32% (12/38). The CR rate of these multineage leukaemias was not significantly different from that of the 24 (39%) who showed granulocyte/monocyte precursor involvement only, 42% (10) of whom achieved CR. The presence of features of differentiation within blast cells such as Auer rods or sudanophilia (greater than 50% positive blasts) was associated with a higher remission rate 47% (18/38) than that of poorly differentiated cases 17% (3/18) (P = 0.04) and thus appeared to be a more important determinant of CR achievement than was lineage involvement. Cases with a normal karyotype had a 33% (7/21) CR rate, while those with chromosomal abnormalities had a 37% (9/24) CR rate. Only 12 of the 45 cases with adequate cytogenetic analysis showed deletions or monosomies involving chromosomes 5 or 7, and seven of these were in Group 1.

Antibody therapy for acute myeloid leukaemia.

PubMed

Gasiorowski, Robin E; Clark, Georgina J; Bradstock, Kenneth; Hart, Derek N J

2014-02-01

Novel therapies with increased efficacy and decreased toxicity are desperately needed for the treatment of acute myeloid leukaemia (AML). The anti CD33 immunoconjugate, gemtuzumab ozogamicin (GO), was withdrawn with concerns over induction mortality and lack of efficacy. However a number of recent trials suggest that, particularly in AML with favourable cytogenetics, GO may improve overall survival. This data and the development of alternative novel monoclonal antibodies (mAb) have renewed interest in the area. Leukaemic stem cells (LSC) are identified as the subset of AML blasts that reproduces the leukaemic phenotype upon transplantation into immunosuppressed mice. AML relapse may be caused by chemoresistant LSC and this has refocused interest on identifying and targeting antigens specific for LSC. Several mAb have been developed that target LSC effectively in xenogeneic models but only a few have begun clinical evaluation. Antibody engineering may improve the activity of potential new therapeutics for AML. The encouraging results seen with bispecific T cell-engaging mAb-based molecules against CD19 in the treatment of B-cell acute lymphobalstic leukaemia, highlight the potential efficacy of engineered antibodies in the treatment of acute leukaemia. Potent engineered mAb, possibly targeting novel LSC antigens, offer hope for improving the current poor prognosis for AML. © 2013 John Wiley & Sons Ltd.

Impact of Early Cytomegalovirus Reactivation in Cord Blood Stem Cell Recipients in the Current Era

PubMed Central

Ramanathan, Muthalagu; Teira, Pierre; Battiwalla, Minoo; Barrett, John; Ahn, Kwang Woo; Chen, Min; Green, Jamie; Laughlin, Mary; Lazarus, Hillard M.; Marks, David; Saad, Ayman; Seftel, Matthew; Saber, Wael; Savani, Bipin; Waller, Edmund; Wingard, John; Auletta, Jeffery J.; Lindemans, Caroline A.; Boeckh, Michael; Riches, Marcie L.

2016-01-01

Several studies have reported an association between cytomegalovirus (CMV) reactivation and a decreased incidence of relapse for acute myeloid leukemia (AML) after adult donor allogeneic hematopoietic cell transplantation (HCT). Limited data, however, are available on the impact of CMV reactivation on relapse after cord blood stem cell (CB) transplantation. The unique combination of higher incidence of CMV reactivation in the seropositive recipient and lower incidence of graft versus host disease (GvHD) in CB HCT allows for a valuable design to analyze the impact of CMV reactivation. Data from 1684 patients transplanted with cord blood (CB) between 2003 and 2010 for AML and acute lymphoblastic leukemia (ALL) were analyzed. The median time to CMV reactivation was 34 days (range: 2 – 287). CMV reactivation and positive CMV serology were associated with increased non-relapse mortality (NRM) amongst both AML and ALL CB recipients [Reactivation, AML: RR 1.41 (1.07–1.85); ALL: 1.60 (1.14 – 2.23); Serology, AML: RR 1.39 (1.05 – 1.85), ALL: RR 1.61 (1.18 – 2.19)]. For patients with ALL, but not those with AML, this yielded inferior overall survival (p<0.005). Risk of relapse was not impacted by CMV reactivation or positive CMV serostatus for either disease. PMID:27042847

Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients

PubMed Central

Fucikova, Jitka; Truxova, Iva; Hensler, Michal; Becht, Etienne; Kasikova, Lenka; Moserova, Irena; Vosahlikova, Sarka; Klouckova, Jana; Church, Sarah E.; Cremer, Isabelle; Kepp, Oliver; Kroemer, Guido; Galluzzi, Lorenzo; Salek, Cyril

2016-01-01

Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called “damage-associated molecular patterns,” DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response. PMID:27802968

The change of nuclear LC3 distribution in acute myeloid leukemia cells.

PubMed

Guo, Wenjian; Jin, Jingrui; Pan, Jiajia; Yao, Rongxing; Li, Xia; Huang, Xin; Ma, Zhixing; Huang, Sujuan; Yan, Xiao; Jin, Jie; Dong, Aishu

2018-05-09

Making sure the change of nuclear LC3 distribution in the autophagy of acute myeloid leukemia (AML) cell and finding out the regulation mechanism may lead to a breakthrough for killing AML cells. Western blots were performed to assess the expression of autophagy proteins. Changes in the LC3 distribution were monitored by immunofluorescence assays together with western blots, and the expression levels of Sirt1, DOR, Beclin1, HMGB1, and AMPK mRNA were detected via fluorescent quantitative PCR. The effects of Sirt1 and DOR on cell proliferation and survival were analyzed by MTT, flow cytometry, and western blotting assays. We found that treating AML cells with Ara-c or Sorafenib resulted in autophagy enhancement, and when autophagy was enhanced, nuclear LC3 moved into the cytoplasm. Notably, when autophagy was inhibited by blocking the nuclear LC3 shift, the cytotoxicity of drugs was enhanced. Our results also identified Sirt1 and DOR as regulatory molecules for the observed nuclear LC3 shift, and these molecules further affected the expression of Beclin1, HMGB1, and AMPK. Our results suggest the distribution of nuclear LC3 can be a novel way for further studying death of AML cells,and the regulatory molecules may be new targets for treating AML. Copyright © 2018 Elsevier Inc. All rights reserved.

1,25-DIHYDROXYVITAMIN D3 INDUCES MONOCYTIC DIFFERENTIATION OF HUMAN MYELOID LEUKEMIA CELLS BY REGULATING C/EBPβ EXPRESSION THROUGH MEF2C

PubMed Central

Zheng, Ruifang; Wang, Xuening; Studzinski, George P.

2015-01-01

Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/Enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27Kip1 and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (Activating Transcription Factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, is mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression. PMID:25448741

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

PubMed Central

Fenouille, Nina; Bassil, Christopher F.; Ben-Sahra, Issam; Benajiba, Lina; Alexe, Gabriela; Ramos, Azucena; Pikman, Yana; Conway, Amy S.; Burgess, Michael R.; Li, Qing; Luciano, Frédéric; Auberger, Patrick; Galinsky, Ilene; DeAngelo, Daniel J.; Stone, Richard M.; Zhang, Yi; Perkins, Archibald S.; Shannon, Kevin; Hemann, Michael T.; Puissant, Alexandre; Stegmaier, Kimberly

2017-01-01

Expression of the EVI1 proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A pooled shRNA screen identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a metabolic dependency in EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted cell cycle arrest and apoptosis of human EVI1-positive AML cells, and prolonged survival in human orthotopic and mouse primary AML models. CKMT1 inhibition alters mitochondrial respiration and ATP production, an effect that is abrogated by phospho-creatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens. PMID:28191887

The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1.

PubMed

Yoneda-Kato, N; Look, A T; Kirstein, M N; Valentine, M B; Raimondi, S C; Cohen, K J; Carroll, A J; Morris, S W

1996-01-18

A t(3;5)(q25.1;q34) chromosomal translocation associated with myelodysplastic syndrome and acute myeloid leukemia (AML) was found to rearrange part of the nucleophosmin (NPM) gene on chromosome 5 with sequences from a novel gene on chromosome 3. Chimeric transcripts expressed by these cells contain 5' NPM coding sequences fused in-frame to those of the new gene, which we named myelodysplasia/myeloid leukemia factor 1 (MLF1). RNA-based polymerase chain reaction analysis revealed identical NPM-MLF1 mRNA fusions in each of the three t(3;5)-positive cases of AML examined. The predicted MLF1 amino acid sequence lacked homology to previously characterized proteins and did not contain known functional motifs. Normal MLF1 transcripts were expressed in a variety of tissues, most abundantly in testis, ovary, skeletal muscle, heart, kidney and colon. Anti-MLF1 antibodies detected the wild-type 31 kDa protein in K562 and HEL erythroleukemia cell lines, but not in HL-60, U937 or KG-1 myeloid leukemia lines. By contrast, t(3;5)-positive leukemia cells expressed a 54 kDa NPM-MLF1 protein, but not normal MLF1. Immunostaining experiments indicated that MLF1 is normally located in the cytoplasm, whereas NPM-MLF1 is targeted to the nucleus, with highest levels in the nucleolus. The nuclear/nucleolar localization of NPM-MLF1 mirrors that of NPM, indicating that NPM trafficking signals direct MLF1 to an inappropriate cellular compartment in myeloid leukemia cells.

The significance of trilineage myelodysplasia in de novo acute myeloblastic leukemia: clinical and laboratory features.

PubMed

Lima, C S; Vassalo, J; Lorand-Metze, I; Bechelli, A P; Souza, C A

1997-01-01

A prospective study was undertaken to elucidate the clinical and laboratory differences between de novo acute myeloid leukemia (AML) and AML with trilineage myelodysplasia (AML-TMDS). One hundred and seven patients with AML were diagnosed at the University Hospital between January 1987 and July 1992, and were followed until July 1995. TMDS was identified in 17 of them (16%). With regard to age and sex distribution no difference was found between AML patients with and without TMDS (p = 0.43, p = 0.54, respectively). The duration of symptoms at presentation in AML-TMDS was similar to those observed in de novo AML (p = 0.29). Hemoglobin values and platelet counts were similar in both groups of patients (p = 0.45, p = 0.44, respectively). However, peripheral white blood cell and neutrophil counts, as well as blast counts in AML-TMDS patients were lower than those observed in AML without TMDS patients (p < 0.001 for all of them). Bone marrow blast counts in de novo AML were higher than the values observed in AML-TMDS patients (p < 0.001). TMDS occurred predominantly in M2 and M6 FAB types, and was absent in the M3 type. Bone marrow histology showed no particular feature that could be of diagnostic relevance. The remission rates were similar in both groups of patients (p = 0.55). The same was true for the probability of disease-free survival and overall survival during the period of study (p = 0.50, p = 0.33, respectively). These results suggest that: 1) in AML-TMDS patients, leukemia transformation occurs in a more undifferentiated pluripotent stem cell, leading to a dysplastic residual hemopoiesis besides the blast proliferation; 2) the incidence of TMDS in our group of patients did not influence the clinical outcome after treatment of the disease.

Expression profile analysis of long non-coding RNA in acute myeloid leukemia by microarray and bioinformatics.

PubMed

Feng, Yuandong; Shen, Ying; Chen, Hongli; Wang, Xiaman; Zhang, Ru; Peng, Yue; Lei, Xiaoru; Liu, Tian; Liu, Jing; Gu, Liufang; Wang, Fangxia; Yang, Yun; Bai, Ju; Wang, Jianli; Zhao, Wanhong; He, Aili

2018-02-01

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt that are involved in tumorigenesis and play a key role in cancer progression. To determine whether lncRNAs are involved in acute myeloid leukemia (AML), we analyzed the expression profile of lncRNAs and mRNAs in AML. Five pairs of AML patients and iron deficiency anemia (IDA) controls were screened by microarray. Through coexpression analysis, differently expressed transcripts were divided into modules, and lncRNAs were functionally annotated. We further analyzed the clinical significance of crucial lncRNAs from modules in public data. Finally, the expression of three lncRNAs, RP11-222K16.2, AC092580.4, and RP11-305O.6, were validated in newly diagnosed AML, AML relapse, and IDA patient groups by quantitative RT-PCR, which may be associated with AML patients' overall survival. Further analysis showed that RP11-222K16.2 might affect the differentiation of natural killer cells, and promote the immunized evasion of AML by regulating Eomesodermin expression. Analysis of this study revealed that dysregulated lncRNAs and mRNAs in AML vs IDA controls could affect the immune system and hematopoietic cell differentiation. The biological functions of those lncRNAs need to be further validated. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Acute myeloid leukemia targets for bispecific antibodies

PubMed Central

Hoseini, S S; Cheung, N K

2017-01-01

Despite substantial gains in our understanding of the genomics of acute myelogenous leukemia (AML), patient survival remains unsatisfactory especially among the older age group. T cell-based therapy of lymphoblastic leukemia is rapidly advancing; however, its application in AML is still lagging behind. Bispecific antibodies can redirect polyclonal effector cells to engage chosen targets on leukemia blasts. When the effector cells are natural-killer cells, both antibody-dependent and antibody-independent mechanisms could be exploited. When the effectors are T cells, direct tumor cytotoxicity can be engaged followed by a potential vaccination effect. In this review, we summarize the AML-associated tumor targets and the bispecific antibodies that have been studied. The potentials and limitations of each of these systems will be discussed. PMID:28157217

Kefir induces apoptosis and inhibits cell proliferation in human acute erythroleukemia.

PubMed

Jalali, Fatemeh; Sharifi, Mohammadreza; Salehi, Rasoul

2016-01-01

Acute erythroleukemia is an uncommon subtype of acute myeloid leukemia which has been considered to be a subtype of AML with a worse prognosis. Intensive chemotherapy is the first line of treatment. In recent years, the effect of kefir on some malignancies has been experimented. Kefir is a kind of beverage, which obtained by incubation of kefir grains with raw milk. Kefir grains are a symbiotic complex of different kinds of yeasts and bacteria, especially lactic acid bacteria which gather in a mostly carbohydrate matrix, named kefiran. We investigated the effect of kefir on acute erythroleukemia cell line (KG-1) and peripheral blood mononuclear cells (PBMCs). The cell line and PBMCs were treated with different doses of kefir and milk and incubated for three different times. We used Polymixin B to block the lipopolysaccharide and NaOH (1 mol/l) to neutralize the acidic media. Viability was detected by MTT assay. Apoptosis and necrosis were assessed by annexin-propidium iodide staining. Our results showed that kefir induced apoptosis and necrosis in KG-1 cell line. It was revealed that kefir decreased proliferation in erythroleukemia cell line. We did not observe a remarkable effect of kefir on PBMCs. Our study suggested that kefir may have potential to be an effective treatment for erythroleukemia.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia

PubMed Central

Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2015-01-01

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML. PMID:26450903

[Detection of heterogeneity and evolution of subclones in t(8;21) AML by QM-FISH].

PubMed

Wang, Ying-chan; Hu, Lin-ping; Lin, Dong; Li, Cheng-wen; Yuan, Tian; Jia, Yu-jiao; Tian, Zheng; Tang, Ke-jing; Wang, Min; Wang, Jian-xiang

2013-10-01

To explore the heterogeneous subclones in acute myeloid leukemia (AML) with t(8;21) by quantitative multicolor- fluorescence in situ hybridization (QM-FISH), and to figure out whether there is putative ancestral relationship among different subclones. Bacterial artificial chromosomes (BAC) clones that contain the targeted genes including AML1, ETO, WT1, p27 and c-kit were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP or dCTP to targeted genes by nick translation. Bone marrow mononuclear cells from t (8;21) AML patients are dropped on to the wet surface of glass slides after hypotonic treatment and fixation. After hybridization, the fluorescence signals were captured by Zeiss fluorescence microscope. The copy number of AML1, ETO, WT1, p27, c- kit and the AML1-ETO fusion gene in AML1-ETO positive cells was counted. The cells with same signals were defined as a subclone. Various subclones were recorded and their proportions were calculated, and their evolutionary relationship was deduced. The subclones in matched primary and relapsed samples were compared, the evolution of dominant clones were figured out and the genomic abnormality that is associated with relapse and drug resistance were speculated. In this study, 36 primary AML with t(8;21) cases and 1 relapsed case paired with the primary case were detected. In these 36 primary cases, 4 cases (11.1%) acquired additional AML1-ETO fusion signal, 3(8.3%) had additional AML1 signal, 4(11.1%) had additional ETO signal, 20(55.6%) had additional WT1 signal, 15(41.7%) had additional p27 signal and 14(38.9%) had additional c-kit signal. In addition, 10(27.8%) displayed AML1 signal deletion, and such an aberration represents statistic significance in male patients. It seems that male patients usually accompany AML1 signal deletion. Of 36 cases, 28(77.8 %) harbored at least 2 subclones (ranged from 2 to 10). According to the genetic signature of subclones, we can assemble a putative ancestral tree, and the genetic architecture is linear or branching. In particular, the clonal architecture of the relapsed sample exhibited significant clonal evolution compared to its paired sample at diagnosis, including proportion changes in dominant clone, subclone disappearance and appearance of new dominant clones. Genomic abnormality is very diverse in t(8;21) AML. Subclones have linear or complex branching evolutionary histories, and clonal architecture is dynamic.

Chemical shift magnetic resonance imaging for distinguishing minimal-fat renal angiomyolipoma from renal cell carcinoma: a meta-analysis.

PubMed

Chen, Ling-Shan; Zhu, Zheng-Qiu; Wang, Zhi-Tao; Li, Jing; Liang, Li-Feng; Jin, Ji-Yang; Wang, Zhong-Qiu

2018-05-01

To determine the performance of chemical shift signal intensity index (CS-SII) values for distinguishing minimal-fat renal angiomyolipoma (mfAML) from renal cell carcinoma (RCC) and to assess RCC subtype characterisation. We identified eligible studies on CS magnetic resonance imaging (CS-MRI) of focal renal lesions via PubMed, Embase, and the Cochrane Library. CS-SII values were extracted by lesion type and evaluated using linear mixed model-based meta-regression. RCC subtypes were analysed. Two-sided p value <0.05 indicated statistical significance. Methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. Eleven articles involving 850 patients were included. Minimal-fat AML had significantly higher CS-SII value than RCC (p < 0.05); there were no significant differences between mfAML and clear cell RCC (cc-RCC) (p = 0.112). Clear cell RCC had a significantly higher CS-SII value than papillary RCC (p-RCC) (p < 0.001) and chromophobe RCC (ch-RCC) (p = 0.045). The methodological quality was relatively high, and Begg's test data points indicated no obvious publication bias. The CS-SII value for differentiating mfAML from cc-RCC remains unproven, but is a promising method for differentiating cc-RCC from p-RCC and ch-RCC. • RCC CS-SII values are significantly lower than those of mfAML overall. • CS-SII values cannot aid differentiation between mfAML and cc-RCC. • CS-SII values might help characterise RCC subtypes.

MUC1-C induces DNA methyltransferase 1 and represses tumor suppressor genes in acute myeloid leukemia.

PubMed

Tagde, Ashujit; Rajabi, Hasan; Stroopinsky, Dina; Gali, Reddy; Alam, Maroof; Bouillez, Audrey; Kharbanda, Surender; Stone, Richard; Avigan, David; Kufe, Donald

2016-06-28

Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38- population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML.

Clonal Architecture of Secondary Acute Myeloid Leukemia

PubMed Central

Walter, Matthew J.; Shen, Dong; Ding, Li; Shao, Jin; Koboldt, Daniel C.; Chen, Ken; Larson, David E.; McLellan, Michael D.; Dooling, David; Abbott, Rachel; Fulton, Robert; Magrini, Vincent; Schmidt, Heather; Kalicki-Veizer, Joelle; O’Laughlin, Michelle; Fan, Xian; Grillot, Marcus; Witowski, Sarah; Heath, Sharon; Frater, John L.; Eades, William; Tomasson, Michael; Westervelt, Peter; DiPersio, John F.; Link, Daniel C.; Mardis, Elaine R.; Ley, Timothy J.; Wilson, Richard K.; Graubert, Timothy A.

2012-01-01

BACKGROUND The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood. METHODS We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations. RESULTS Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene. CONCLUSIONS Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.) PMID:22417201

Acute Myeloid Leukemia With Central Nervous System Involvement in Children: Experience From the French Protocol Analysis ELAM02.

PubMed

Felix, Arthur; Leblanc, Thierry; Petit, Arnaud; Nelkem, Brigitte; Bertrand, Yves; Gandemer, Virginie; Sirvent, Anne; Paillard, Catherine; Schmitt, Claudine; Rohrlich, Pierre Simon; Fenneteau, Odile; Ragu, Christine; Michel, Gerard; Auvrignon, Anne; Baruchel, André; Leverger, Guy

2018-01-01

Central nervous system (CNS) involvement at diagnosis of pediatric acute myeloid leukemia (AML) is not considered as an independent prognostic factor. This study describes the prognostic value of pediatric AML with CNS involvement at diagnosis. Pediatric patients were treated for de novo AML in the French multicenter trial ELAM02. Lumbar puncture was carried out in the first week, and the treatment was adapted to the CNS status. No patient received CNS radiotherapy. The patients were classified into 2 groups: CNS+ and CNS-. Of the 438 patients, 16% (n=70) had CNS involvement at diagnosis, and 29% showed clinical signs. The patients with CNS disease were younger (40% were below 2 y old), had a higher white blood cell count (median of 45 vs. 13 G/L), and had M4 and M5 morphologies. The complete remission rate was similar at 92.8% for CNS+ and 88.5% for CNS-. There was no significant difference between the CNS+ and the CNS- group in overall survival (76% and 71%, respectively) and event-free survival (57% and 52%, respectively). Regarding the occurrence of first relapse, the CNS+ group had a higher combined relapse rate of 26.1% compared with 10% for the CNS- group. The results indicate that CNS involvement at diagnosis of pediatric AML is not an independent prognostic factor. Triple intrathecal chemotherapy combined with high-dose intravenous cytarabine should be the first-line treatment for CNS disease.

Hierarchy in gene expression is predictive of risk, progression, and outcome in adult acute myeloid leukemia

NASA Astrophysics Data System (ADS)

Tripathi, Shubham; Deem, Michael W.

2015-02-01

Cancer progresses with a change in the structure of the gene network in normal cells. We define a measure of organizational hierarchy in gene networks of affected cells in adult acute myeloid leukemia (AML) patients. With a retrospective cohort analysis based on the gene expression profiles of 116 AML patients, we find that the likelihood of future cancer relapse and the level of clinical risk are directly correlated with the level of organization in the cancer related gene network. We also explore the variation of the level of organization in the gene network with cancer progression. We find that this variation is non-monotonic, which implies the fitness landscape in the evolution of AML cancer cells is non-trivial. We further find that the hierarchy in gene expression at the time of diagnosis may be a useful biomarker in AML prognosis.

The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers

PubMed Central

Russler-Germain, David A.; Spencer, David H.; Young, Margaret A.; Lamprecht, Tamara L.; Miller, Christopher A.; Fulton, Robert; Meyer, Matthew R.; Erdmann-Gilmore, Petra; Townsend, R. Reid; Wilson, Richard K.; Ley, Timothy J.

2014-01-01

Summary Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in ~30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 within the catalytic domain (most commonly R882H), suggesting the possibility of dominant negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by ~80% compared to the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity but co-expression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes. PMID:24656771

UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

PubMed Central

Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T. C.; Imren, Suzan; Lam, Vivian; Poon, Grace F. T.; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William

2016-01-01

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

Stage-Specific Human Induced Pluripotent Stem Cells Map the Progression of Myeloid Transformation to Transplantable Leukemia.

PubMed

Kotini, Andriana G; Chang, Chan-Jung; Chow, Arthur; Yuan, Han; Ho, Tzu-Chieh; Wang, Tiansu; Vora, Shailee; Solovyov, Alexander; Husser, Chrystel; Olszewska, Malgorzata; Teruya-Feldstein, Julie; Perumal, Deepak; Klimek, Virginia M; Spyridonidis, Alexandros; Rampal, Raajit K; Silverman, Lewis; Reddy, E Premkumar; Papaemmanuil, Elli; Parekh, Samir; Greenbaum, Benjamin D; Leslie, Christina S; Kharas, Michael G; Papapetrou, Eirini P

2017-03-02

Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of induced pluripotent stem cell (iPSC) lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS, and secondary AML. Upon their differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9 and that this iPSC-based approach can be used to uncover disease-stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

The role of HOXB2 and HOXB3 in acute myeloid leukemia.

PubMed

Lindblad, Oscar; Chougule, Rohit A; Moharram, Sausan A; Kabir, Nuzhat N; Sun, Jianmin; Kazi, Julhash U; Rönnstrand, Lars

2015-11-27

Acute myeloid leukemia (AML) is a heterogeneous aggressive disease and the most common form of adult leukemia. Mutations in the type III receptor tyrosine kinase FLT3 are found in more than 30% of AML patients. Drugs against FLT3 have been developed for the treatment of AML, but they lack specificity, show poor response and lead to the development of a resistant phenotype upon treatment. Therefore, a deeper understanding of FLT3 signaling will facilitate identification of additional pharmacological targets in FLT3-driven AML. In this report, we identify HOXB2 and HOXB3 as novel regulators of oncogenic FLT3-ITD-driven AML. We show that HOXB2 and HOXB3 expression is upregulated in a group of AML patients carrying FLT3-ITD. Overexpression of HOXB2 or HOXB3 in mouse pro-B cells resulted in decreased FLT3-ITD-dependent cell proliferation as well as colony formation and increased apoptosis. Expression of HOXB2 or HOXB3 resulted in a significant decrease in FLT3-ITD-induced AKT, ERK, p38 and STAT5 phosphorylation. Our data suggest that HOXB2 and HOXB3 act as tumor suppressors in FLT3-ITD driven AML. Copyright © 2015 Elsevier Inc. All rights reserved.

The role of HOXB2 and HOXB3 in acute myeloid leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindblad, Oscar; Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund; Department of Hematology and Vascular Disorders, Skåne University Hospital, Lund

2015-11-27

Acute myeloid leukemia (AML) is a heterogeneous aggressive disease and the most common form of adult leukemia. Mutations in the type III receptor tyrosine kinase FLT3 are found in more than 30% of AML patients. Drugs against FLT3 have been developed for the treatment of AML, but they lack specificity, show poor response and lead to the development of a resistant phenotype upon treatment. Therefore, a deeper understanding of FLT3 signaling will facilitate identification of additional pharmacological targets in FLT3-driven AML. In this report, we identify HOXB2 and HOXB3 as novel regulators of oncogenic FLT3-ITD-driven AML. We show that HOXB2more » and HOXB3 expression is upregulated in a group of AML patients carrying FLT3-ITD. Overexpression of HOXB2 or HOXB3 in mouse pro-B cells resulted in decreased FLT3-ITD-dependent cell proliferation as well as colony formation and increased apoptosis. Expression of HOXB2 or HOXB3 resulted in a significant decrease in FLT3-ITD-induced AKT, ERK, p38 and STAT5 phosphorylation. Our data suggest that HOXB2 and HOXB3 act as tumor suppressors in FLT3-ITD driven AML.« less

Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients.

PubMed

Rapin, Nicolas; Bagger, Frederik Otzen; Jendholm, Johan; Mora-Jensen, Helena; Krogh, Anders; Kohlmann, Alexander; Thiede, Christian; Borregaard, Niels; Bullinger, Lars; Winther, Ole; Theilgaard-Mönch, Kim; Porse, Bo T

2014-02-06

Gene expression profiling has been used extensively to characterize cancer, identify novel subtypes, and improve patient stratification. However, it has largely failed to identify transcriptional programs that differ between cancer and corresponding normal cells and has not been efficient in identifying expression changes fundamental to disease etiology. Here we present a method that facilitates the comparison of any cancer sample to its nearest normal cellular counterpart, using acute myeloid leukemia (AML) as a model. We first generated a gene expression-based landscape of the normal hematopoietic hierarchy, using expression profiles from normal stem/progenitor cells, and next mapped the AML patient samples to this landscape. This allowed us to identify the closest normal counterpart of individual AML samples and determine gene expression changes between cancer and normal. We find the cancer vs normal method (CvN method) to be superior to conventional methods in stratifying AML patients with aberrant karyotype and in identifying common aberrant transcriptional programs with potential importance for AML etiology. Moreover, the CvN method uncovered a novel poor-outcome subtype of normal-karyotype AML, which allowed for the generation of a highly prognostic survival signature. Collectively, our CvN method holds great potential as a tool for the analysis of gene expression profiles of cancer patients.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

PubMed

Müller-Tidow, Carsten; Klein, Hans-Ulrich; Hascher, Antje; Isken, Fabienne; Tickenbrock, Lara; Thoennissen, Nils; Agrawal-Singh, Shuchi; Tschanter, Petra; Disselhoff, Christine; Wang, Yipeng; Becker, Anke; Thiede, Christian; Ehninger, Gerhard; zur Stadt, Udo; Koschmieder, Steffen; Seidl, Matthias; Müller, Frank U; Schmitz, Wilhelm; Schlenke, Peter; McClelland, Michael; Berdel, Wolfgang E; Dugas, Martin; Serve, Hubert

2010-11-04

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2more » activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular fractions from control, MVAL or propiconazole-treated cells revealed increased Ras protein in the cytoplasmic fraction of L-744,832-treated cells, while propiconazole or MVAL reversed these effects. Western blot analysis indicated that phosphorylation of Erk1/2, a protein downstream of Ras, was increased by propiconazole. These data indicate that propiconazole increases cell proliferation by increasing the levels of cholesterol biosynthesis intermediates presumably through a negative feedback mechanism within the pathway, a result of CYP51 inhibition. This feedback mechanism increases Erk1/2 signaling through mevalonate-mediated Ras activation. These results provide an explanation for the observed effects of propiconazole on hepatic cholesterol pathways and on the increased hepatic cell proliferation induced by propiconazole in mice. Highlights: ► Propiconazole increases cell proliferation in AML12 mouse hepatocytes. ► Propiconazole increases Ras farnesylation and alters Ras membrane localization. ► Propiconazole increases Erk1/2 phosphorylation. ► Dysregulation of the cholesterol biosynthesis pathway can explain these results. ► These results can explain similar effects induced by propiconazole in mice.« less

Coexpression of the genes for interleukin 6 and its receptor without apparent involvement in the proliferation of acute myeloid leukemia cells.

PubMed

Lopez, M; Maroc, N; Kerangueven, F; Bardin, F; Courcoul, M; Lavezzi, C; Birg, F; Mannoni, P

1991-09-01

Leukemic cells isolated from patients with either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) were screened for their capacity to express the interleukin 6 (IL-6) and IL-6 receptor genes, both at the RNA and protein levels. Variable levels (10 to greater than 600 U/ml) of an IL-6 activity, inhibited by neutralizing anti-IL-6 antibodies, were detected in AML cell supernatants using the B9 cell bioassay. High levels (greater than 100 U/ml) were observed in differentiated (M4 and M5 stages) AML, as well as in less mature (M1 and M2 stages) AML. Detection of the IL-6 transcript correlated with the biological activity. In addition, both IL-6 activity and IL-6 mRNA were detected in "fresh" leukemic cells, indicating that the glycoprotein was actually synthesized in vivo. In contrast, the IL-6 gene was less frequently expressed in ALL. The IL-6 receptor gene was transcribed in both AML and ALL; binding experiments showed that the protein was present at the cell surface. The spontaneous in vitro proliferation of leukemic cells coexpressing the transcripts for IL-6 and its receptor was not significantly inhibited by a neutralizing anti-IL-6 antibody, suggesting that IL-6 is not primarily implicated in the proliferation of the leukemic clone via an autocrine loop. Synthesis of IL-6 could, however, confer on leukemic cells a selective growth advantage through activation of the cytokine cascade.

Classification of Acute Myelogenous Leukemia (AML M2 and AML M3) using Momentum Back Propagation from Watershed Distance Transform Segmented Images

NASA Astrophysics Data System (ADS)

Suryani, Esti; Wiharto; Palgunadi, Sarngadi; Nurcahya Pradana, TP

2017-01-01

This study uses image processing to analyze white blood cell with leukemia indicated that includes the identification, analysis of shapes and sizes, as well as white blood cell count indicated the symptoms of leukemia. A case study in this research was blood cells, from the type of leukemia Acute Myelogenous Leukemia (AML), M2 and M3 in particular. Image processing operations used for segmentation by utilizing the color conversion from RGB (Red, Green dab Blue) to obtain white blood cell candidates. Furthermore, the white blood cells candidates are separated by other cells with active contour without edge. WBC (White Blood Cell) results still have intersected or overlap condition. Watershed distance transform method can separate overlap of WBC. Furthermore, the separation of the nucleus from the cytoplasm using the HSI (Hue Saturation Intensity). The further characteristic extraction process is done by calculating the area WBC, WBC edge, roundness, the ratio of the nucleus, the mean and standard deviation of pixel intensities. The feature extraction results are used for training and testing in determining the classification of AML: M2 and M3 by using the momentum backpropagation algorithm. The classification process is done by testing the numeric data input from the feature extraction results that have been entered in the database. K-Fold validation is used to divide the amount of training data and to test the classification of AML M2 and M3. The experiment results of eight images trials, the result, was 94.285% per cell accuracy and 75% per image accuracy

Maintenance of telomere length in AML.

PubMed

Lansdorp, Peter M

2017-11-28

The importance of telomere length to human health, aging, and cancer continues to be underappreciated. This review examines some basics of telomere biology and relates how telomere function, telomerase activity, and mutations in TERC or TERT are involved in bone marrow failure, leukemias, and other cancers. Given the challenge to obtain accurate data on telomerase activity and telomere length in specific cell types, the situation in acute myeloid leukemia (AML) remains puzzling. In most cancers, telomerase levels are increased after cells have encountered a "telomere crisis," which is typically associated with poor prognosis. Cells emerging from "telomere crisis" have defective DNA damage responses, resulting, for example, from loss of p53. Such cells often express elevated telomerase levels as a result of point mutations in the TERT promoter or amplification of the TERT gene. While telomeres in AML blasts are typically shorter than expected for normal leukocytes, most AML cells do not show evidence of having gone through a "telomere crisis." In chronic myeloid leukemia (CML), the difference between the telomere length in nonmalignant T cells and malignant blasts from the same patient was found to correlate with the remaining duration of the chronic phase. This observation supports that a mitotic clock is ticking in CML stem cells and that disease progression in CML heralds the onset of a "telomere crisis." The presence of very short telomeres in tumor cells was found to predict disease progression in chronic lymphocytic leukemia, myeloma, and various solid tumors. In view of these findings longitudinal studies of telomere length in AML appear worthwhile.

Diagnostic and Prognostic Utility of Fluorescence In situ Hybridization (FISH) Analysis in Acute Myeloid Leukemia.

PubMed

Gonzales, Patrick R; Mikhail, Fady M

2017-12-01

Acute myeloid leukemia (AML) is a hematologic neoplasia consisting of incompletely differentiated hematopoietic cells of the myeloid lineage that proliferate in the bone marrow, blood, and/or other tissues. Clinical implementation of fluorescence in situ hybridization (FISH) in cytogenetic laboratories allows for high-resolution analysis of recurrent structural chromosomal rearrangements specific to AML, especially in AML with normal karyotypes, which comprises approximately 33-50% of AML-positive specimens. Here, we review the use of several FISH probe strategies in the diagnosis of AML. We also review the standards and guidelines currently in place for use by clinical cytogenetic laboratories in the evaluation of AML. Updated standards and guidelines from the WHO, ACMG, and NCCN have further defined clinically significant, recurring cytogenetic anomalies in AML that are detectable by FISH. FISH continues to be a powerful technique in the diagnosis of AML, with higher resolution than conventional cytogenetic analysis, rapid turnaround time, and a considerable diagnostic and prognostic utility.

Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells.

PubMed

Weber, Christoph; Schreiber, Thiemo B; Daub, Henrik

2012-02-02

Small molecule inhibitors of protein kinases have emerged as a major class of therapeutic agents for the treatment of hematological malignancies. Both in vitro studies and patient case reports suggest therapeutic potential of the clinical kinase inhibitors erlotinib and gefitinib in acute myeloid leukemia (AML). The drugs' cellular modes of action in AML warrant further investigation as their primary therapeutic target, the epidermal growth factor receptor, is not expressed. We therefore performed SILAC-based quantitative mass spectrometry analyses to a depth of 10,975 distinct phosphorylation sites to characterize the phosphoproteome of KG1 AML cells and its regulation upon erlotinib and gefitinib treatment. Less than 50 site-specific phosphorylations changed significantly, indicating rather specific interference with AML cell signaling. Many drug-induced changes occurred within a network of tyrosine phosphorylated proteins that included Src family kinases (SFKs) and the tyrosine kinases Btk and Syk. We further performed quantitative chemical proteomics in KG1 cell extracts and identified SFKs and Btk as direct cellular targets of both erlotinib and gefitinib. Taken together, our data suggest that cellular perturbation of SFKs and/or Btk translates into rather specific signal transduction inhibition, which in turn contributes to the antileukemic activity of erlotinib and gefitinib in AML. Copyright © 2011 Elsevier B.V. All rights reserved.

Outcome after intensive reinduction therapy and allogeneic stem cell transplant in paediatric relapsed acute myeloid leukaemia.

PubMed

Karlsson, Lene; Forestier, Erik; Hasle, Henrik; Jahnukainen, Kirsi; Jónsson, Ólafur G; Lausen, Birgitte; Norén Nyström, Ulrika; Palle, Josefine; Tierens, Anne; Zeller, Bernward; Abrahamsson, Jonas

2017-08-01

Given that 30-40% of children with acute myeloid leukaemia (AML) relapse after primary therapy it is important to define prognostic factors and identify optimal therapy. From 1993 to 2012, 543 children from the Nordic countries were treated according to two consecutive protocols: 208 children relapsed. The influence of disease characteristics, first line treatment, relapse therapy and duration of first remission on outcome was analysed. Second complete remission (CR2) was achieved in 146 (70%) patients. Estimated 5-year overall survival (OS 5y ) was 39 ± 4% for the whole group and 43 ± 4% for the 190 patients given re-induction therapy, of whom 76% received regimens that included fludarabine, cytarabine (FLA) ± anthracyclines, 18% received Nordic Society for Paediatric Haematology and Oncology (NOPHO) upfront blocks and 5% received other regimens. Late relapse ≥1 year from diagnosis, no allogeneic stem cell transplantation (SCT) in first remission and core binding factor AML were independent favourable prognostic factors for survival. For the 128 children (124 in CR2) that received SCT as consolidation therapy after relapse, OS 5y was 61 ± 5%. Four of 19 children (21%) survived without receiving SCT as part of relapse therapy. Our data show that intensive re-induction followed by SCT can give cure rates of 40% in children with relapsed AML. © 2017 John Wiley & Sons Ltd.

MPL expression on AML blasts predicts peripheral blood neutropenia and thrombocytopenia.

PubMed

Rauch, Philipp J; Ellegast, Jana M; Widmer, Corinne C; Fritsch, Kristin; Goede, Jeroen S; Valk, Peter J M; Löwenberg, Bob; Takizawa, Hitoshi; Manz, Markus G

2016-11-03

Although the molecular pathways that cause acute myeloid leukemia (AML) are increasingly well understood, the pathogenesis of peripheral blood cytopenia, a major cause of AML mortality, remains obscure. A prevailing assumption states that AML spatially displaces nonleukemic hematopoiesis from the bone marrow. However, examining an initial cohort of 223 AML patients, we found no correlation between bone marrow blast content and cytopenia, questioning the displacement theory. Measuring serum concentration of thrombopoietin (TPO), a key regulator of hematopoietic stem cells and megakaryocytes, revealed loss of physiologic negative correlation with platelet count in AML cases with blasts expressing MPL, the thrombopoietin (scavenging) receptor. Mechanistic studies demonstrated that MPL hi blasts could indeed clear TPO, likely therefore leading to insufficient cytokine levels for nonleukemic hematopoiesis. Microarray analysis in an independent multicenter study cohort of 437 AML cases validated MPL expression as a central predictor of thrombocytopenia and neutropenia in AML. Moreover, t(8;21) AML cases demonstrated the highest average MPL expression and lowest average platelet and absolute neutrophil counts among subgroups. Our work thus explains the pathophysiology of peripheral blood cytopenia in a relevant number of AML cases. © 2016 by The American Society of Hematology.

Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO.

PubMed

Roh, Soung-Hun; Kasembeli, Moses; Galaz-Montoya, Jesús G; Trnka, Mike; Lau, Wilson Chun-Yu; Burlingame, Alma; Chiu, Wah; Tweardy, David J

2016-02-26

AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease with a 5-year survival rate of only 24%. AML1-ETO functions as a rogue transcription factor, altering the expression of genes critical for myeloid cell development and differentiation. Currently, there are no specific therapies for AML1-ETO-positive AML. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the biosynthesis and folding of the AML1-ETO protein is facilitated by interaction with the essential eukaryotic chaperonin TRiC (or CCT). We demonstrate that a folding intermediate of AML1-ETO binds to TRiC directly, mainly through its β-strand rich, DNA-binding domain (AML-(1-175)), with the assistance of HSP70. Our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions between the oncoprotein's DNA-binding domain, which may be targeted for therapeutic benefit. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A vicious loop of fatty acid-binding protein 4 and DNA methyltransferase 1 promotes acute myeloid leukemia and acts as a therapeutic target

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhao, N; Zhang, YW; Bode, AM; Al-Kali, A; Litzow, MR; Li, B; Liu, SJ

2017-01-01

Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMT) is a key hallmark of acute myeloid leukemia (AML), yet efforts to target DNMT deregulation for drug development have lagged. We previously demonstrated that upregulation of fatty acid-binding protein 4 (FABP4) promotes AML aggressiveness through enhanced DNMT1-dependent DNA methylation. Here we demonstrate that FABP4 upregulation in AML cells occurs through vascular endothelial growth factor (VEGF) signaling, thus elucidating a crucial FABP4-DNMT1 regulatory feedback loop in AML biology. We show that FABP4 dysfunction by its selective inhibitor BMS309403 leads to downregulation of DNMT1, decrease of global DNA methylation and re-expression of p15INK4B tumor suppressor gene by promoter DNA hypomethylation in vitro, ex vivo and in vivo. Functionally, BMS309403 suppresses cell colony formation, induces cell differentiation, and, importantly, impairs leukemic disease progression in mouse models of leukemia. Our findings highlight AML-promoting properties of the FABP4-DNMT1 vicious loop, and identify an attractive class of therapeutic agents with a high potential for clinical use in AML patients. The results will also assist in establishing the FABP4-DNMT1 loop as a target for therapeutic discovery to enhance the index of current epigenetic therapies. PMID:28993705

MiR-100 regulates cell differentiation and survival by targeting RBSP3, a phosphatase-like tumor suppressor in acute myeloid leukemia

PubMed Central

Zheng, Y-S; Zhang, H; Zhang, X-J; Feng, D-D; Luo, X-Q; Zeng, C-W; Lin, K-Y; Zhou, H; Qu, L-H; Zhang, P; Chen, Y-Q

2012-01-01

Acute myeloblastic leukemia (AML) is characterized by the accumulation of abnormal myeloblasts (mainly granulocyte or monocyte precursors) in the bone marrow and blood. Though great progress has been made for improvement in clinical treatment during the past decades, only minority with AML achieve long-term survival. Therefore, further understanding mechanisms of leukemogenesis and exploring novel therapeutic strategies are still crucial for improving disease outcome. MicroRNA-100 (miR-100), a small non-coding RNA molecule, has been reported as a frequent event aberrantly expressed in patients with AML; however, the molecular basis for this phenotype and the statuses of its downstream targets have not yet been elucidated. In the present study, we found that the expression level of miR-100 in vivo was related to the stage of the maturation block underlying the subtypes of myeloid leukemia. In vitro experiments further demonstrated that miR-100 was required to promote the cell proliferation of promyelocytic blasts and arrest them differentiated to granulocyte/monocyte lineages. Significantly, we identified RBSP3, a phosphatase-like tumor suppressor, as a bona fide target of miR-100 and validated that RBSP3 was involved in cell differentiation and survival in AML. Moreover, we revealed a new pathway that miR-100 regulates G1/S transition and S-phase entry and blocks the terminal differentiation by targeting RBSP3, which partly in turn modulates the cell cycle effectors pRB/E2F1 in AML. These events promoted cell proliferation and blocked granulocyte/monocyte differentiation. Our data highlight an important role of miR-100 in the molecular etiology of AML, and implicate the potential application of miR-100 in cancer therapy. PMID:21643017

Secondary pure erythroid leukaemia in relapsed acute lymphoblastic leukaemia: lineage switch or chemotherapy effect?

PubMed

Gupta, Sanjeev Kumar; Kumar, Rajive; Chharchhodawala, Taher; Kumar, Lalit

2014-05-19

Pure erythroid leukaemia is a rare subtype of acute myeloid leukaemia (AML) and its occurrence at acute lymphoblastic leukaemia (ALL) relapse has not been reported earlier. A 39-year-old man received chemotherapy for Philadelphia-negative B cell ALL. Subsequently, he developed pure erythroid leukaemia with >80% immature erythroid precursors in bone marrow showing block positivity on periodic acid-Schiff stain, expressing CD71, CD34 but lacking CD235a. The interval between exposure to multidrug chemotherapy including cyclophosphamide and AML diagnosis was 2 years and 9 months. No cytogenetic abnormality was detected at the time of relapse. The patient died 2 weeks after starting AML chemotherapy. The relatively narrow time interval (usually 5-10 years) between chemotherapy and AML development and normal karyotype at relapse raises a possibility of lineage switch besides therapy-related AML as the likely pathogenesis. Further exploration of such cases may unravel the pathways responsible for lineage assignment in pluripotent stem cells. 2014 BMJ Publishing Group Ltd.

Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia

PubMed Central

Ford, Anthony M.; Bennett, Caroline A.; Price, Cathy M.; Bruin, M. C. A.; Van Wering, Elisabeth R.; Greaves, Mel

1998-01-01

The TEL (ETV6)−AML1 (CBFA2) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in ≈25% of the most predominant subtype of leukemia— common acute lymphoblastic leukemia. The TEL-AML1 genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive TEL-AML1 fusion sequence. The most plausible explanation for this finding is a single cell origin of the TEL-AML fusion in one fetus in utero, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged IGH allele. These data have implications for the etiology and natural history of childhood leukemia. PMID:9539781

Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling

PubMed Central

Pulikkan, John Anto; Madera, Dmitri; Xue, Liting; Bradley, Paul; Landrette, Sean Francis; Kuo, Ya-Huei; Abbas, Saman; Zhu, Lihua Julie; Valk, Peter

2012-01-01

Oncogenic mutations in components of cytokine signaling pathways elicit ligand-independent activation of downstream signaling, enhancing proliferation and survival in acute myeloid leukemia (AML). The myeloproliferative leukemia virus oncogene, MPL, a homodimeric receptor activated by thrombopoietin (THPO), is mutated in myeloproliferative disorders but rarely in AML. Here we show that wild-type MPL expression is increased in a fraction of human AML samples expressing RUNX1-ETO, a fusion protein created by chromosome translocation t(8;21), and that up-regulation of Mpl expression in mice induces AML when coexpressed with RUNX1-ETO. The leukemic cells are sensitive to THPO, activating survival and proliferative responses. Mpl expression is not regulated by RUNX1-ETO in mouse hematopoietic progenitors or leukemic cells. Moreover, we find that activation of PI3K/AKT but not ERK/MEK pathway is a critical mediator of the MPL-directed antiapoptotic function in leukemic cells. Hence, this study provides evidence that up-regulation of wild-type MPL levels promotes leukemia development and maintenance through activation of the PI3K/AKT axis, and suggests that inhibitors of this axis could be effective for treatment of MPL-positive AML. PMID:22613795

Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling.

PubMed

Pulikkan, John Anto; Madera, Dmitri; Xue, Liting; Bradley, Paul; Landrette, Sean Francis; Kuo, Ya-Huei; Abbas, Saman; Zhu, Lihua Julie; Valk, Peter; Castilla, Lucio Hernán

2012-07-26

Oncogenic mutations in components of cytokine signaling pathways elicit ligand-independent activation of downstream signaling, enhancing proliferation and survival in acute myeloid leukemia (AML). The myeloproliferative leukemia virus oncogene, MPL, a homodimeric receptor activated by thrombopoietin (THPO), is mutated in myeloproliferative disorders but rarely in AML. Here we show that wild-type MPL expression is increased in a fraction of human AML samples expressing RUNX1-ETO, a fusion protein created by chromosome translocation t(8;21), and that up-regulation of Mpl expression in mice induces AML when coexpressed with RUNX1-ETO. The leukemic cells are sensitive to THPO, activating survival and proliferative responses. Mpl expression is not regulated by RUNX1-ETO in mouse hematopoietic progenitors or leukemic cells. Moreover, we find that activation of PI3K/AKT but not ERK/MEK pathway is a critical mediator of the MPL-directed antiapoptotic function in leukemic cells. Hence, this study provides evidence that up-regulation of wild-type MPL levels promotes leukemia development and maintenance through activation of the PI3K/AKT axis, and suggests that inhibitors of this axis could be effective for treatment of MPL-positive AML.

Accelerate Genomic Aging in Congenital Neutropenia

DTIC Science & Technology

2017-10-01

syndrome (MDS) or acute myeloid leukemia (AML) in patients with congenital neutropenia. We hypothesize that replicative stress and/or changes in the...neutropenia; Shwachman Diamond syndrome ; Cyclic neutropenia; Hematopoietic stem cells; Granulocyte colony-stimulating factor; Acute myeloid leukemia... syndrome (MDS) or acute myeloid leukemia (AML). The cumulative incidence of MDS/AML in patients with SCN treated with G-CSF is 22%. Likewise, the

Transglutaminase 2 expression in acute myeloid leukemia: Association with adhesion molecule expression and leukemic blast motility

PubMed Central

Meyer, Stefan; Ravandi-Kashani, Farhad; Borthakur, Gautam; Coombes, Kevin R.; Zhang, Nianxiang; Kornblau, Steven

2016-01-01

Acute myeloid leukemia (AML) is a heterogenous disease with differential oncogene association, outcome and treatment regimens. Treatment strategies for AML have improved outcome but despite increased molecular biological information AML is still associated with poor prognosis. Proteomic analysis on the effects of a range of leukemogenic oncogenes showed that the protein transglutaminase 2 (TG2) is expressed at greater levels as a consequence of oncogenic transformation. Further analysis of this observation was performed with 511 AML samples using reverse phase proteomic arrays, demonstrating that TG2 expression was higher at relapse than diagnosis in many cases. In addition elevated TG2 expression correlated with increased expression of numerous adhesion proteins and many apoptosis regulating proteins, two processes related to leukemogenesis. TG2 has previously been linked to drug resistance in cancer and given the negative correlation between TG2 levels and peripheral blasts observed increased TG2 levels may lead to the protection of the leukemic stem cell due to increased adhesion/reduced motility. TG2 may therefore form part of a network of proteins that define poor outcome in AML patients and potentially offer a target to sensitize AML stem cells to drug treatment. PMID:23576428

Smoking Adversely Affects Survival in Acute Myeloid Leukemia Patients

PubMed Central

Varadarajan, Ramya; Licht, Andrea S; Hyland, Andrew J; Ford, Laurie A.; Sait, Sheila N.J.; Block, Annemarie W.; Barcos, Maurice; Baer, Maria R.; Wang, Eunice S.; Wetzler, Meir

2011-01-01

Summary Smoking adversely affects hematopoietic stem cell transplantation outcome. We asked whether smoking affected outcome of newly diagnosed acute myeloid leukemia (AML) patients treated with chemotherapy. Data were collected on 280 AML patients treated with high-dose cytarabine and idarubicin-containing regimens at Roswell Park Cancer Institute who had smoking status data at diagnosis. Patients’ gender, age, AML presentation (de novo vs. secondary), white blood cell (WBC) count at diagnosis, karyotype and smoking status (never vs. ever) were analyzed. Among the 161 males and 119 females with a median follow-up of 12.9 months, 101 (36.1%) had never smoked and 179 (63.9%) were ever smokers. The proportion of patients between never and ever smokers was similar with respect to age, AML presentation, WBC count at diagnosis or karyotype based on univariate analysis of these categorical variables. Never smokers had a significantly longer overall survival (60.32 months) compared to ever smokers (30.89; p=0.005). In multivariate analysis incorporating gender, age, AML presentation, WBC count, karyotype, and smoking status as covariates, age, karyotype and smoking status retained prognostic value for overall survival. In summary, cigarette smoking has a deleterious effect on overall survival in AML. PMID:21520043

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia

PubMed Central

Klein, Hans-Ulrich; Hascher, Antje; Isken, Fabienne; Tickenbrock, Lara; Thoennissen, Nils; Agrawal-Singh, Shuchi; Tschanter, Petra; Disselhoff, Christine; Wang, Yipeng; Becker, Anke; Thiede, Christian; Ehninger, Gerhard; zur Stadt, Udo; Koschmieder, Steffen; Seidl, Matthias; Müller, Frank U.; Schmitz, Wilhelm; Schlenke, Peter; McClelland, Michael; Berdel, Wolfgang E.; Dugas, Martin; Serve, Hubert

2010-01-01

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34+ cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML. PMID:20498303

Dawn of Aurora kinase inhibitors as anticancer drugs.

PubMed

Doggrell, Sheila A

2004-09-01

With the current standard chemotherapy regimens only approximately 25% of acute myelogenous leukaemia (AML) patients survive > 5 years. Aurora kinases are overexpressed in many human cancers. VX-680 inhibited Aurora-A, -B, -C and the FMS-like tyrosine kinase-3 with apparent inhibitory constants of 0.6, 18, 4.6 and 30 nM, respectively. In primary leukaemia cells from patients with AML, which were refractory to standard therapies, VX-680 inhibited colony formation. In nude mice, VX-680 markedly reduced human AML tumours. The development of VX-680 for use in AML should continue.

FLT3-ITD induces expression of Pim kinases through STAT5 to confer resistance to the PI3K/Akt pathway inhibitors on leukemic cells by enhancing the mTORC1/Mcl-1 pathway.

PubMed

Okada, Keigo; Nogami, Ayako; Ishida, Shinya; Akiyama, Hiroki; Chen, Cheng; Umezawa, Yoshihiro; Miura, Osamu

2018-02-06

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously reported that activation of STAT5 confers resistance to PI3K/Akt inhibitors on the FLT3-ITD-positive AML cell line MV4-11 and 32D cells driven by FLT3-ITD (32D/ITD) but not by FLT3 mutated in the tyrosine kinase domain (32D/TKD). Here, we report the involvement of Pim kinases expressed through STAT5 activation in acquisition of this resistance. The specific pan-Pim kinase inhibitor AZD1208 as well as PIM447 in combination with the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 cooperatively downregulated the mTORC1/4EBP1 pathway, formation of the eIF4E/eIF4G complex, and Mcl-1 expression leading to activation of Bak and Bax to induce caspase-dependent apoptosis synergistically in these cells. These cooperative effects were enhanced or inhibited by knock down of mTOR or expression of its activated mutant, respectively. Overexpression of Mcl-1 conferred the resistance on 32D/ITD cells to combined inhibition of the PI3K/Akt pathway and Pim kinases, while the Mcl-1-specific BH3 mimetic A-1210477 conquered the resistance of MV4-11 cells to GDC-0941. Furthermore, overexpression of Pim-1 in 32D/TKD enhanced the mTORC1/Mcl-1 pathway and partially protected it from the PI3K/Akt inhibitors or the FLT3 inhibitor gilteritinib to confer the resistance to PI3K/Akt inhibitors. Finally, AZD1208 and GDC-0941 cooperatively inhibited the mTORC1/Mcl-1 pathway and reduced viable cell numbers of primary AML cells from some FLT3-ITD positive cases. Thus, Pim kinases may protect the mTORC1/4EBP1/Mcl-1 pathway to confer the resistance to the PI3K/Akt inhibitors on FLT3-ITD cells and represent promising therapeutic targets.

Autophagy is an important event for low-dose cytarabine treatment in acute myeloid leukemia cells.

PubMed

Chen, Liyun; Guo, Pei; Zhang, Yunxiang; Li, Xiaoyang; Jia, Peimin; Tong, Jianhua; Li, Junmin

2017-09-01

Cytarabine (Ara-c) has been an important agent in acute myeloid leukemia (AML) treatment for more than 40 years. While, the mechanisms underlying low dose cytarabine (LD Ara-c) is poorly understood. In this study, we investigated the therapeutic effect of LD Ara-C in vitro. U937 and HEL cell lines were treated with increasing dose of Ara-C and showed growth inhibition rates in a time and dose-dependent manner. Treatment with LD Ara-C (50nM) induced a time-dependent increase in expression of microtubule-associated protein light chain 3 (LC3) and beclin1, but degradation of sequestosome1 (p62) in both U937 and HEL cells. Characteristic of autophagosomes appeared after 24h treatment. Meanwhile, deregulation of Akt-mTOR pathway was also detected. When cultured in presence of autophagy inhibitors, autophagy and differentiation was reversed, and cell growth inhibition was also attenuated. Similar phenomenon could also be seen when beclin1 expression was down-regulated. Taken together, we concluded that LD Ara-C can induce autophagy in AML cells and appeared to play an important role in differentiation and death. Down-regulation of Akt-mTOR pathway is involved in these processes. We suggest that cytarabine-induced autophagy is not a pro-survival mechanism, but accounts for its antineoplastic effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

BCL2 Inhibition by Venetoclax: Targeting the Achilles' Heel of the Acute Myeloid Leukemia Stem Cell?

PubMed

Pullarkat, Vinod A; Newman, Edward M

2016-10-01

Venetoclax is an oral drug with an excellent side-effect profile that has the potential to revolutionize acute myeloid leukemia (AML) therapy in two areas. Venetoclax-based combination therapies could be a bridge to hematopoietic cell transplant with curative intent for patients with refractory/relapsed AML, and venetoclax-based therapy could provide meaningful survival prolongation for older patients with AML who are not candidates for more aggressive therapies. Cancer Discov; 6(10); 1082-3. ©2016 AACR.See related article by Konopleva and colleagues, p. 1106. ©2016 American Association for Cancer Research.

Acute myeloid leukaemia at an early age: Reviewing the interaction between pesticide exposure and KMT2A-rearrangement

PubMed Central

Pombo-de-Oliveira, Maria S; Andrade, Francianne Gomes; Brisson, Gisele Dallapicola; dos Santos Bueno, Filipe Vicente; Cezar, Ingrid Sardou; Noronha, Elda Pereira

2017-01-01

Acute myeloid leukaemia (AML) in early childhood is characterised by a high frequency of recurrent genomic aberrations associated with distinct myeloid subtypes, clinical outcomes and pathogenesis. Genomic instability is the first step of pathogenic mechanism in early childhood AML. A sum of adverse events is necessary to the development of infant AML (i-AML), which includes latency of biochemical-molecular and cellular effects. Inherited genetic susceptibility associated with exposures to biotransformation substances can modulate the risk of DNA damage and it is a very important piece in the pathogenic puzzle. In this review, we have aimed to explore the chain of events in the time-points of the natural history of i-AML, which includes maternal exposures during pregnancy, the speculations about the formation of somatic mutations during foetal life and the secondary genomic aberrations associated with i-AML. The modulation of risk conferred by xenobiotic metabolism´s genes variants is the bottom line of the pathogenic process. Since we have conducted observational and molecular investigations in early childhood leukaemia, the data focused here is based on Brazilian findings with summarised results of our experience with epidemiological and molecular studies in early-age leukaemia. PMID:29225689

Schizandrin inhibits fibrosis and epithelial-mesenchymal transition in transforming growth factor-β1-stimulated AML12 cells.

PubMed

Park, Ji-hyun; Yoon, Jaewoo

2015-04-01

The transforming growth factor (TGF)-β1 plays a crucial role in the induction of the epithelial-to-mesenchymal transition (EMT) in hepatocytes, which contributes to the pathogenesis of liver fibrosis. The inhibition of the TGF-β1 cascade suppresses EMT and the resultant fibrosis. Schizandrin (Sch) has various therapeutic effects on a range of medical conditions such as anti-asthmatic, anti-cancer, and anti-inflammatory effects. However, the effect of Sch on TGF-β1-stimulated hepatic fibrosis and EMT is still unknown. In the present investigation, we evaluated the anti-fibrotic and anti-EMT properties of Sch and its underlying mechanisms in murine hepatocyte AML12 cells. Overall, we found that Sch inhibited the pro-fibrotic activity of TGF-β1 in AML12 cells; thus, it suppressed the accumulation of ECM proteins. Also, Sch inhibited the EMT as assessed by reduced expression of vimentin and fibronectin, and increased E-cadherin and ZO-1 in TGF-β1 induced AML12 cells. Sch reduced TGF-β1-mediated phosphorylation of Smad2/3 and Smad3/4 DNA binding activity. On the other hand, Sch reduced TGF-β1-induced ERK1/2 and PI3K/Akt phosphorylation in the non-Smad pathway. In conclusion, Sch can antagonize TGF-β1-mediated fibrosis and EMT in AML12 cells. Sch may possess potential as an anti-fibrotic molecule in the treatment of liver fibrosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Integrative analysis of RUNX1 downstream pathways and target genes

PubMed Central

Michaud, Joëlle; Simpson, Ken M; Escher, Robert; Buchet-Poyau, Karine; Beissbarth, Tim; Carmichael, Catherine; Ritchie, Matthew E; Schütz, Frédéric; Cannon, Ping; Liu, Marjorie; Shen, Xiaofeng; Ito, Yoshiaki; Raskind, Wendy H; Horwitz, Marshall S; Osato, Motomi; Turner, David R; Speed, Terence P; Kavallaris, Maria; Smyth, Gordon K; Scott, Hamish S

2008-01-01

Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications. PMID:18671852

Measurable residual disease testing in acute myeloid leukaemia.

PubMed

Hourigan, C S; Gale, R P; Gormley, N J; Ossenkoppele, G J; Walter, R B

2017-07-01

There is considerable interest in developing techniques to detect and/or quantify remaining leukaemia cells termed measurable or, less precisely, minimal residual disease (MRD) in persons with acute myeloid leukaemia (AML) in complete remission defined by cytomorphological criteria. An important reason for AML MRD-testing is the possibility of estimating the likelihood (and timing) of leukaemia relapse. A perfect MRD-test would precisely quantify leukaemia cells biologically able and likely to cause leukaemia relapse within a defined interval. AML is genetically diverse and there is currently no uniform approach to detecting such cells. Several technologies focused on immune phenotype or cytogenetic and/or molecular abnormalities have been developed, each with advantages and disadvantages. Many studies report a positive MRD-test at diverse time points during AML therapy identifies persons with a higher risk of leukaemia relapse compared with those with a negative MRD-test even after adjusting for other prognostic and predictive variables. No MRD-test in AML has perfect sensitivity and specificity for relapse prediction at the cohort- or subject levels and there are substantial rates of false-positive and -negative tests. Despite these limitations, correlations between MRD-test results and relapse risk have generated interest in MRD-test result-directed therapy interventions. However, convincing proof that a specific intervention will reduce relapse risk in persons with a positive MRD-test is lacking and needs testing in randomized trials. Routine clinical use of MRD-testing requires further refinements and standardization/harmonization of assay platforms and results reporting. Such data are needed to determine whether results of MRD-testing can be used as a surrogate end point in AML therapy trials. This could make drug-testing more efficient and accelerate regulatory approvals. Although MRD-testing in AML has advanced substantially, much remains to be done.

miR-125b promotes MLL-AF9–driven murine acute myeloid leukemia involving a VEGFA-mediated non–cell-intrinsic mechanism

PubMed Central

Liu, Jun; Guo, Bo; Chen, Zhuo; Wang, Nayi; Iacovino, Michelina; Cheng, Jijun; Roden, Christine; Pan, Wen; Khan, Sajid; Chen, Suning; Kyba, Michael; Fan, Rong; Guo, Shangqin

2017-01-01

The hematopoietic stem cell–enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9. By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9–driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b. Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non–cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2. Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b. In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non–cell-intrinsic leukemia-promoting effects by an oncogenic miRNA. PMID:28053194

Haploidentical Stem Cell Transplant for Treatment Refractory Hematological Malignancies

ClinicalTrials.gov

2009-02-12

Acute Lymphoblastic Leukemia (ALL); Acute Myeloid Leukemia (AML); Secondary AML; Myelodysplastic Syndrome (MDS); Secondary MDS; Chronic Myeloid Leukemia; Juvenile Myelomonocytic Leukemia (JMML); Paroxysmal Nocturnal Hemoglobinuria (PNH); Lymphoma, Non-Hodgkin; Hodgkin Disease

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome

PubMed Central

Alachkar, Houda; Santhanam, Ramasamy; Maharry, Kati; Metzeler, Klaus H.; Huang, Xiaomeng; Kohlschmidt, Jessica; Mendler, Jason H.; Benito, Juliana M.; Hickey, Christopher; Neviani, Paolo; Dorrance, Adrienne M.; Anghelina, Mirela; Khalife, Jihane; Tarighat, Somayeh S.; Volinia, Stefano; Whitman, Susan P.; Paschka, Peter; Hoellerbauer, Pia; Wu, Yue-Zhong; Han, Lina; Bolon, Brad N.; Blum, William; Mrózek, Krzysztof; Carroll, Andrew J.; Perrotti, Danilo; Andreeff, Michael; Caligiuri, Michael A.; Konopleva, Marina; Garzon, Ramiro; Bloomfield, Clara D.; Marcucci, Guido

2014-01-01

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML. PMID:24590286

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome.

PubMed

Alachkar, Houda; Santhanam, Ramasamy; Maharry, Kati; Metzeler, Klaus H; Huang, Xiaomeng; Kohlschmidt, Jessica; Mendler, Jason H; Benito, Juliana M; Hickey, Christopher; Neviani, Paolo; Dorrance, Adrienne M; Anghelina, Mirela; Khalife, Jihane; Tarighat, Somayeh S; Volinia, Stefano; Whitman, Susan P; Paschka, Peter; Hoellerbauer, Pia; Wu, Yue-Zhong; Han, Lina; Bolon, Brad N; Blum, William; Mrózek, Krzysztof; Carroll, Andrew J; Perrotti, Danilo; Andreeff, Michael; Caligiuri, Michael A; Konopleva, Marina; Garzon, Ramiro; Bloomfield, Clara D; Marcucci, Guido

2014-04-01

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

Granulocyte, monocyte and blast immunophenotype abnormalities in acute myeloid leukemia with myelodysplasia-related changes.

PubMed

Ayar, Sonali P; Ravula, Sreelakshmi; Polski, Jacek M

2014-01-01

Little literature exists regarding granulocyte and monocyte immunophenotype abnormalities in Acute Myeloid Leukemia (AML). We hypothesized that granulocyte and monocyte immunophenotype abnormalities are common in AML, and especially in AML with myelodysplasia-related changes (AMLMRC). Bone marrow or peripheral blood specimens from 48 cases of AML and 22 cases of control specimens were analyzed by flow cytometric immunophenotyping. Granulocyte, monocyte, and blast immunophenotype abnormalities were compared between cases of AML versus controls and AMLMRC versus AML without myelodysplasia. The results revealed that granulocyte, monocyte, and blast abnormalities were more common in AMLMRC than in AML without myelodysplasia or control cases. The difference reached statistical significance for abnormalities of granulocytes and abnormalities in all cells of interest. From the numerous individual abnormalities, only CD25 expression in blasts was significantly more prevalent in AMLMRC in this study. We conclude that detection of granulocyte, monocyte, and blast immunophenotype abnormalities can contribute to the diagnosis of AMLMRC.

Acute myeloid leukemia with leukemic pleural effusion.

PubMed

Chang, Hung

2013-10-01

Acute myeloid leukemia (AML) may be associated with extramedullary tumor growth, which is commonly known as myeloid sarcoma. Although AML with leukemic pleural effusion is considered rare, the true incidence is not clear. We report three cases of AML involving pleural effusion in this study. The cases were encountered in a single institute within two years, suggesting that leukemic effusion is more common than previously reported. Leukemic cells showed evidence of monocytic differentiation in all cases. Two patients presented with advanced AML. Both had concurrent myeloid sarcoma. Both were ineligible for intensive treatment and died soon after diagnosis of myeloid sarcoma. The third patient had pleural effusion upon diagnosis of AML. Remission was achieved and the effusion disappeared after treatment. We conclude leukemic effusion may become more common in an era of improved care and prolonged survival for AML patients. The prognostic impact is unclear and patients should be given standard AML treatment whenever possible. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

High resolution, low cost solar cell contact development

NASA Technical Reports Server (NTRS)

Mardesich, N.

1979-01-01

The experimental work demonstrating the feasibility of the MIDFILM process as a low cost means of applying solar cell collector metallization as reported. Cell efficiencies of above 14% (AMl, 28 C) were achieved with fritted silver metallization. Environmental tests suggest that the metallization is slightly humidity sensitive and degradation is observed on cells with high series resistance. The major yield loss in the fabrication of cells was due to discontinuous grid lines, resulting in high series resitance. Standard lead-tin solder plated interconnections do not appear compatible with the MIDFILM contact. Copper, nickel and molybdemun base powder were investigated as low cost metallization systems. The copper based powder degraded the cell response. The nickel and molybdenum base powders oxidized when sintered in the oxidizing atmosphere necessary to ash the photoresin.

Bone marrow T-cell percentage: A novel prognostic indicator in acute myeloid leukemia.

PubMed

Ismail, Manar M; Abdulateef, Nahla A B

2017-04-01

Acute myeloid leukemia (AML) is an aggressive malignancy for which overall disease-free survival is less than 50%. Manipulation of the immune system is an interesting and promising therapy for AML patients. We aimed to characterize the immune system of AML patients, highlighting the clinical relevance of total bone marrow (BM) lymphocytes and subpopulations. Sixty-six new AML cases diagnosed according to WHO criteria from King Abdullah Medical City, KSA, from October 2012 to February 2015. Analysis of BM lymphocytes and subpopulations was done by flowcytometry. Significantly, high percentages of BM lymphocytes, T cells, and natural killer (NK) cells were detected in the group that achieved complete remission (P values = 0.004, <0.001, and <0.001, respectively). Overall survival (OS) was significantly prolonged in patients with high BM lymphocytes and T cells (P values = 0.047 and P 0.002, respectively). Multivariate analysis indicated that BM T-cell percentage and cytogenetics were independent prognostic factors predictive of OS (HR 4.7, P value = 0.011). BM T-cell percentage constitutes a novel host factor that can be used in combination with cytogenetics to better predict OS. Large-scale multicenter studies are recommended to clarify its role as a predictor of OS and leukemia-free survival.

Examining the Origins of Myeloid Leukemia | Center for Cancer Research

Cancer.gov

Acute myeloid leukemia or AML, a cancer of the white blood cells, is the most common type of rapidly-growing leukemia in adults. The over-production of white blood cells in the bone marrow inhibits the development of other necessary blood components including red blood cells, which carry oxygen throughout the body, and platelets, which are required for clot formation. The cellular changes that lead to AML disease initiation and progression, however, are not clear. Because of the aging of the U.S. population and AML’s increasing incidence with age, cases of this disease are likely to rise significantly in the near future. Thus, understanding what causes AML should lead to the identification of novel targets and the enhanced treatment of patients.

Receptor tyrosine kinase alterations in AML - biology and therapy.

PubMed

Stirewalt, Derek L; Meshinchi, Soheil

2010-01-01

Acute myeloid leukemia (AML) is the most common form of leukemia in adults, and despite some recent progress in understanding the biology of the disease, AML remains the leading cause of leukemia-related deaths in adults and children. AML is a complex and heterogeneous disease, often involving multiple genetic defects that promote leukemic transformation and drug resistance. The cooperativity model suggests that an initial genetic event leads to maturational arrest in a myeloid progenitor cell, and subsequent genetic events induce proliferation and block apoptosis. Together, these genetic abnormalities lead to clonal expansion and frank leukemia. The purpose of this chapter is to review the biology of receptor tyrosine kinases (RTKs) in AML, exploring how RTKs are being used as novel prognostic factors and potential therapeutic targets.

Acute myeloid leukemia presenting with panhypopituitarism or diabetes insipidus: a case series with molecular genetic analysis and review of the literature.

PubMed

Cull, Elizabeth H; Watts, Justin M; Tallman, Martin S; Kopp, Peter; Frattini, Mark; Rapaport, Franck; Rampal, Raajit; Levine, Ross; Altman, Jessica K

2014-09-01

Central diabetes insipidus (DI) is a rare finding in patients with acute myeloid leukemia (AML), usually occurring in patients with chromosome 3 or 7 abnormalities. We describe four patients with AML and concurrent DI and a fifth patient with AML and panhypopituitarism. Four of five patients had monosomy 7. Three patients had chromosome 3q21q26/EVI-1 gene rearrangements. The molecular genotype of patients with AML and DI is not known. Therefore, we performed gene sequencing of 30 genes commonly mutated in AML in three patients with available leukemia cell DNA. One patient had no identifiable mutations, and two had RUNX1 F158S mutations.

Indole-3-carbinol induces G1 cell cycle arrest and apoptosis through aryl hydrocarbon receptor in THP-1 monocytic cell line.

PubMed

Mohammadi, Saeed; Seyedhosseini, Fakhri Sadat; Behnampour, Nasser; Yazdani, Yaghoub

2017-10-01

The role of aryl hydrocarbon receptor (AhR) in carcinogenesis has been studied recently. Indole-3-carbinol (I3C) is an AhR agonist and a potential anticancer agent. Here, we investigated the effects of I3C on cell cycle progression and apoptosis through activation of AhR on THP-1 acute myeloid leukemia (AML) cell line. MTT viability assay was used to measure the cytotoxic effects of I3C on THP-1 cells. Apoptosis and cell cycle assays were investigated using flow cytometry. Real time RT-PCR was conducted to measure the alterations in the expression of AhR gene, key genes associated with AhR activation (IL1β and CYP1A1) and major genes involved in cell cycle regulation and apoptosis including P27, P21, CDK2, P53, BCL2 and FasR. Our findings revealed that I3C inhibits the proliferation of THP-1 cells in a dose- and time-dependent manner with minimal toxicity over normal monocytes. The AhR target genes (CYP1A1, IL1β) were overexpressed upon I3C treatment (p < .05 to p < .001). The antiproliferative effects of I3C were in association with programed cell death. I3C downregulated BCL2 and upregulated FasR in THP-1 cells (p < .05 to p < .001). G1 cell cycle arrest was also observed using flow cytometry. G1-acting cell cycle genes (P21, P27 and P53) were overexpressed (p < .05 to p < .001), while CDK2 was downregulated upon I3C treatment (p < .01 to p < .001). I3C could exert its antileukemic effects through AhR activation which is associated with programed cell death and G1 cell cycle arrest in a dose- and time-dependent manner. Therefore, AhR could be targeted as a novel treatment possibility in AML.

Adult Acute Myeloid Leukemia Treatment (PDQ®)—Health Professional Version

Cancer.gov

Acute myeloid (myelogenous) leukemia (AML) treatment options include chemotherapy, radiation therapy, stem cell transplant, and other medications. Cytogenetic analysis helps predict treatment outcomes. Get detailed information about AML in this summary for clinicians.

The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO

PubMed Central

Chou, Fu-Sheng; Griesinger, Andrea; Wunderlich, Mark; Lin, Shan; Link, Kevin A.; Shrestha, Mahesh; Goyama, Susumu; Mizukawa, Benjamin; Shen, Shuhong; Marcucci, Guido

2012-01-01

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO–induced MPL. PMID:22337712

TdT activity in acute myeloid leukemias defined by monoclonal antibodies.

PubMed

San Miguel, J F; González, M; Cañizo, M C; Anta, J P; Portero, J A; López-Borrasca, A

1986-09-01

Blast cells from eight out of 71 patients diagnosed with acute myeloid leukemia (AML) by morphological, cytochemical, and immunological criteria showed TdT activity. Their distribution according to the FAB classification was one M1, one M2, one M4, two M5a, one M5b, one M6, and one undifferentiated case. The TdT+ AML cases did not show major clinical and hematological differences when compared with the classical TdT- AML patients. Other phenotypical aberrations in the expression of membrane antigens, apart from the presence of nuclear TdT, were not observed in these TdT+ cases after study with a large panel of monoclonal antibodies. A higher incidence of TdT+ cases was found among the monocytic variants of AML (M4 and M5)--four cases--than in the granulocytic variants (M1, M2, and M3)--2 cases. These TdT+ cases should be distinguished from mixed leukemias by double labeling techniques, assessing in the TdT+ AML the coexpression of TdT and myeloid markers in individual cells as shown in four of our cases.

MDS and secondary AML display unique patterns and abundance of aberrant DNA methylation

PubMed Central

Figueroa, Maria E.; Skrabanek, Lucy; Li, Yushan; Jiemjit, Anchalee; Fandy, Tamer E.; Paietta, Elisabeth; Fernandez, Hugo; Tallman, Martin S.; Greally, John M.; Carraway, Hetty; Licht, Jonathan D.; Gore, Steven D.

2009-01-01

Increasing evidence shows aberrant hypermethylation of genes occurring in and potentially contributing to pathogenesis of myeloid malignancies. Several of these diseases, such as myelodysplastic syndromes (MDSs), are responsive to DNA methyltransferase inhibitors. To determine the extent of promoter hypermethylation in such tumors, we compared the distribution of DNA methylation of 14 000 promoters in MDS and secondary acute myeloid leukemia (AML) patients enrolled in a phase 1 trial of 5-azacytidine and the histone deacetylase inhibitor entinostat against de novo AML patients and normal CD34+ bone marrow cells. The MDS and secondary AML patients displayed more extensive aberrant DNA methylation involving thousands of genes than did the normal CD34+ bone marrow cells or de novo AML blasts. Aberrant methylation in MDS and secondary AML tended to affect particular chromosomal regions, occurred more frequently in Alu-poor genes, and included prominent involvement of genes involved in the WNT and MAPK signaling pathways. DNA methylation was also measured at days 15 and 29 after the first treatment cycle. DNA methylation was reversed at day 15 in a uniform manner throughout the genome, and this effect persisted through day 29, even without continuous administration of the study drugs. This trial was registered at www.clinicaltrials.gov as J0443. PMID:19652201

Epigenetic Guardian: A Review of the DNA Methyltransferase DNMT3A in Acute Myeloid Leukaemia and Clonal Haematopoiesis.

PubMed

Chaudry, Sabah F; Chevassut, Timothy J T

2017-01-01

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by clonal stem cell proliferation and aberrant block in differentiation. Dysfunction of epigenetic modifiers contributes significantly to the pathogenesis of AML. One frequently mutated gene involved in epigenetic modification is DNMT3A (DNA methyltransferase-3-alpha), a DNA methyltransferase that alters gene expression by de novo methylation of cytosine bases at CpG dinucleotides. Approximately 22% of AML and 36% of cytogenetically normal AML cases carry DNMT3A mutations and around 60% of these mutations affect the R882 codon. These mutations have been associated with poor prognosis and adverse survival outcomes for AML patients. Advances in whole-exome sequencing techniques have recently identified a large number of DNMT3A mutations present in clonal cells in normal elderly individuals with no features of haematological malignancy. Categorically distinct from other preleukaemic conditions, this disorder has been termed clonal haematopoiesis of indeterminate potential (CHIP). Further insight into the mutational landscape of CHIP may illustrate the consequence of particular mutations found in DNMT3A and identify specific "founder" mutations responsible for clonal expansion that may contribute to leukaemogenesis. This review will focus on current research and understanding of DNMT3A mutations in both AML and CHIP.

Hematopoietic cell transplantation in patients with intermediate and high-risk AML: results from the randomized Study Alliance Leukemia (SAL) AML 2003 trial.

PubMed

Schetelig, J; Schaich, M; Schäfer-Eckart, K; Hänel, M; Aulitzky, W E; Einsele, H; Schmitz, N; Rösler, W; Stelljes, M; Baldus, C D; Ho, A D; Neubauer, A; Serve, H; Mayer, J; Berdel, W E; Mohr, B; Oelschlägel, U; Parmentier, S; Röllig, C; Kramer, M; Platzbecker, U; Illmer, T; Thiede, C; Bornhäuser, M; Ehninger, G

2015-05-01

The optimal timing of allogeneic hematopoietic stem cell transplantation (HCT) in acute myeloid leukemia (AML) is controversial. We report on 1179 patients with a median age of 48 years who were randomized upfront. In the control arm, sibling HCT was scheduled in the first complete remission for intermediate-risk or high-risk AML and matched unrelated HCT in complex karyotype AML. In the experimental arm, matched unrelated HCT in first remission was offered also to patients with an FLT3-ITD (FMS-like tyrosine kinase 3-internal tandem duplication) allelic ratio >0.8, poor day +15 marrow blast clearance and adverse karyotypes. Further, allogeneic HCT was recommended in high-risk AML to be performed in aplasia after induction chemotherapy. In the intent-to-treat (ITT) analysis, superiority of the experimental transplant strategy could not be shown with respect to overall survival (OS) or event-free survival. As-treated analyses suggest a profound effect of allogeneic HCT on OS (HR 0.73; P=0.002) and event-free survival (HR 0.67; P<0.001). In high-risk patients, OS was significantly improved after allogeneic HCT in aplasia (HR 0.64; P=0.046) and after HCT in remission (HR 0.74; P=0.03). Although superiority of one study arm could not be demonstrated in the ITT analysis, secondary analyses suggest that early allogeneic HCT is a promising strategy for patients with high-risk AML.

Acute myeloid leukaemia with myelodysplastic features in children: a report of Japanese Paediatric Leukaemia/Lymphoma Study Group.

PubMed

Kinoshita, Akitoshi; Miyachi, Hayato; Matsushita, Hiromichi; Yabe, Miharu; Taki, Tomohiko; Watanabe, Tomoyuki; Saito, Akiko M; Tomizawa, Daisuke; Taga, Takashi; Takahashi, Hiroyuki; Matsuo, Hidemasa; Kodama, Kumi; Ohki, Kentaro; Hayashi, Yasuhide; Tawa, Akio; Horibe, Keizo; Adachi, Souichi

2014-10-01

The clinical characteristics and prognostic relevance of acute myeloid leukaemia (AML) with myelodysplastic features remains to be clarified in children. We prospectively examined 443 newly diagnosed patients in a multicentre clinical trial for paediatric de novo AML, and found 'AML with myelodysplasia-related changes' (AML-MRC) according to the 2008 World Health Organization classification in 93 (21·0%), in whom 59 were diagnosed from myelodysplasia-related cytogenetics alone, 28 from multilineage dysplasia alone and six from a combination of both. Compared with 111 patients with 'AML, not otherwise specified' (AML-NOS), patients with 'AML-MRC' presented at a younger age, with a lower white blood cell count, higher incidence of 20-30% bone marrow blasts, unfavourable cytogenetics and a lower frequency of Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), NPM1 and CEBPA mutations. Complete remission rate and 3-year probability of event-free survival were significantly worse in 'AML-MRC' patients (67·7 vs. 85·6%, P < 0·01, 37·1% vs. 53·8%, P = 0·02, respectively), but 3-year overall survival and relapse-free survival were comparable with 'AML-NOS' patients. By multivariate analysis, FLT3-ITD was solely associated with worse overall survival. These results support the distinctive features of the category 'AML-MRC' even in children. © 2014 John Wiley & Sons Ltd.

Loss of RUNX1/AML1 arginine-methylation impairs in peripheral T cell homeostasis

PubMed Central

Mizutani, Shinsuke; Yoshida, Tatsushi; Zhao, Xinyang; Nimer, Stephen D.; Taniwaki, Masafumi; Okuda, Tsukasa

2016-01-01

Summary RUNX1 (previously termed AML1) is a frequent target of human leukaemia-associated gene aberrations, and it encodes the DNA-binding subunit of the Core-Binding Factor transcription factor complex. RUNX1 expression is essential for the initiation of definitive haematopoiesis, for steady-state thrombopoiesis, and for normal lymphocytes development. Recent studies revealed that protein arginine methyltransferase 1 (PRMT1), which accounts for the majority of the type I PRMT activity in cells, methylates two arginine residues in RUNX1 (R206 and R210), and these modifications inhibit corepressor-binding to RUNX1 thereby enhancing its transcriptional activity. In order to elucidate the biological significance of these methylations, we established novel knock-in mouse lines with non-methylable, double arginine-to-lysine (RTAMR-to-KTAMK) mutations in RUNX1. Homozygous Runx1KTAMK/KTAMK mice are born alive and appear normal during adulthood. However, Runx1KTAMK/KTAMK mice showed a reduction in CD3+ T lymphoid cells and a decrease in CD4+ T cells in peripheral lymphoid organs, in comparison to their wild-type littermates, leading to a reduction in the CD4+ to CD8+ T-cell ratio. These findings suggest that arginine-methylation of RUNX1 in the RTAMR-motif is dispensable for the development of definitive haematopoiesis and for steady-state platelet production, however this modification affects the role of RUNX1 in the maintenance of the peripheral CD4+ T-cell population. PMID:26010396

Fanconi anemia and the development of leukemia.

PubMed

Alter, Blanche P

2014-01-01

Fanconi anemia (FA) is a rare autosomal recessive cancer-prone inherited bone marrow failure syndrome, due to mutations in 16 genes, whose protein products collaborate in a DNA repair pathway. The major complications are aplastic anemia, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and specific solid tumors. A severe subset, due to mutations in FANCD1/BRCA2, has a cumulative incidence of cancer of 97% by age 7 years; the cancers are AML, brain tumors, and Wilms tumor; several patients have multiple events. Patients with the other genotypes (FANCA through FANCQ) have cumulative risks of more than 50% of marrow failure, 20% of AML, and 30% of solid tumors (usually head and neck or gynecologic squamous cell carcinoma), by age 40, and they too are at risk of multiple adverse events. Hematopoietic stem cell transplant may cure AML and MDS, and preemptive transplant may be appropriate, but its use is a complicated decision. Published by Elsevier Ltd.

A novel HLA-A*0201 restricted peptide derived from cathepsin G is an effective immunotherapeutic target in acute myeloid leukemia.

PubMed

Zhang, Mao; Sukhumalchandra, Pariya; Enyenihi, Atim A; St John, Lisa S; Hunsucker, Sally A; Mittendorf, Elizabeth A; Sergeeva, Anna; Ruisaard, Kathryn; Al-Atrache, Zein; Ropp, Patricia A; Jakher, Haroon; Rodriguez-Cruz, Tania; Lizee, Gregory; Clise-Dwyer, Karen; Lu, Sijie; Molldrem, Jeffrey J; Glish, Gary L; Armistead, Paul M; Alatrash, Gheath

2013-01-01

Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients. CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL-mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.

A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Xiao-Jian; Wang, Zhanxin; Wang, Lan

2013-06-30

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1–ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1–ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that ‘read’ the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1–ETO resides in and functions through a stable AML1–ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalentmore » interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1–ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2–N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1–ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1–ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.« less

When the good go bad: Mutant NPM1 in acute myeloid leukemia.

PubMed

Kunchala, Preethi; Kuravi, Sudhakiranmayi; Jensen, Roy; McGuirk, Joseph; Balusu, Ramesh

2018-05-01

Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein that performs diverse biological functions including molecular chaperoning, ribosome biogenesis, DNA repair, and genome stability. Acute myeloid leukemia (AML) is a heterogeneous disease, more than half of the AML cases exhibit normal karyotype (NK). Approximately 50-60 percent of patients with NK-AML carry NPM1 mutations which are characterized by cytoplasmic dislocation of the NPM1 protein. In AML, mutant NPM1 (NPM1c+) acts in a dominant negative fashion and also blocks the differentiation of myeloid cells through gain-of-function for the AML phenotype. Currently, there is limited knowledge on the gain-of-function mechanism of mutant NPM1. Here, we review the known mechanisms of mutant NPM1 in the pathogenesis of AML. We describe genetic abnormalities, the clinical significance of exon-12 mutations in the NPM1 gene, and chromosomal translocations including the recently discovered NPM1-TYK2, and NPM1-HAUS1. Also, we outline the possible therapeutic interventions for the treatment of AML by targeting NPM1. Overall, the review will summarize present knowledge on mutant NPM1 origin, pathogenesis, and therapy in AML. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.

2014-10-02

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction ofmore » phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.« less

Deletion of RUNX1 exons 1 and 2 associated with familial platelet disorder with propensity to acute myeloid leukemia.

PubMed

Cavalcante de Andrade Silva, Marcela; Krepischi, Ana Cristina Victorino; Kulikowski, Leslie Domenici; Zanardo, Evelin Aline; Nardinelli, Luciana; Leal, Aline Medeiros; Costa, Silvia Souza; Muto, Nair Hideki; Rocha, Vanderson; Velloso, Elvira Deolinda Rodrigues Pereira

2018-04-01

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) associated with RUNX1 mutations is an autosomal dominant disorder included in the group of the myeloid neoplasms with germ line predisposition. We describe two brothers who were diagnosed with hematological malignancies (one with AML and the other with T-cell lymphoblastic lymphoma). There was a history of leukemia in the paternal family and two of their siblings presented with low platelet counts and no history of significant bleeding. A microdeletion encompassing exons 1-2 of RUNX1 (outside the cluster region of the Runt Homology domain and the transactivation domain) was detected in six family members using array-CGH and MLPA validation. A low platelet count was not present in all deletion carriers and, therefore, it should not be used as an indication for screening in suspected families and family members. Copyright © 2018 Elsevier Inc. All rights reserved.

[Study of radiation dose to the eye lens by multi-detector row computed tomography of the temporal bone].

PubMed

Hirakuri, Ayaka; Numasawa, Kanako; Takeishi, Hideki; Satomura, Minato; Takeda, Hiromitsu; Harada, Kuniaki; Asanuma, Osamu; Sakata, Motomichi

2012-01-01

The exposure of the eye lens caused by multi-detector row computed tomography (MDCT) of the temporal bone is a serious problem. Our aim was to evaluate the radiation dose to the eye lens by different scan baselines (orbitomeatal line; OML, acanthiomeatal line; AML) and examine the difference of the depiction of the temporal bone structures. Measurement of the exposure to the eye lens was performed by means of MDCT of the temporal bone with a radio-photoluminescence glass dosimeter using a rand phantom. Moreover, we studied only one volunteer (58-year-old male) who had no symptom and was not suspected of having any ear abnormalities with a two scan baseline. Visualization of the major anatomical structures of the temporal bone (the tympanic portion of the facial nerve canal, the body of the incus, stapes superstructures, vestibule etc.) was performed on the volunteer. The average absorbed dose was 6.42 mGy by the OML and 1.59 mGy by the AML, respectively. With regard to visualization of the temporal bone structures, all structures were of equal quality with the two scan baseline. With the AML line, the radiation dose to the eye lens was reduced to 75%. Therefore, the authors recommended an AML for use for MDCT of the temporal bone. In clinical practice, the optimization of scanning factor (kVp, mAs etc.) and the use of the radio-protection should be implemented for radiation dose reduction of the eye lens by MDCT of the temporal bone.

CD25 expression status improves prognostic risk classification in AML independent of established biomarkers: ECOG phase 3 trial, E1900

PubMed Central

Gönen, Mithat; Sun, Zhuoxin; Figueroa, Maria E.; Patel, Jay P.; Abdel-Wahab, Omar; Racevskis, Janis; Ketterling, Rhett P.; Fernandez, Hugo; Rowe, Jacob M.; Tallman, Martin S.; Melnick, Ari; Levine, Ross L.

2012-01-01

We determined the prognostic relevance of CD25 (IL-2 receptor-α) expression in 657 patients (≤ 60 years) with de novo acute myeloid leukemia (AML) treated in the Eastern Cooperative Oncology Group trial, E1900. We identified CD25POS myeloblasts in 87 patients (13%), of whom 92% had intermediate-risk cytogenetics. CD25 expression correlated with expression of stem cell antigen CD123. In multivariate analysis, controlled for prognostic baseline characteristics and daunorubicin dose, CD25POS patients had inferior complete remission rates (P = .0005) and overall survival (P < .0001) compared with CD25NEG cases. In a subset of 396 patients, we integrated CD25 expression with somatic mutation status to determine whether CD25 impacted outcome independent of prognostic mutations. CD25 was positively correlated with internal tandem duplications in FLT3 (FLT3-ITD), DNMT3A, and NPM1 mutations. The adverse prognostic impact of FLT3-ITDPOS AML was restricted to CD25POS patients. CD25 expression improved AML prognostication independent of integrated, cytogenetic and mutational data, such that it reallocated 11% of patients with intermediate-risk disease to the unfavorable-risk group. Gene expression analysis revealed that CD25POS status correlated with the expression of previously reported leukemia stem cell signatures. We conclude that CD25POS status provides prognostic relevance in AML independent of known biomarkers and is correlated with stem cell gene-expression signatures associated with adverse outcome in AML. PMID:22855599

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

PubMed Central

Cesano, Alessandra; Putta, Santosh; Rosen, David B.; Cohen, Aileen C.; Gayko, Urte; Mathi, Kavita; Woronicz, John; Hawtin, Rachael E.; Cripe, Larry; Sun, Zhuoxin; Tallman, Martin S.; Paietta, Elisabeth

2013-01-01

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy. PMID:23431389

Expression of microRNA-181 determines response to treatment with azacitidine and predicts survival in elderly patients with acute myeloid leukaemia.

PubMed

Butrym, Aleksandra; Rybka, Justyna; Baczyńska, Dagmara; Poręba, Rafał; Mazur, Grzegorz; Kuliczkowski, Kazimierz

2016-10-01

MicroRNAs (miRs) are small non-coding RNAs that play important roles in cell differentiation and survival. Abnormal expression of miRs has been demonstrated in numerous types of cancer, including acute myeloid leukaemia (AML). The aim of the present study was to evaluate miR-181 expression at diagnosis and following the completion of chemotherapy in AML patients, with regard to clinical response and outcome, particularly in patients treated with azacitidine. miR-181 expression was analysed using reverse transcription-quantitative polymerase chain reaction in 95 bone marrow specimens from newly diagnosed AML patients and in 20 healthy subjects for comparison. The results revealed upregulated miR-181 expression in the total cohort of AML patients, which was correlated with longer survival. However, in a subset of older AML patients treated with azacitidine, low miR-181 expression at diagnosis was a predictor for complete remission and prolonged survival. The findings indicated that miR-181 has an important role in AML and determines response to azacitidine treatment in older AML patients.

Expression of microRNA-181 determines response to treatment with azacitidine and predicts survival in elderly patients with acute myeloid leukaemia

PubMed Central

Butrym, Aleksandra; Rybka, Justyna; Baczyńska, Dagmara; Poręba, Rafał; Mazur, Grzegorz; Kuliczkowski, Kazimierz

2016-01-01

MicroRNAs (miRs) are small non-coding RNAs that play important roles in cell differentiation and survival. Abnormal expression of miRs has been demonstrated in numerous types of cancer, including acute myeloid leukaemia (AML). The aim of the present study was to evaluate miR-181 expression at diagnosis and following the completion of chemotherapy in AML patients, with regard to clinical response and outcome, particularly in patients treated with azacitidine. miR-181 expression was analysed using reverse transcription-quantitative polymerase chain reaction in 95 bone marrow specimens from newly diagnosed AML patients and in 20 healthy subjects for comparison. The results revealed upregulated miR-181 expression in the total cohort of AML patients, which was correlated with longer survival. However, in a subset of older AML patients treated with azacitidine, low miR-181 expression at diagnosis was a predictor for complete remission and prolonged survival. The findings indicated that miR-181 has an important role in AML and determines response to azacitidine treatment in older AML patients. PMID:27698792

[Value of immunologic phenotyping of acute leukemias in children].

PubMed

Vannier, J P; Bene, M C

1989-10-01

Immunologic typing has demonstrated considerable heterogeneity among the acute leukemias. The most significant recent advance has been development of monoclonal antibody techniques. Some markers identified using these techniques seem to be specific for a given stage of maturation of one lymphoid or myeloid cell line. Most acute lymphoblastic leukemias (ALLs) are malignant proliferations whose differentiation appears to have become 'stuck' at one stage of maturation. Results of immunologic typing correlate well with the other clinical and biological data. For prognostic purposes, several patterns can be identified. Among B line ALLs, four varieties have been differentiated, i.e., CD10 negative ALLs, common ALLs, pre-B ALLs, and B ALLs. T ALLs include a broad spectrum of heterogeneous proliferations whose immunologic classification is made difficult by the large number of phenotypes encountered. Among acute myeloblastic leukemias (AMLs), some highly undifferentiated forms have been recognized, by means of immunologic typing, as originating in one of the myeloid cell lines. However, the nosologic and prognostic significance of these studies is less obvious than in ALLs.

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed Central

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-01-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells. PMID:3855866

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-02-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.

In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering.

PubMed

Chang, Siou Han; Huang, Han Hsiang; Kang, Pei Leun; Wu, Yu Chian; Chang, Ming-Huang; Kuo, Shyh Ming

2017-11-01

Volvox sphere is a biomimetic concept of a natural Volvox, wherein a large outer sphere contains smaller inner spheres, which can encapsulate cells and provide a double-layer three-dimensional environment for culturing cells. This study simultaneously encapsulated rat mesenchymal stem cells (MSCs) and AML12 hepatocytes in volvox spheres and extensively evaluated the effects of various culturing modes on cell functions and fates. The results showed that compared with a static flask culture, MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin (ALB) expression and a 2.5-fold increase in cytokeratin 18 expression in a dynamic bioreactor. Moreover, the restorative effects of volvox spheres encapsulating cells on retrorsine-exposed CCl 4 -induced liver injuries in rats were evaluated. The data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of the new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl 4 . Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury. In this study, we used a volvox sphere, which is a unique design that mimics the natural Volvox, that consists of a large outer sphere that contains smaller inner spheres, which provide a three-dimensional environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in volvox spheres and evaluate two different culture methods, dynamic bioreactor and static culture flask,on the cultured cells. In addition, we aimed to evaluate the restorative effects of volvox spheres encapsulating MSCs and/or AML12 liver cells on rats with retrorsine-exposed CCl 4 -induced liver injuries. The results showed that MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin expression and a 2.5-fold increase in cytokeratin 18 expression ina dynamic bioreactor. Moreover, the data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl 4 . Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The Complexity of Targeting PI3K-Akt-mTOR Signalling in Human Acute Myeloid Leukaemia: The Importance of Leukemic Cell Heterogeneity, Neighbouring Mesenchymal Stem Cells and Immunocompetent Cells.

PubMed

Brenner, Annette K; Andersson Tvedt, Tor Henrik; Bruserud, Øystein

2016-11-11

Therapeutic targeting of PI3K-Akt-mTOR is considered a possible strategy in human acute myeloid leukaemia (AML); the most important rationale being the proapoptotic and antiproliferative effects of direct PI3K/mTOR inhibition observed in experimental studies of human AML cells. However, AML is a heterogeneous disease and these effects caused by direct pathway inhibition in the leukemic cells are observed only for a subset of patients. Furthermore, the final effect of PI3K-Akt-mTOR inhibition is modulated by indirect effects, i.e., treatment effects on AML-supporting non-leukemic bone marrow cells. In this article we focus on the effects of this treatment on mesenchymal stem cells (MSCs) and monocytes/macrophages; both these cell types are parts of the haematopoietic stem cell niches in the bone marrow. MSCs have unique membrane molecule and constitutive cytokine release profiles, and mediate their support through bidirectional crosstalk involving both cell-cell contact and the local cytokine network. It is not known how various forms of PI3K-Akt-mTOR targeting alter the molecular mechanisms of this crosstalk. The effect on monocytes/macrophages is also difficult to predict and depends on the targeted molecule. Thus, further development of PI3K-Akt-mTOR targeting into a clinical strategy requires detailed molecular studies in well-characterized experimental models combined with careful clinical studies, to identify patient subsets that are likely to respond to this treatment.

Daunorubicin and Cytarabine Liposome in Newly Diagnosed Therapy-Related Acute Myeloid Leukemia (AML) or AML With Myelodysplasia-Related Changes.

PubMed

Kim, Miryoung; Williams, Sherry

2018-03-01

To evaluate the efficacy and safety of daunorubicin and cytarabine liposome in older adults with newly diagnosed therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (AML-MRC). A literature search of PubMed and MEDLINE (January 2017 to January 2018) was performed using the terms CPX-351, Vyxeos, daunorubicin and cytarabine liposome, and acute myeloid leukemia. Phase I, II, and III clinical trials evaluating the efficacy and safety of daunorubicin and cytarabine liposome were reviewed with a specific focus on its use in older patients with newly diagnosed AML. All peer-reviewed articles with clinically relevant information were evaluated for inclusion. The phase II trial demonstrated that daunorubicin and cytarabine liposome improved response rates (RR), but there was no difference in event-free survival and overall survival in the overall patient population. However, clinical benefit was most pronounced in secondary AML with an increased RR and survival. The phase III trial illustrated that daunorubicin and cytarabine liposome improved survival and RR with tolerable toxicity compared with standard 7 plus 3 (daunorubicin and cytarabine) in patients 60 to 75 years of age with t-AML or AML-MRC. More patients proceeded to a stem cell transplant, and 30-day and 60-day mortality was lower with daunorubicin and cytarabine liposome. Grade 3 to 5 toxicities were similar between the 2 groups, except daunorubicin and cytarabine liposome had prolonged cytopenia and a higher risk of hemorrhage. Daunorubicin and cytarabine liposome improves RR and survival, with tolerable toxicity in older patients with t-AML or AML-MRC.

Autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells.

PubMed

Morita, Ken; Noura, Mina; Tokushige, Chieko; Maeda, Shintaro; Kiyose, Hiroki; Kashiwazaki, Gengo; Taniguchi, Junichi; Bando, Toshikazu; Yoshida, Kenichi; Ozaki, Toshifumi; Matsuo, Hidemasa; Ogawa, Seishi; Liu, Pu Paul; Nakahata, Tatsutoshi; Sugiyama, Hiroshi; Adachi, Souichi; Kamikubo, Yasuhiko

2017-11-30

Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-β (CBFB) play pivotal roles in leukemogenesis, and inhibition of RUNX1 has now been widely recognized as a novel strategy for anti-leukemic therapies, it has been elusive how leukemic cells could acquire the serious resistance against RUNX1-inhibition therapies and also whether CBFB could participate in this process. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion, and their mutual interaction causes the physiological resistance against chemotherapy for acute myeloid leukemia (AML) cells. Mechanistically, p53 induced by RUNX1 gene silencing directly binds to CBFB promoter and stimulates its transcription as well as its translation, which in turn acts as a platform for the stabilization of RUNX1, thereby creating a compensative RUNX1-p53-CBFB feedback loop. Indeed, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis, and these CBFB expressions were positively correlated to those of p53. Our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted at any sides of this triangle in strategizing anti-leukemia therapies.

Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes.

PubMed

Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O'Connor, Timothy R

2017-01-01

Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient's stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much higher than the 0.65% predicted for such a short time frame, based on ageing results for healthy individuals.

Systematic computation with functional gene-sets among leukemic and hematopoietic stem cells reveals a favorable prognostic signature for acute myeloid leukemia.

PubMed

Yang, Xinan Holly; Li, Meiyi; Wang, Bin; Zhu, Wanqi; Desgardin, Aurelie; Onel, Kenan; de Jong, Jill; Chen, Jianjun; Chen, Luonan; Cunningham, John M

2015-03-24

Genes that regulate stem cell function are suspected to exert adverse effects on prognosis in malignancy. However, diverse cancer stem cell signatures are difficult for physicians to interpret and apply clinically. To connect the transcriptome and stem cell biology, with potential clinical applications, we propose a novel computational "gene-to-function, snapshot-to-dynamics, and biology-to-clinic" framework to uncover core functional gene-sets signatures. This framework incorporates three function-centric gene-set analysis strategies: a meta-analysis of both microarray and RNA-seq data, novel dynamic network mechanism (DNM) identification, and a personalized prognostic indicator analysis. This work uses complex disease acute myeloid leukemia (AML) as a research platform. We introduced an adjustable "soft threshold" to a functional gene-set algorithm and found that two different analysis methods identified distinct gene-set signatures from the same samples. We identified a 30-gene cluster that characterizes leukemic stem cell (LSC)-depleted cells and a 25-gene cluster that characterizes LSC-enriched cells in parallel; both mark favorable-prognosis in AML. Genes within each signature significantly share common biological processes and/or molecular functions (empirical p = 6e-5 and 0.03 respectively). The 25-gene signature reflects the abnormal development of stem cells in AML, such as AURKA over-expression. We subsequently determined that the clinical relevance of both signatures is independent of known clinical risk classifications in 214 patients with cytogenetically normal AML. We successfully validated the prognosis of both signatures in two independent cohorts of 91 and 242 patients respectively (log-rank p < 0.0015 and 0.05; empirical p < 0.015 and 0.08). The proposed algorithms and computational framework will harness systems biology research because they efficiently translate gene-sets (rather than single genes) into biological discoveries about AML and other complex diseases.

A Case of AML Characterized by a Novel t(4;15)(q31;q22) Translocation That Confers a Growth-Stimulatory Response to Retinoid-Based Therapy

PubMed Central

Watts, Justin M.; Pereira, Lutecia; Fan, Yao-Shan; Brown, Geoffrey; Vega, Francisco; Swords, Ronan T.; Zelent, Arthur

2017-01-01

Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all-trans retinoic acid (ATRA) as part of investigational therapy (NCT02273102). The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15)(q31;q22) reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15) primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15) AML revealed that in stark contrast to non-t(4;15) AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15)(q31;q22) reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML. PMID:28696354

NEDD9, an independent good prognostic factor in intermediate-risk acute myeloid leukemia patients

PubMed Central

Pallarès, Victor; Hoyos, Montserrat; Chillón, M. Carmen; Barragán, Eva; Conde, M. Isabel Prieto; Llop, Marta; Céspedes, María Virtudes; Nomdedeu, Josep F.; Brunet, Salut; Sanz, Miguel Ángel; González-Díaz, Marcos; Sierra, Jorge; Casanova, Isolda; Mangues, Ramon

2017-01-01

Intermediate-risk acute myeloid leukemia (IR-AML) is the largest subgroup of AML patients and is highly heterogeneous. Whereas adverse and favourable risk patients have well-established treatment protocols, IR-AML patients have not. It is, therefore, crucial to find novel factors that stratify this subgroup to implement risk-adapted strategies. The CAS (Crk-associated substrate) adaptor protein family regulates cell proliferation, survival, migration and adhesion. Despite its association with metastatic dissemination and prognosis of different solid tumors, the role of these proteins in hematological malignancies has been scarcely evaluated. Nevertheless, previous work has established an important role for the CAS family members NEDD9 or BCAR1 in the migratory and dissemination capacities of myeloid cells. On this basis, we hypothesized that NEDD9 or BCAR1 expression levels could associate with survival in IR-AML patients and become new prognostic markers. To that purpose, we assessed BCAR1 and NEDD9 gene expression in a cohort of 73 adult AML patients validating the results in an independent cohort (n = 206). We have identified NEDD9, but not BCAR1, as a new a marker for longer overall and disease-free survival, and for lower cumulative incidence of relapse. In summary, NEDD9 gene expression is an independent prognostic factor for favourable prognosis in IR-AML patients. PMID:29100287

Monoclonal antibody anti-MPO is useful in recognizing minimally differentiated acute myeloid leukaemia.

PubMed

Praxedes, M K; De Oliveira, L Z; Pereira, W da V; Quintana, I Z; Tabak, D G; De Oliveira, M S

1994-01-01

The enzyme myeloperoxidase (MPO) is the most specific marker of myeloid lineage. The recognition of acute myeloid leukaemia (AML) with minimally differentiation (AML-M0) is established with methods that include myeloid markers CD13/CD33 and detection of MPO in blast cells by immunological techniques or electron microscopy cytochemistry (EM). We have analysed the presence of MPO in leukaemic blast cells by conventional cytochemistry and immunological methods using a monoclonal antibody anti-MPO (CLB-MPO1) in 121 cases of acute leukaemia. The aim of the study was to investigate the sensitivity of this McAb to identify AML-M0, as CD13/CD33 can be expressed in some cases of acute lymphoblastic leukaemia (ALL) and EM cytochemistry is not always available in many laboratories. Anti-MPO was positive in all cases of AML (M1-M5) which were positive by Sudan Black B reaction in similar or higher percentage ratio for each case, although in some of them did not label with CD13/CD33 tested by IF and IPc techniques. Based on the anti-MPO positivity, 5 out of 10 cases called undifferentiated leukaemia (AUL) were reclassified as AML-M0, though 4 cases were CD13/CD33 negative. Furthermore, after analysing the anti-MPO expression among 32 cases of ALL, we had to reclassify four of them as acute biphenotypic leukaemia. We conclude that anti-MPO is a very sensitive and reliable tool in AML diagnosis and has an important role in distinguishing minimally differentiated AML and biphenotypic acute leukaemia from AUL and ALL.

Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance.

PubMed

Vukovic, Milica; Guitart, Amelie V; Sepulveda, Catarina; Villacreces, Arnaud; O'Duibhir, Eoghan; Panagopoulou, Theano I; Ivens, Alasdair; Menendez-Gonzalez, Juan; Iglesias, Juan Manuel; Allen, Lewis; Glykofrydis, Fokion; Subramani, Chithra; Armesilla-Diaz, Alejandro; Post, Annemarie E M; Schaak, Katrin; Gezer, Deniz; So, Chi Wai Eric; Holyoake, Tessa L; Wood, Andrew; O'Carroll, Dónal; Ratcliffe, Peter J; Kranc, Kamil R

2015-12-14

Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance. © 2015 Vukovic et al.

Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance

PubMed Central

Vukovic, Milica; Guitart, Amelie V.; Sepulveda, Catarina; Villacreces, Arnaud; O'Duibhir, Eoghan; Panagopoulou, Theano I.; Ivens, Alasdair; Menendez-Gonzalez, Juan; Iglesias, Juan Manuel; Allen, Lewis; Glykofrydis, Fokion; Subramani, Chithra; Armesilla-Diaz, Alejandro; Post, Annemarie E.M.; Schaak, Katrin; Gezer, Deniz; So, Chi Wai Eric; Holyoake, Tessa L.; Wood, Andrew; O'Carroll, Dónal; Ratcliffe, Peter J.

2015-01-01

Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance. PMID:26642852

PBSCT is associated with poorer survival and increased chronic GvHD than BMT in Japanese paediatric patients with acute leukaemia and an HLA-matched sibling donor.

PubMed

Shinzato, Aki; Tabuchi, Ken; Atsuta, Yoshiko; Inoue, Masami; Inagaki, Jiro; Yabe, Hiromasa; Koh, Katsuyoshi; Kato, Koji; Ohta, Hideaki; Kigasawa, Hisato; Kitoh, Toshiyuki; Ogawa, Atsushi; Takahashi, Yoshiyuki; Sasahara, Yoji; Kato, Shun-Ichi; Adachi, Souichi

2013-09-01

Peripheral blood stem cells (PBSC) may be used as an alternative to bone marrow (BM) for allogeneic transplantation. Since peripheral blood stem cell bank from unrelated volunteer donor has been started in Japan, use of PBSC allografts may be increased. Therefore we surveyed the outcomes of Japanese leukemia children after PBSC and BM transplantation. This retrospective study compared the outcomes of 661 children (0-18 years) with acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML) who received their first allogeneic peripheral blood stem cell transplantation (PBSCT; n = 90) or bone marrow transplantation (BMT; n = 571) from HLA-matched siblings between January 1996 and December 2007. Neutrophil recovery was faster after PBSCT than after BMT (ALL: P < 0.0001; AML: P = 0.0002), as was platelet recovery (ALL: P = 0.0008; AML: P = 0.0848). However, the cumulative incidence of chronic graft-versus-host disease (GvHD) was higher after PBSCT than after BMT (ALL: 26.0% vs. 9.9%, P = 0.0066; AML: 41.6% vs. 11.1%, P < 0.0001). The 5-year disease-free survival (DFS) was lower after PBSCT than after BMT for ALL (40.6% vs. 57.1%, P = 0.0257). The 5-year overall survival (OS) was lower after PBSCT than after BMT for ALL (42.4% vs. 63.7%, P = 0.0032) and AML (49.8% vs. 71.8%, P = 0.0163). Multivariate analysis revealed the use of PBSC was a significant risk factor for DFS and OS. PBSCT and BMT did not differ in relapse rate, acute GvHD for ALL and AML, or in DFS for AML. PBSC allografts in Japanese children engraft faster but are associated with poorer survival and increased chronic GvHD. Copyright © 2013 Wiley Periodicals, Inc.

Single agent and synergistic combinatorial efficacy of first-in-class small molecule imipridone ONC201 in hematological malignancies.

PubMed

Prabhu, Varun V; Talekar, Mala K; Lulla, Amriti R; Kline, C Leah B; Zhou, Lanlan; Hall, Junior; Van den Heuvel, A Pieter J; Dicker, David T; Babar, Jawad; Grupp, Stephan A; Garnett, Mathew J; McDermott, Ultan; Benes, Cyril H; Pu, Jeffrey J; Claxton, David F; Khan, Nadia; Oster, Wolfgang; Allen, Joshua E; El-Deiry, Wafik S

2018-01-01

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1-8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.

High incidence of biallelic point mutations in the Runt domain of the AML1/PEBP2 alpha B gene in Mo acute myeloid leukemia and in myeloid malignancies with acquired trisomy 21.

PubMed

Preudhomme, C; Warot-Loze, D; Roumier, C; Grardel-Duflos, N; Garand, R; Lai, J L; Dastugue, N; Macintyre, E; Denis, C; Bauters, F; Kerckaert, J P; Cosson, A; Fenaux, P

2000-10-15

The AML1 gene, situated in 21q22, is often rearranged in acute leukemias through t(8;21) translocation, t(12;21) translocation, or less often t(3;21) translocation. Recently, point mutations in the Runt domain of the AML1 gene have also been reported in leukemia patients. Observations for mutations of the Runt domain of the AML1 gene in bone marrow cells were made in 300 patients, including 131 with acute myeloid leukemia (AML), 94 with myelodysplastic syndrome (MDS), 28 with blast crisis chronic myeloid leukemia (CML), 3 with atypical CML, 41 with acute lymphoblastic leukemia (ALL), and 3 with essential thrombocythemia (ET). Forty-one of the patients had chromosome 21 abnormalities, including t(8;21) in 6 of the patients with AML, t(12;21) in 8 patients with ALL, acquired trisomy 21 in 17 patients, tetrasomy 21 in 7 patients, and constitutional trisomy 21 (Down syndrome) in 3 patients. A point mutation was found in 14 cases (4.7%), including 9 (22%) of the 41 patients with AML of the Mo type (MoAML) (none of them had detectable chromosome 21 rearrangement) and 5 (38%) of the 13 myeloid malignancies with acquired trisomy 21 (1 M1AML, 2 M2AML, 1 ET, and 1 atypical CML). In at least 8 of 9 mutated cases of MoAML, both AML alleles were mutated: 3 patients had different stop codon mutations of the 2 AML1 alleles, and 5 patients had the same missense or stop codon mutation in both AML1 alleles, which resulted in at least 3 of the patients having duplication of the mutated allele and deletion of the normal residual allele, as shown by FISH analysis and by comparing microsatellite analyses of several chromosome 21 markers on diagnosis and remission samples. In the remaining mutated cases, with acquired trisomy 21, a missense mutation of AML1, which involved 2 of the 3 copies of the AML1 gene, was found. Four of the 7 mutated cases could be reanalyzed in complete remission, and no AML1 mutation was found, showing that mutations were acquired in the leukemic clone. In conclusion, these findings confirm the possibility of mutations of the Runt domain of the AML1 gene in leukemias, mainly in MoAML and in myeloid malignancies with acquired trisomy 21. AML1 mutations, in MoAML, involved both alleles and probably lead to nonfunctional AML1 protein. As AML1 protein regulates the expression of the myeloperoxidase gene, the relationship between AML1 mutations and Mo phenotype in AML will have to be further explored. (Blood. 2000;96:2862-2869)

AMPK/FIS1-Mediated Mitophagy Is Required for Self-Renewal of Human AML Stem Cells.

PubMed

Pei, Shanshan; Minhajuddin, Mohammad; Adane, Biniam; Khan, Nabilah; Stevens, Brett M; Mack, Stephen C; Lai, Sisi; Rich, Jeremy N; Inguva, Anagha; Shannon, Kevin M; Kim, Hyunmin; Tan, Aik-Choon; Myers, Jason R; Ashton, John M; Neff, Tobias; Pollyea, Daniel A; Smith, Clayton A; Jordan, Craig T

2018-06-06

Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation. Copyright © 2018 Elsevier Inc. All rights reserved.

Impaired health-related quality of life in acute myeloid leukemia survivors: a single-center study.

PubMed

Leunis, Annemieke; Redekop, William K; Uyl-de Groot, Carin A; Löwenberg, Bob

2014-09-01

The purpose of this study was to assess the impact of acute myeloid leukemia (AML) and its treatment on health-related quality of life (HRQOL) by comparing the HRQOL of AML survivors with the HRQOL in the general population. Two HRQOL questionnaires (EQ-5D and QLQ-C30) were sent to patients diagnosed with AML between 1999 and 2011 at a single academic hospital and still alive in 2012. HRQOL in AML survivors was compared with general population reference values. Multivariate analysis was used to identify factors associated with HRQOL in AML survivors. Questionnaires were returned by 92 of the 103 patients (89%). AML survivors reported significantly worse functioning, more fatigue, pain, dyspnea, appetite loss, and financial difficulties and lower EQ-VAS scores than the general population (P < 0.05). Impaired HRQOL in AML survivors was mainly found in survivors without a paid job. Other factors associated with a poor HRQOL were allogeneic hematopoietic stem cell transplantation and the absence of social support. This single-center study showed that the HRQOL in AML survivors is worse than the HRQOL in the general population. HRQOL in these patients can be improved by adequately treating and preventing fatigue, pain, dyspnea, and appetite loss. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rapid expansion of preexisting nonleukemic hematopoietic clones frequently follows induction therapy for de novo AML.

PubMed

Wong, Terrence N; Miller, Christopher A; Klco, Jeffery M; Petti, Allegra; Demeter, Ryan; Helton, Nichole M; Li, Tiandao; Fulton, Robert S; Heath, Sharon E; Mardis, Elaine R; Westervelt, Peter; DiPersio, John F; Walter, Matthew J; Welch, John S; Graubert, Timothy A; Wilson, Richard K; Ley, Timothy J; Link, Daniel C

2016-02-18

There is interest in using leukemia-gene panels and next-generation sequencing to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy influences the clonal evolution of a patient's nonleukemic hematopoietic population. Here, we report that 5 of 15 patients with genetic clearance of their founding AML clone after induction chemotherapy had a concomitant expansion of a hematopoietic population unrelated to the initial AML. These populations frequently harbored somatic mutations in genes recurrently mutated in AML or myelodysplastic syndromes and were detectable at very low frequencies at the time of AML diagnosis. These results suggest that nonleukemic hematopoietic stem and progenitor cells, harboring specific aging-acquired mutations, may have a competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these "rising" clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy. © 2016 by The American Society of Hematology.

Azacitidine and lenalidomide as an alternative treatment for refractory acute myeloid leukemia: a case report.

PubMed

Todaro, Juliana; Bollmann, Patrícia Weinschenker; Rother, Edna Terezinha; del Giglio, Auro

2015-01-01

Refractory acute myeloid leukemia (AML) is a difficult disease to control with second or third-line chemotherapy regimens. In this report, we describe using azacitidine in combination with lenalidomide as salvage therapy. 52-year-old female was diagnosed with refractory AML and high-risk cytogenetics: complex monosomal karyotype consisting of t (3, 3) in association with monosomy 7 and del 5q. Morphological remission associated with maintenance of the cytogenetic abnormality of chromosome 3 and disappearance of the abnormalities relating to chromosomes 5 and 7 was achieved after three cycles of combination therapy with azacitidine and lenalidomide. Azacitidine plus lenalidomide can be a therapeutic option for patients with refractory AML, as illustrated in this case.

Low CLL-1 Expression Is a Novel Adverse Predictor in 123 Patients with De Novo CD34+ Acute Myeloid Leukemia.

PubMed

Wang, Yan-Yu; Chen, Wen-Lian; Weng, Xiang-Qin; Sheng, Yan; Wu, Jing; Hao, Jie; Liu, Zhan-Yun; Zhu, Yong-Mei; Chen, Bing; Xiong, Shu-Min; Chen, Yu; Chen, Qiu-Sheng; Sun, Hui-Ping; Li, Jun-Min; Wang, Jin

2017-10-15

Recent reports state that C-type lectin-like molecule-1 (CLL-1) in acute myeloid leukemia (AML) is expressed primarily on myeloid cells, but there is still no investigation about its prognostic significance on leukemic blast compartment. Hence, this study aimed to evaluate the prognostic value of CLL-1 in 123 patients with de novo CD34 + Non-M3 AML. Multiparameter flow cytometry was used to assess the expression of CLL-1 on immature compartment in AML and control groups. We found that CLL-1 expression level on blast compartment was closely linked to clinical characteristics, treatment response, and survival outcome of patients. Decreased expression of CLL-1 was observed on immature compartment from AML patients as compared with controls (62.6% vs. 86.5%, P < 0.05). Logistic model exhibited that CLL-1 low independently predicted low complete remission rate with an odds ratio of 4.57 (2.53-6.61, P < 0.05). Additionally, CLL-1 expression level at diagnosis was inversely correlated to the residual blast cells (residual leukemia cell) after induction chemotherapy (r = -0.423, P < 0.05). Furthermore, multivariate Cox regression model demonstrated that CLL-1 low was still an independent adverse predictor (P < 0.05 for event-free survival, P < 0.05 for overall survival). Notably, CLL-1 low was able to discriminate poor survival patients from intermediate- and favorable-risk groups. Taken together, CLL-1 is a novel prognostic predictor that could be exploited to supplement the current AML prognostic risk stratification system, and potentially optimize the clinical management of AML.

Elevated MLF1 expression correlates with malignant progression from myelodysplastic syndrome.

PubMed

Matsumoto, N; Yoneda-Kato, N; Iguchi, T; Kishimoto, Y; Kyo, T; Sawada, H; Tatsumi, E; Fukuhara, S

2000-10-01

MLF1 is a novel protein identified as the NPM-MLF1 chimeric protein produced by a t(3;5)(q25.1;q34) chromosomal translocation, which is associated with myelodysplastic syndrome (MDS), often prior to acute myeloid leukemia (AML), except for M3. The clinical features of t(3;5)-positive myeloid disorders suggest that this chimeric protein is involved in dysregulation of progenitor cells with the capability to differentiate into multiple lineages. So far, involvement of wild-type MLF1 in hematopoiesis or in leukemogenesis has not been fully investigated. In the present study, 65 patients with AML and 44 patients with MDS were tested for the expression of MLF1 using the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. A significantly higher level of MLF1 expression (ratio of MLF1/beta-actin mRNA >0.4) was readily detected in seven of 65 patients with de novo AML, three of 12 with post-MDS AML and seven of 44 with MDS, but not in any patients with ALL (n = 18). According to the FAB classification, high levels of MLF1 were found in patients with relatively immature subtypes of AML (M1, M2, M6 and M7) and high risk MDS (RAEB and RAEB-T). These findings indicate that the pattern of MLF1 expression is identical to the clinical morphology appearing in the t(3;5)-positive myeloid disorders and is correlated to the MDS-associated AML and transformation phase of MDS in t(3;5)-negative myeloid disorders. A CD34+ population of normal bone marrow cells preferentially expressed MLF1 with obviously decreasing levels of expression during maturation. Therefore, MLF1 normally functions in multi-potent progenitor cells and its dysregulation may take part in leukemogenesis from MDS.

Contrasting in vitro effects for the combination of fludarabine, cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (FLAG) compared with daunorubicin and Ara-C in P-glycoprotein-positive and P-glycoprotein-negative acute myeloblastic leukaemia.

PubMed

Higashi, Y; Turzanski, J; Pallis, M; Russell, N H

2000-11-01

It has been suggested that the FLAG remission induction regimen comprising fludarabine (F-ara), cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (G-CSF) may be capable of overcoming P-glycoprotein (P-gp)-related multidrug resistance (MDR) in patients with acute myeloblastic leukaemia (AML). We have investigated the in vitro response of P-gp-positive and -negative AML clones to FLAG and compared this with their response to treatment with Ara-C and daunorubicin (DNR). Twenty-four cryopreserved samples from patients with AML were studied using a flow cytometric technique for the enumeration of viable (7-amino actinomycin D negative) cells. Samples consisted of 12 P-gp-positive and 12 P-gp-negative cases, as measured by the MRK16 antibody. The results were analysed by calculating the comparative drug resistance (CDR), i.e. the percentage cell death caused by Ara-C + DNR subtracted from the percentage cell death, caused by FLAG after 48 h incubation in suspension culture. P-gp-positive clones were shown to have a significantly higher CDR than P-gp-negative clones (P = 0. 001). Furthermore, a significant positive correlation (r2 = 0.40, P < 0.01) was found between P-gp protein expression and CDR. However, P-gp function, measured using cyclosporin modulation of rhodamine 123 (R123) uptake, was not associated with the CDR, demonstrating that there are other properties of P-gp, besides its role in drug efflux, that modulate the responsiveness of AML blasts to chemotherapy. These results are consistent with a potential benefit for FLAG in P-gp-positive AML, but not P-gp-negative AML, compared with standard anthracycline and Ara-C therapy.

Concise review: preleukemic stem cells: molecular biology and clinical implications of the precursors to leukemia stem cells.

PubMed

Pandolfi, Ashley; Barreyro, Laura; Steidl, Ulrich

2013-02-01

Recent experimental evidence has shown that acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) arise from transformed immature hematopoietic cells following the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells and committed progenitors. The series of transforming events initially gives rise to preleukemic stem cells (pre-LSC), preceding the formation of fully transformed leukemia stem cells (LSC). Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS. The therapeutic elimination of all LSC, as well as pre-LSC, which provide a silent reservoir for the re-formation of LSC, will be essential for achieving lasting cures. Conventional sequencing and next-generation genome sequencing have allowed us to describe many of the recurrent mutations in the bulk cell populations in AML and MDS, and recent work has also focused on identifying the initial molecular changes contributing to leukemogenesis. Here we review recent and ongoing advances in understanding the roles of pre-LSC, and the aberrations that lead to pre-LSC formation and subsequent LSC transformation.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

PubMed

Klöß, Stephan; Oberschmidt, Olaf; Morgan, Michael; Dahlke, Julia; Arseniev, Lubomir; Huppert, Volker; Granzin, Markus; Gardlowski, Tanja; Matthies, Nadine; Soltenborn, Stephanie; Schambach, Axel; Koehl, Ulrike

2017-10-01

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56 + CD3 - ) was carried out with the CliniMACS Prodigy ® in a single process, starting with approximately 1.2 × 10 9 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 6 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO ™ 10, CellGro ® , TexMACS ™ , and NK MACS ® ). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56 + CD3 - target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56 dim CD16 pos and CD56 bright CD16 dim&neg NK subsets on day 14 with cells cultivated in NK MACS ® media. Moreover, effector cell expansion in manually performed experiments with NK MACS ® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123 pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Dendritic cells (DCs) can be successfully generated from leukemic blasts in individual patients with AML or MDS: an evaluation of different methods.

PubMed

Kremser, Andreas; Dressig, Julia; Grabrucker, Christine; Liepert, Anja; Kroell, Tanja; Scholl, Nina; Schmid, Christoph; Tischer, Johanna; Kufner, Stefanie; Salih, Helmut; Kolb, Hans Jochem; Schmetzer, Helga

2010-01-01

Myeloid-leukemic cells (AML, MDS, CML) can be differentiated to leukemia-derived dendritic cell [DC (DCleu)] potentially presenting the whole leukemic antigen repertoire without knowledge of distinct leukemia antigens and are regarded as promising candidates for a vaccination strategy. We studied the capability of 6 serum-free DC culture methods, chosen according to different mechanisms, to induce DC differentiation in 137 cases of AML and 52 cases of MDS. DC-stimulating substances were cytokines ("standard-medium", "MCM-Mimic", "cytokine-method"), bacterial lysates ("Picibanil"), double-stranded RNA ["Poly (I:C)"] or a cytokine bypass method ("Ca-ionophore"). The quality/quantity of DC generated was estimated by flow cytometry studying (co) expressions of "DC"antigens, costimulatory, maturation, and blast-antigens. Comparing these methods on average 15% to 32% DC, depending on methods used, could be obtained from blast-containing mononuclear cells (MNC) in AML/MDS cases with a DC viability of more than 60%. In all, 39% to 64% of these DC were mature; 31% to 52% of leukemic blasts could be converted to DCleu and DCleu-proportions in the suspension were 2% to 70% (13%). Average results of all culture methods tested were comparable, however not every given case of AML could be differentiated to DC with 1 selected method. However performing a pre-analysis with 3 DC-generating methods (MCM-Mimic, Picibanil, Ca-ionophore) we could generate DC in any given case. Functional analyses provided proof, that DC primed T cells to antileukemia-directed cytotoxic cells, although an anti-leukemic reaction was not achieved in every case. In summary our data show that a successful, quantitative DC/DCleu generation is possible with the best of 3 previously tested methods in any given case. Reasons for different functional behaviors of DC-primed T cells must be evaluated to design a practicable DC-based vaccination strategy.

Metal Occupancy of Zinc Finger Motifs as Determinants for Zn2+-Mediated Chemosensitization of Prostate Cancer Cells

DTIC Science & Technology

2013-12-01

leukemia (AML) and glioblastoma ( GBM ). Our laboratory is interested in the potential of F10 for improved treatment of prostate cancer based upon...displays strong anti-cancer activity and minimal systemic toxicity in pre-clinical models of AML and GBM and that in previous studies demonstrated...of the low toxicity and strong anti-cancer activity of F10 in animal models of AML and GBM this combination is likely to be effective and well

Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy

PubMed Central

Meyer, Mona; Rübsamen, Daniela; Slany, Robert; Illmer, Thomas; Stabla, Kathleen; Roth, Petra; Stiewe, Thorsten

2009-01-01

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells. PMID:19890398

Risk factors for bacteremia and central line-associated blood stream infections in children with acute myelogenous leukemia: A single-institution report.

PubMed

Rogers, Ashley E J; Eisenman, Kristen M; Dolan, Susan A; Belderson, Kristin M; Zauche, Jocelyn R; Tong, Suhong; Gralla, Jane; Hilden, Joanne M; Wang, Michael; Maloney, Kelly W; Dominguez, Samuel R

2017-03-01

Central line-associated blood stream infections (CLABSIs) are a source of high morbidity and mortality in children with acute myelogenous leukemia (AML). To understand the epidemiology and risk factors associated with the development of CLABSI in children with AML. We retrospectively reviewed all patients with AML over a 5-year period between 2007 and 2011 at the Children's Hospital Colorado. Cases and controls were classified on the basis of the presence of a CLABSI as defined by the National Healthcare Safety Network. Of 40 patients in the study, 25 (62.5%) developed at least one CLABSI during therapy. The majority of CLABSIs were due to oral or gastrointestinal organisms (83.0%). Skin organisms accounted for 8.5%. In a multivariable analysis, the strongest risk factors associated with CLABSI were diarrhea (odds ratio [OR] 6.7, 95% confidence interval [CI] 1.6-28.7), receipt of blood products in the preceding 4-7 days (OR 10.0, 95%CI 3.2-31.0), not receiving antibiotics (OR 8.3, 95%CI 2.8-25.0), and chemotherapy cycle (OR 3.5, 95%CI 1.4-8.9). CLABSIs led to increased morbidity, with 13 cases (32.5%) versus two controls (1.9%) requiring transfer to the pediatric intensive care unit (P < 0.001). Three (7.5%) of 40 CLABSI events resulted in or contributed to death. Intensified line care efforts cannot eliminate all CLABSIs in the patients with AML. Exploring the role of mucosal barrier breakdown and/or the use of antibiotic prophylaxis may be effective strategies for further prevention of CLABSIs, supporting ongoing trials in this patient population. © 2016 Wiley Periodicals, Inc.

Comprehensive discovery of noncoding RNAs in acute myeloid leukemia cell transcriptomes.

PubMed

Zhang, Jin; Griffith, Malachi; Miller, Christopher A; Griffith, Obi L; Spencer, David H; Walker, Jason R; Magrini, Vincent; McGrath, Sean D; Ly, Amy; Helton, Nichole M; Trissal, Maria; Link, Daniel C; Dang, Ha X; Larson, David E; Kulkarni, Shashikant; Cordes, Matthew G; Fronick, Catrina C; Fulton, Robert S; Klco, Jeffery M; Mardis, Elaine R; Ley, Timothy J; Wilson, Richard K; Maher, Christopher A

2017-11-01

To detect diverse and novel RNA species comprehensively, we compared deep small RNA and RNA sequencing (RNA-seq) methods applied to a primary acute myeloid leukemia (AML) sample. We were able to discover previously unannotated small RNAs using deep sequencing of a library method using broader insert size selection. We analyzed the long noncoding RNA (lncRNA) landscape in AML by comparing deep sequencing from multiple RNA-seq library construction methods for the sample that we studied and then integrating RNA-seq data from 179 AML cases. This identified lncRNAs that are completely novel, differentially expressed, and associated with specific AML subtypes. Our study revealed the complexity of the noncoding RNA transcriptome through a combined strategy of strand-specific small RNA and total RNA-seq. This dataset will serve as an invaluable resource for future RNA-based analyses. Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Evidence for a pre-existing telomere deficit in non-clonal hematopoietic stem cells in patients with acute myeloid leukemia.

PubMed

Ventura Ferreira, Mónica S; Crysandt, Martina; Ziegler, Patrick; Hummel, Sebastian; Wilop, Stefan; Kirschner, Martin; Schemionek, Mirle; Jost, Edgar; Wagner, Wolfgang; Brümmendorf, Tim H; Beier, Fabian

2017-09-01

Telomere shortening represents an established mechanism connecting aging and cancer development. We sequentially analyzed telomere length (TL) of 49 acute myeloid leukemia (AML) patients at diagnosis (n = 24), once they achieved complete cytological remission (CCR) and/or during refractory disease or relapse and after 1-year follow-up, with all patients having at least two sequential samples. TL was analyzed by monochrome multiplex quantitative polymerase chain reaction. We have observed substantially shortened TL in the cells of patients at diagnosis compared to age-adjusted controls. In patients reaching CCR after chemotherapy, telomere shortening was less pronounced than in persistence or relapse but still significantly shortened compared to controls. We estimate patients harboring approximately 20 years of premature telomere loss compared to healthy aged-matched subjects at the time of AML onset. Our data indicate a pre-existing telomere deficit in non-clonal hematopoiesis of AML patients providing a link between age and AML development.

Enhancement of the cytotoxic effects of Cytarabine in synergism with Hesperidine and Silibinin in Acute Myeloid Leukemia: An in-vitro approach.

PubMed

Desai, Urja N; Shah, Krupa P; Mirza, Sheefa H; Panchal, Darshil K; Parikh, Sonia K; Rawal, Rakesh M

2015-01-01

Acute Myeloid Leukemia (AML) therapy continues to be a daunting challenge. Cytosine Arabinoside (Ara-C) is widely used to treat hematological malignancy in humans, but often becomes ineffective because of increased resistance to the drug which may lead to a worse prognosis. Therefore new strategies are needed to understand the mechanism responsible for drug resistance and to develop new therapies to overcome it. Research evidence based on natural compounds used alone or in combination with current chemotherapeutic agents proved their efficacy to treat and prevent cancer. Hesperidin and Silibinin displayed anti-cancer activity against various types of cancers and cell lines and can be used in combination with Cytarabine with the aim to increase cytotoxicy profile and reduction in drug resistance. Experimental Work: Primary cells obtained from AML patient's bone marrow were used to develop in-vitro model and further exposed to various concentration of Cytarabine (10 nM-5000 nM), Hesperidin (0.5 μM-100 μM) and Silibinin (0.5 μM-100 μM) alone and in combination with Cytarabine (Hesperidin-25 μM, Silibinin10 μM) to check cytotoxicity using MTT assay. Synergistic effect was evaluated by Combination Index method. In-vitro study of Hesperidin and Silibinin indicated their cytotoxicity at IC 50 value 50.12 μM and 16.2 μM, respectively. Combination Index study revealed Hesperidin and Silibinin both showed synergistic potential and decreased the IC 50 value of Cytarabine by ~5.9 and ~4.5 folds, respectively. Both natural compounds showed potential anti-leukemic activity hence may be used for AML therapy alone or in combination with other chemotherapeutic agents.

Combined cistrome and transcriptome analysis of SKI in AML cells identifies SKI as a co-repressor for RUNX1

PubMed Central

Feld, Christine; Sahu, Peeyush; Frech, Miriam; Finkernagel, Florian; Nist, Andrea; Stiewe, Thorsten; Bauer, Uta-Maria; Neubauer, Andreas

2018-01-01

Abstract SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here, we use ChIP-seq and RNA-seq analysis to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which >40% harbor SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI contributes to a myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets upon SKI deletion. RUNX1 ChIP-seq displays that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time the predominant co-repressive function of SKI in AML cells on a genome-wide scale and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression. PMID:29471413

Acute myeloid leukemia in children: Current status and future directions.

PubMed

Taga, Takashi; Tomizawa, Daisuke; Takahashi, Hiroyuki; Adachi, Souichi

2016-02-01

Acute myeloid leukemia (AML) accounts for 25% of pediatric leukemia and affects approximately 180 patients annually in Japan. The treatment outcome for pediatric AML has improved through advances in chemotherapy, hematopoietic stem cell transplantation (HSCT), supportive care, and optimal risk stratification. Currently, clinical pediatric AML studies are conducted separately according to the AML subtypes: de novo AML, acute promyelocytic leukemia (APL), and myeloid leukemia with Down syndrome (ML-DS). Children with de novo AML are treated mainly with anthracyclines and cytarabine, in some cases with HSCT, and the overall survival (OS) rate now approaches 70%. Children with APL are treated with an all-trans retinoic acid (ATRA)-combined regimen with an 80-90% OS. Children with ML-DS are treated with a less intensive regimen compared with non-DS patients, and the OS is approximately 80%. HSCT in first remission is restricted to children with high-risk de novo AML only. To further improve outcomes, it will be necessary to combine more accurate risk stratification strategies using molecular genetic analysis with assessment of minimum residual disease, and the introduction of new drugs in international collaborative clinical trials. © 2015 Japan Pediatric Society.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

PubMed

Kadono, Moe; Kanai, Akinori; Nagamachi, Akiko; Shinriki, Satoru; Kawata, Jin; Iwato, Koji; Kyo, Taiichi; Oshima, Kumi; Yokoyama, Akihiko; Kawamura, Takeshi; Nagase, Reina; Inoue, Daichi; Kitamura, Toshio; Inaba, Toshiya; Ichinohe, Tatsuo; Matsui, Hirotaka

2016-08-01

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to cause AML. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

PubMed

Thokala, Radhika; Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E; Laskowski, Tamara; McNamara, George; Cooper, Laurence J N

2016-01-01

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

Development and in vitro evaluations of new decitabine nanocarriers for the treatment of acute myeloid leukemia

PubMed Central

Briot, Thomas; Roger, Emilie; Lautram, Nolwenn; Verger, Alexis; Clavreul, Anne; Lagarce, Frederic

2017-01-01

Decitabine is a hydrophilic drug that acts by hypomethylating DNA. Decitabine is used in Europe for the treatment of acute myeloid leukemia (AML) in patients aged ≥65 years. However, it can only be administered intravenously due to very low oral bioavailability and a large distribution volume. Oral administration would allow outpatient treatment, improving quality of life and reducing treatment costs. The present study proposes to develop lipid nanocapsules (LNCs), originally designed for lipophilic drugs, to encapsulate decitabine. Two different formulations of LNCs were designed: LNCs based on a high proportion of Transcutol® HP (THP-LNCs) and LNCs associated with a mixture of Transcutol® HP and Tween® 80 (THP-T80-LNCs). The second formulation had a diameter of 26.5±0.5 nm, high encapsulation efficiency (>85%), and a drug payload of 472±64 µg/mL. Decitabine-loaded THP-T80-LNC cytotoxicity was evaluated on two AML cell lines depending on their decitabine resistance: HEL (not resistant) and HL-60 (resistant). The permeability of decitabine-loaded THP-T80-LNCs was also evaluated on Caco-2 cell monolayers. Decitabine cytotoxicity against HEL and HL-60 was higher when decitabine was loaded in THP-T80-LNCs than when free. Apparent permeability on Caco-2 cell monolayers was also increased, suggesting a potentially useful formulation to increase the oral bioavailability of decitabine. PMID:29200853

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model

PubMed Central

Cabezas-Wallscheid, Nina; Eichwald, Victoria; de Graaf, Jos; Löwer, Martin; Lehr, Hans-Anton; Kreft, Andreas; Eshkind, Leonid; Hildebrandt, Andreas; Abassi, Yasmin; Heck, Rosario; Dehof, Anna Katharina; Ohngemach, Svetlana; Sprengel, Rolf; Wörtge, Simone; Schmitt, Steffen; Lotz, Johannes; Meyer, Claudius; Kindler, Thomas; Zhang, Dong-Er; Kaina, Bernd; Castle, John C; Trumpp, Andreas; Sahin, Ugur; Bockamp, Ernesto

2013-01-01

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option. PMID:24124051

AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia.

PubMed

Zhou, Fengbiao; Liu, Yi; Rohde, Christian; Pauli, Cornelius; Gerloff, Dennis; Köhn, Marcel; Misiak, Danny; Bäumer, Nicole; Cui, Chunhong; Göllner, Stefanie; Oellerich, Thomas; Serve, Hubert; Garcia-Cuellar, Maria-Paz; Slany, Robert; Maciejewski, Jaroslaw P; Przychodzen, Bartlomiej; Seliger, Barbara; Klein, Hans-Ulrich; Bartenhagen, Christoph; Berdel, Wolfgang E; Dugas, Martin; Taketo, Makoto Mark; Farouq, Daneyal; Schwartz, Schraga; Regev, Aviv; Hébert, Josée; Sauvageau, Guy; Pabst, Caroline; Hüttelmaier, Stefan; Müller-Tidow, Carsten

2017-07-01

Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2'-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2'-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis.

Expression profiling of snoRNAs in normal hematopoiesis and AML

PubMed Central

Warner, Wayne A.; Spencer, David H.; Trissal, Maria; White, Brian S.; Helton, Nichole; Ley, Timothy J.

2018-01-01

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis. PMID:29365324

Venetoclax and low-dose cytarabine induced complete remission in a patient with high-risk acute myeloid leukemia: a case report.

PubMed

Liu, Bingshan; Narurkar, Roshni; Hanmantgad, Madhura; Zafar, Wahib; Song, Yongping; Liu, Delong

2018-05-21

Conventional combination therapies have not resulted in considerable progress in the treatment of acute myeloid leukemia (AML). Elderly patients with AML and poor risk factors have grave prognosis. Midostaurin has been recently approved for the treatment of FLT-3-mutated AML. Venetoclax, a BCL-2 inhibitor, has been approved for the treatment of relapsed and/or refractory chronic lymphoid leukemia. Clinical trials on applying venetoclax in combination with cytarabine and other agents to treat various hematological malignancies are currently underway. Here, we present a case of a male patient with poor performance status and who developed AML following allogeneic hematopoietic stem cell transplant for high-risk myelodysplasia. The patient with high risk AML achieved complete response to the combined treatment regimen of low-dose cytarabine and venetoclax. Furthermore, we reviewed current clinical trials on the use of venetoclax for hematological malignancies.

Enhanced Fructose Utilization Mediated by SLC2A5 Is a Unique Metabolic Feature of Acute Myeloid Leukemia with Therapeutic Potential.

PubMed

Chen, Wen-Lian; Wang, Yue-Ying; Zhao, Aihua; Xia, Li; Xie, Guoxiang; Su, Mingming; Zhao, Linjing; Liu, Jiajian; Qu, Chun; Wei, Runmin; Rajani, Cynthia; Ni, Yan; Cheng, Zhen; Chen, Zhu; Chen, Sai-Juan; Jia, Wei

2016-11-14

Rapidly proliferating leukemic progenitor cells consume substantial glucose, which may lead to glucose insufficiency in bone marrow. We show that acute myeloid leukemia (AML) cells are prone to fructose utilization with an upregulated fructose transporter GLUT5, which compensates for glucose deficiency. Notably, AML patients with upregulated transcription of the GLUT5-encoding gene SLC2A5 or increased fructose utilization have poor outcomes. Pharmacological blockage of fructose uptake ameliorates leukemic phenotypes and potentiates the cytotoxicity of the antileukemic agent, Ara-C. In conclusion, this study highlights enhanced fructose utilization as a metabolic feature of AML and a potential therapeutic target. Copyright © 2016 Elsevier Inc. All rights reserved.

Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML

PubMed Central

Carter, Bing Z.; Mak, Duncan H.; Schober, Wendy D.; Koller, Erich; Pinilla, Clemencia; Vassilev, Lyubomir T.; Reed, John C.

2010-01-01

Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a–induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic. PMID:19897582

Is Acute Myeloid Leukemia a Liquid Tumor?

PubMed Central

Ohanian, Maro; Faderl, Stefan; Ravandi, Farhad; Pemmaraju, Naveen; Garcia-Manero, Guillermo; Cortes, Jorge; Estrov, Zeev

2014-01-01

Extramedullary manifestations of acute myeloid leukemia (AML) were described as early as the 19th century. However, the incidence, clinical significance, and pathobiology of extramedullary AML remain ill defined. We reviewed case reports, retrospective case series, pilot studies, and imaging studies of extramedullary leukemia (EML) to determine its frequency, characteristics, clinical presentation, and significance. EML precedes or accompanies development of AML and occurs during or following treatment, even during remission. Although imaging studies are rarely conducted and the true incidence of EML has yet to be verified, authors have reported several estimates based on retrospective and autopsy studies. The incidence of EML in patients with AML of all ages is estimated to be about 9% and EML in children with AML was detected in 40% of patients at diagnosis. The combination of positron emission tomography and computed tomography were the most sensitive and reliable techniques of detecting and monitoring EML. Based on our literature review, the frequency of EML is likely underreported. The well-documented nature of EML in AML patients suggests that AML can manifest as a solid tumor. The extent to which EML accompanies AML and whether EML is derived from bone marrow are unknown. Furthermore, questions remain regarding the role of the microenvironment, which may or may not facilitate the survival and proliferation of EML, and the implications of these interactions with regard to minimal residual disease, tumor cell quiescence, and relapse. Therefore, prospective studies of detection and characterization of EML in AML patients are warranted. PMID:23280377

Is acute myeloid leukemia a liquid tumor?

PubMed

Ohanian, Maro; Faderl, Stefan; Ravandi, Farhad; Pemmaraju, Naveen; Garcia-Manero, Guillermo; Cortes, Jorge; Estrov, Zeev

2013-08-01

Extramedullary manifestations of acute myeloid leukemia (AML) were described as early as the 19th century. However, the incidence, clinical significance and pathobiology of extramedullary AML remain ill defined. We reviewed case reports, retrospective case series, pilot studies and imaging studies of extramedullary leukemia (EML) to determine its frequency, characteristics, clinical presentation and significance. EML precedes or accompanies development of AML and occurs during or following treatment, even during remission. Although imaging studies are rarely conducted and the true incidence of EML has yet to be verified, authors have reported several estimates based on retrospective and autopsy studies. The incidence of EML in patients with AML of all ages is estimated to be about 9% and EML in children with AML was detected in 40% of patients at diagnosis. The combination of positron emission tomography and computed tomography were the most sensitive and reliable techniques of detecting and monitoring EML. Based on our literature review, the frequency of EML is likely underreported. The well-documented nature of EML in patients with AML suggests that AML can manifest as a solid tumor. The extent to which EML accompanies AML and whether EML is derived from bone marrow are unknown. Furthermore, questions remain regarding the role of the microenvironment, which may or may not facilitate the survival and proliferation of EML, and the implications of these interactions with regard to minimal residual disease, tumor cell quiescence and relapse. Therefore, prospective studies of detection and characterization of EML in patients with AML are warranted. Copyright © 2013 UICC.

Adult Acute Myeloid Leukemia Treatment (PDQ®)—Health Professional Version

Cancer.gov

Acute myeloid leukemia (AML; also called acute myelogenous leukemia, acute nonlymphocytic leukemia) treatment advances have resulted in substantially improved CR rates. Cytogenetic analysis helps predict outcomes of treatment which includes chemotherapy, radiation, and stem cell transplant. Get detailed information about AML in this clinician summary.

Discordant detection of monosomy 7 by GTG-banding and FISH in a patient with Shwachman-Diamond syndrome without evidence of myelodysplastic syndrome or acute myelogenous leukemia.

PubMed

Sokolic, R A; Ferguson, W; Mark, H F

1999-12-01

The myelodysplastic syndromes (MDS) are a group of hematologic disorders commonly affecting elderly persons and often leading to acute myelogenous leukemia (AML). Although rare in children, when MDS does occur, it is frequently part of a congenital disorder such as Shwachman-Diamond syndrome (SDS). Monosomy 7 and/or deletion of part or all of 7q are poor prognostic signs in MDS and AML, although the pathophysiologic relationship between this finding and MDS or AML is unclear. Shwachman-Diamond syndrome is an inherited illness characterized by exocrine pancreatic insufficiency and by congenital neutropenia. Patients with SDS are at increased risk of developing myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Because monosomy 7 is a poor prognostic sign in MDS and AML, establishing its presence is important. However, different methods of detection of monosomy 7 may lead to different results in some patients. We present the case of a 10-year-old girl known to have SDS, who had a bone marrow aspiration and biopsy done to rule out MDS and AML. By light microscopy, the patient's bone marrow was unremarkable. GTG-banding showed the following karyotype: 45,XX,-C[3]/47,XX,+C[1]/46,XX[45]. Fluorescence in situ hybridization (FISH) was performed with a chromosome 7-specific alpha-satellite probe (D7Z1). Almost all (373 of 376) cells exhibited only one chromosome 7 signal. A second marrow aspiration done 6 months later showed an essentially normal karyotype by GTG-banding. Fluorescence in situ hybridization with the same chromosome 7 probe showed 230 of 250 cells to be monosomic for chromosome 7. A whole chromosome 7 painting probe demonstrated disomy for chromosome 7 in 90 of 90 cells; however, subtle heteromorphism in the centromeric regions of the 2 copies of chromosome 7 was noted in some cells. This case demonstrates that FISH and GTG-banding can give discordant results, that the two should be viewed as complementary technologies, and that both have a place in a full karyotypic analysis. Furthermore, this case demonstrates for the first time that heteromorphism and/or subtle structural abnormalities of chromosome 7, previously associated with MDS and AML, can exist without clinical or morphologic signs of these illnesses. It will be of interest to further study the relationship, if any, between SDS and various structural abnormalities of chromosome 7 in MDS and AML, and to elucidate the molecular mechanisms of pathogenesis, physiology, and treatment of these disorders.

FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia

PubMed Central

Kim, Hyung-Gyoon; Kojima, Kyoko; Swindle, C. Scott; Cotta, Claudiu V.; Huo, Yongliang; Reddy, Vishnu

2008-01-01

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML. PMID:17967943

Allogeneic Stem Cell Transplantation Improves Survival in Patients with Acute Myeloid Leukemia Characterized by a High Allelic Ratio of Mutant FLT3-ITD.

PubMed

Ho, Anthony D; Schetelig, Johannes; Bochtler, Tilmann; Schaich, Markus; Schäfer-Eckart, Kerstin; Hänel, Mathias; Rösler, Wolf; Einsele, Hermann; Kaufmann, Martin; Serve, Hubert; Berdel, Wolfgang E; Stelljes, Matthias; Mayer, Jiri; Reichle, Albrecht; Baldus, Claudia D; Schmitz, Norbert; Kramer, Michael; Röllig, Christoph; Bornhäuser, Martin; Thiede, Christian; Ehninger, Gerhard

2016-03-01

Allogeneic hematopoietic cell transplantation (alloHCT) as a postremission therapy in patients with FLT3-ITD-positive intermediate-risk acute myeloid leukemia (AML) remains controversial. FLT3-ITD mutations are heterogeneous with respect to allelic ratio, location, and length of the insertion, with a high mutant-to-wild-type ratio consistently associated with inferior prognosis. We retrospectively analyzed the role of alloHCT in first remission in relationship to the allelic ratio and presence or absence of nucleophosmin 1 mutations (NPM1) in the Study Alliance Leukemia AML2003 trial. FLT3-ITD mutations were detected in 209 patients and concomitant NPM1 mutations in 148 patients. Applying a predefined cutoff ratio of .8, AML was grouped into high- and low-ratio FLT3-ITD AML (HR(FLT3-ITD) and LR(FLT3-ITD)). Sixty-one patients (29%) were transplanted in first remission. Overall survival (OS) (HR, .3; 95% CI, .16 to .7; P = .004) and event-free survival (EFS) (HR, .4; 95% CI, .16 to .9; P = .02) were significantly increased in patients with HR(FLT3-ITD) AML who received alloHCT as consolidation treatment compared with patients who received consolidation chemotherapy. Patients with LR(FLT3-ITD) AML and wild-type NPM1 who received alloHCT in first remission had increased OS (HR, .3; 95% CI, .1 to .8; P = .02) and EFS (HR, .2; 95% CI, .1 to .8; P = .02), whereas alloHCT in first remission did not have a significant impact on OS and EFS in patients with LR(FLT3-ITD) AML and concomitant NPM1 mutation. In conclusion, our results provide additional evidence that alloHCT in first remission improves EFS and OS in patients with HR(FLT3-ITD) AML and in patients with LR(FLT3-ITD) AML and wild-type NPM1. Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

PubMed Central

Ishizawa, Jo; Kojima, Kensuke; Chachad, Dhruv; Ruvolo, Peter; Ruvolo, Vivian; Jacamo, Rodrigo O.; Borthakur, Gautam; Mu, Hong; Zeng, Zhihong; Tabe, Yoko; Allen, Joshua E.; Wang, Zhiqiang; Ma, Wencai; Lee, Hans C.; Orlowski, Robert; Sarbassov, Dos D.; Lorenzi, Philip L.; Huang, Xuelin; Neelapu, Sattva S.; McDonnell, Timothy; Miranda, Roberto N.; Wang, Michael; Kantarjian, Hagop; Konopleva, Marina; Davis, R. Eric.; Andreeff, Michael

2016-01-01

The clinical challenge posed by p53 abnormalities in hematological malignancies requires therapeutic strategies other than standard genotoxic chemotherapies. ONC201 is a first-in-class small molecule that activates p53-independent apoptosis, has a benign safety profile, and is in early clinical trials. We found that ONC201 caused p53-independent apoptosis and cell cycle arrest in cell lines and in mantle cell lymphoma (MCL) and acute myeloid leukemia (AML) samples from patients; these included samples from patients with genetic abnormalities associated with poor prognosis or cells that had developed resistance to the nongenotoxic agents ibrutinib and bortezomib. Moreover, ONC201 caused apoptosis in stem and progenitor AML cells and abrogated the engraftment of leukemic stem cells in mice while sparing normal bone marrow cells. ONC201 caused changes in gene expression similar to those caused by the unfolded protein response (UPR) and integrated stress responses (ISRs), which increase the translation of the transcription factor ATF4 through an increase in the phosphorylation of the translation initiation factor eIF2α. However, unlike the UPR and ISR, the increase in ATF4 abundance in ONC201-treated hematopoietic cells promoted apoptosis and did not depend on increased phosphorylation of eIF2α. ONC201 also inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling, likely through ATF4-mediated induction of the mTORC1 inhibitor DDIT4. Overexpression of BCL-2 protected against ONC201-induced apoptosis, and the combination of ONC201 and the BCL-2 antagonist ABT-199 synergistically increased apoptosis. Thus, our results suggest that by inducing an atypical ISR and p53-independent apoptosis, ONC201 has clinical potential in hematological malignancies. PMID:26884599

Antiproliferative and proapoptotic effects of topotecan in combination with thymoquinone on acute myelogenous leukemia.

PubMed

Khalife, Rana; El-Hayek, Stephany; Stephany, El-Hayek; Tarras, Omayr; Hodroj, Mohammad Hassan; Rizk, Sandra

2014-09-01

Topotecan has shown promising antineoplastic activity in solid tumors and acute leukemia. Because of the primary dose-limiting toxicity of topotecan, it is necessary to identify other agents that can work synergistically with topotecan, potentially increasing its efficacy while limiting its toxicity. Many studies showed synergism in combination of topotecan with gemcitabine and bortezomib. Other studies report the increase in growth inhibition of gemcitabine or oxaliplatin when cells were preexposed to naturally occurring drugs such as thymoquinone. The aim of this project was to study the mode of action of topotecan along with thymoquinone, on survival and apoptosis pathways in acute myelogenous leukemia (AML) cell lines, and to investigate the potential synergistic effect of thymoquinone on topotecan. U937 cells were incubated with different topotecan and thymoquinone concentrations for 24 and 48 hours, separately and in combination. Cell proliferation was determined using WST-1 (Roche) reagent. The effect of the compounds on protein expression of Bax, Bcl2, p53, caspase-9, -8, and -3 was determined using Western blot analysis. Cell cycle analysis was performed in addition to annexin/propidium iodide staining. Thymoquinone and topotecan exhibited antiproliferative effects on U937 cells when applied separately. In combination, the reduction in proliferation was extremely significant with a major increase in the expression levels of Bax/Bcl2, p53, and caspase-3 and -9. Preexposure with thymoquinone resulted in an increase in cell growth inhibition compared with topotecan treatment. Thymoquinone, when combined with topotecan in noncytotoxic doses, produced synergistic antiproliferative and proapoptotic effects in AML cells. Preexposure to thymoquinone seems to be more effective than simultaneous application with topotecan. Copyright © 2014 Elsevier Inc. All rights reserved.

ZFX controls propagation and prevents differentiation of acute T-lymphoblastic and myeloid leukemia

PubMed Central

Weisberg, Stuart P.; Smith-Raska, Matthew R.; Esquilin, Jose M.; Zhang, Ji; Arenzana, Teresita L.; Lau, Colleen M.; Churchill, Michael; Pan, Haiyan; Klinakis, Apostolos; Dixon, Jack E.; Mirny, Leonid A.; Mukherjee, Siddhartha; Reizis, Boris

2014-01-01

Summary Tumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem cell-related genetic regulator. PMID:24485662

Therapy-related acute myeloid leukemia and myelodysplastic syndromes in patients with Hodgkin lymphoma: a report from the German Hodgkin Study Group.

PubMed

Eichenauer, Dennis A; Thielen, Indra; Haverkamp, Heinz; Franklin, Jeremy; Behringer, Karolin; Halbsguth, Teresa; Klimm, Beate; Diehl, Volker; Sasse, Stephanie; Rothe, Achim; Fuchs, Michael; Böll, Boris; von Tresckow, Bastian; Borchmann, Peter; Engert, Andreas

2014-03-13

Therapy-related acute myeloid leukemia and myelodysplastic syndromes (t-AML/MDS) represent severe late effects in patients treated for Hodgkin lymphoma (HL). Because more recent data are scarce, we retrospectively analyzed incidence, outcome, and risk factors for the development of t-AML/MDS after HL. A total of 11,952 patients treated for newly diagnosed HL within German Hodgkin Study Group trials between 1993 and 2009 were considered. At a median follow-up of 72 months, t-AML/MDS was diagnosed in 106/11,952 patients (0.9%). Median time from HL treatment to t-AML/MDS was 31 months. The median age of patients with t-AML/MDS was higher than in the whole patient group (43 vs 34 years, P < .0001). Patients who received 4 or more cycles of BEACOPP(escalated) had an increased risk to develop t-AML/MDS when compared with patients treated with less than 4 cycles of BEACOPP(escalated) or no BEACOPP chemotherapy (1.7% vs 0.7% vs 0.3%, P < .0001). The median overall survival (OS) for all t-AML/MDS patients was 7.2 months. However, t-AML/MDS patients proceeding to allogeneic stem cell transplantation had a significantly better outcome with a median OS not reached after a median follow-up of 41 months (P < .001).

A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice

PubMed Central

Illendula, Anuradha; Pulikkan, John A.; Zong, Hongliang; Grembecka, Jolanta; Xue, Liting; Sen, Siddhartha; Zhou, Yunpeng; Boulton, Adam; Kuntimaddi, Aravinda; Gao, Yan; Rajewski, Roger A.; Guzman, Monica L.; Castilla, Lucio H.; Bushweller, John H.

2015-01-01

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers. PMID:25678665

Tuberin-deficiency downregulates N-cadherin and upregulates vimentin in kidney tumor of TSC patients

PubMed Central

Liang, Sitai; Salas, Tiffanie; Gencaslan, Emre; Li, Baojie; Habib, Samy L.

2014-01-01

Angiomyolipomas (AMLs) are associated with cell fibrosis in kidney of Tuberous Sclerosis Complex patients. The mechanism by which the fibrotic proteins accumulated in AMLs has not been explored. In the present study, we investigated the role of Akt/tuberin/mTOR pathway in the regulation cell fibrosis proteins. AML cells that expressed low levels of tuberin showed less expression of N-cadherin and higher of vimentin proteins compared to HEK293 cells. AML cells infected with Ad-tuberin showed a significant decrease in vimentin and an increase in N-cadherin protein expression. In addition, cells treated with rapamycin showed a significant increase in p-Akt and a decrease in p-p70S6K that was associated with a decrease expression of vimentin and a slight increase expression in N-cadherin. On the other hand, cells treated with Akt inhibitor revealed a significant decrease in p-Akt and p-p70S6K that was associated with a significant decrease in vimentin and an increase in N-cadherin expression. In addition, cells transfected with DN-Akt or DN-S6K show significant increase expression in N-cadherin and a decrease in vimentin. Moreover, cells transfected with siRNA against rictor or siRNA against raptor resulted in a decrease in vimentin and an increase N-cadherin expression. Kidney tumors from TSC patients showed significant decrease in N-cadherin and significant increased in vimentin protein expression compared to control kidney tissues. These data comprise the first report to provide the role of Akt/tuberin/mTORC1/2 in the regulation of N-cadherin and vimentin that are involved in the progression of fibrosis in kidney tumor of TSC patients. PMID:25149531

Enhancement of chemosensitivity by simultaneously silencing of Mcl-1 and Survivin genes using small interfering RNA in human myelomonocytic leukaemia.

PubMed

Jafarlou, Mahdi; Shanehbandi, Dariush; Dehghan, Parvin; Mansoori, Behzad; Othman, F; Baradaran, Behzad

2017-11-07

Acute myeloid leukaemia (AML) is a genetically heterogeneous, severe and rapidly progressing disease triggered by blocking granulocyte or monocyte differentiation and maturation. Overexpression of myeloid cell leukaemia-1 (Mcl-1) and Survivin is associated with drug resistance, tumour progression and inhibition of apoptotic mechanisms in leukaemia and several cancers. In the present study, we examined the combined effect of etoposide and dual siRNA-mediated silencing of Mcl-1 and Survivin on U-937 AML cells. The AML cells were co-transfected with Mcl-1 and Survivin-specific siRNAs and genes silencing were confirmed by quantitative real-time PCR and Western blotting. Subsequently, MTT assay was used for the evaluation of cytotoxic effects by dual siRNA and etoposide on their own and in combination. For the studying of apoptosis, DNA-histone ELISA and annexin-V/FITC assays were performed. Co-transfection of Mcl-1 and Survivin siRNA significantly blocked their expression at the mRNA and protein levels, leading to the induction of apoptosis and strong inhibition of growth (p < .05). Besides, combined treatment of etoposide with Mcl-1 and Survivin siRNAs co-transfection leads to synergistically enhance etoposide-induced cytotoxic and apoptotic effects (p < .05). The results showed that Mcl-1 and Survivin play a major role in the U937 cells survival and their resistance relative to etoposide. Thus, Mcl-1 and Survivin can be considered as promising molecular targets for the treatment of AML. The combination treatment with etoposide, and siRNA-mediated silencing of corresponding genes may be a novel strategy in chemoresistance AML treatment.

Comparison of childhood myelodysplastic syndrome, AML FAB M6 or M7, CCG 2891: report from the Children's Oncology Group.

PubMed

Barnard, Dorothy R; Alonzo, Todd A; Gerbing, Robert B; Lange, Beverly; Woods, William G

2007-07-01

Myelodysplastic syndromes (MDS), acute erythroleukemia (FAB M6), and acute megakaryocytic leukemia (FAB M7) have overlapping features. Children without Down syndrome or acute promyelocytic leukemia who were newly diagnosed with primary myelodysplastic syndrome or acute myeloid leukemia (AML) M6 or M7 were compared to children with de novo AML M0-M5. All children were entered on the Children's Cancer Group therapeutic research study CCG 2891. The presentation and outcomes of the 132 children diagnosed with MDS (60 children), AML FAB M6 (19 children), or AML FAB M7 (53 children) were similar. Children with AML FAB M7 were diagnosed at a significantly younger age (P = 0.001). Children with MDS, M6, or M7 had significantly lower white blood cell (WBC) counts (P = 0.001), lower peripheral blast counts (P < 0.001), and an increased frequency of -7/7q- (P = 0.003) at presentation. All three groups had significantly inferior overall survival (OS) (P < 0.001) and event free survival (P < 0.001) compared with the 748 children diagnosed with AML FAB M0-M5 when assessed from entry on study. This poor survival was largely attributable to induction death and failure. However, when assessed from successful completion of induction therapy, the 5-year OS (P = 0.090)(49.1 vs. 56.9%) and disease-free survival (DFS) (P = 0.113)(38.0 vs. 46.3%) therapy were not significantly different from other children with AML. Childhood AML FAB M6 and AML M7 resemble MDS in presentation, poor induction success rates, and outcomes.

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway

PubMed Central

Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K.; Kishore, Uday

2018-01-01

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway.

PubMed

Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K; Kishore, Uday

2018-01-01

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53 mt ), MiaPaCa-2 (p53 mt ), and Capan-2 (p53 wt ) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

Angiomyolipoma with Minimal Fat: Can It Be Differentiated from Clear Cell Renal Cell Carcinoma by Using Standard MR Techniques?

PubMed Central

Hindman, Nicole; Ngo, Long; Genega, Elizabeth M.; Melamed, Jonathan; Wei, Jesse; Braza, Julia M.; Rofsky, Neil M.

2012-01-01

Purpose: To retrospectively assess whether magnetic resonance (MR) imaging with opposed-phase and in-phase gradient-echo (GRE) sequences and MR feature analysis can differentiate angiomyolipomas (AMLs) that contain minimal fat from clear cell renal cell carcinomas (RCCs), with particular emphasis on small (<3-cm) masses. Materials and Methods: Institutional review board approval and a waiver of informed consent were obtained for this HIPAA-compliant study. MR images from 108 pathologically proved renal masses (88 clear cell RCCs and 20 minimal fat AMLs from 64 men and 44 women) at two academic institutions were evaluated. The signal intensity (SI) of each renal mass and spleen on opposed-phase and in-phase GRE images was used to calculate an SI index and tumor-to-spleen SI ratio. Two radiologists who were blinded to the pathologic results independently assessed the subjective presence of intravoxel fat (ie, decreased SI on opposed-phase images compared with that on in-phase images), SI on T1-weighted and T2-weighted images, cystic degeneration, necrosis, hemorrhage, retroperitoneal collaterals, and renal vein thrombosis. Results were analyzed by using the Wilcoxon rank sum test, two-tailed Fisher exact test, and multivariate logistic regression analysis for all renal masses and for small masses. A P value of less than .05 was considered to indicate a statistically significant difference. Results: There were no differences between minimal fat AMLs and clear cell RCCs for the SI index (8.05% ± 14.46 vs 14.99% ± 19.9; P = .146) or tumor-to-spleen ratio (−8.96% ± 16.6 and −15.8% ± 22.4; P = .227) when all masses or small masses were analyzed. Diagnostic accuracy (area under receiver operating characteristic curve) for the SI index and tumor-to-spleen ratio was 0.59. Intratumoral necrosis and larger size were predictive of clear cell RCC (P < .001) for all lesions, whereas low SI (relative to renal parenchyma SI) on T2-weighted images, smaller size, and female sex correlated with minimal fat AML (P < .001) for all lesions. Conclusion: The diagnostic accuracy of opposed-phase and in-phase GRE MR imaging for the differentiation of minimal fat AML and clear cell RCC is poor. In this cohort, low SI on T2-weighted images relative to renal parenchyma and small size suggested minimal fat AML, whereas intratumoral necrosis and large size argued against this diagnosis. © RSNA, 2012 PMID:23012463

Heterogeneity of Clonal Expansion and Maturation-Linked Mutation Acquisition in Hematopoietic Progenitors in Human Acute Myeloid Leukemia

PubMed Central

Walter, Roland B.; Laszlo, George S.; Lionberger, Jack M.; Pollard, Jessica A.; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Othus, Megan; Rafii, Shahin; Meshinchi, Soheil; Appelbaum, Frederick R.; Bernstein, Irwin D.

2014-01-01

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML) but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition, and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34+/CD33− cells revealed polyclonal growth in highly curable AMLs, suggesting mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34+/CD33− cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications. PMID:24721792

Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP)

PubMed Central

Rosen, David B.; Minden, Mark D.; Kornblau, Steven M.; Cohen, Aileen; Gayko, Urte; Putta, Santosh; Woronicz, John; Evensen, Erik; Fantl, Wendy J.; Cesano, Alessandra

2010-01-01

Background Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative. Methodology/Principal Findings Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management. Conclusions/Significance These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations. PMID:21048955

Requirement for CDK6 in MLL-rearranged acute myeloid leukemia

PubMed Central

Placke, Theresa; Faber, Katrin; Nonami, Atsushi; Putwain, Sarah L.; Salih, Helmut R.; Heidel, Florian H.; Krämer, Alwin; Root, David E.; Barbie, David A.; Krivtsov, Andrei V.; Armstrong, Scott A.; Hahn, William C.; Huntly, Brian J.; Sykes, Stephen M.; Milsom, Michael D.; Scholl, Claudia

2014-01-01

Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9–driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. PMID:24764564

Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

PubMed Central

Dorsey, W. C.; Ford, B. D.; Roane, L.; Haynie, D. T.; Tchounwou, P. B.

2005-01-01

Ultra–wideband (UWB) technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR) could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM) cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5–20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8–24 h post exposure. UWBR exerted a statistically significant (p < 0.05) dose-dependent response in cell viability in both serum-treated and serum free medium (SFM) -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma. PMID:16705798

Delineation of yet unknown cryptic subtelomere aberrations in 50% of acute myeloid leukemia with normal GTG-banding karyotype.

PubMed

Gross, Madeleine; Mkrtchyan, Hasmik; Glaser, Melanie; Fricke, Hans Jörg; Höffken, Klaus; Heller, Anita; Weise, Anja; Liehr, Thomas

2009-02-01

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to clinical prognosis and acquired chromosomal aberrations. After routine banding cytogenetic analysis 45% of AML patients show a normal karyotype (NK-AML). For a better understanding of development and progression in AML, it is important to find markers which could be primary genetic aberrations. Therefore, in this study 31 patients with NK-AML were analyzed by new high resolution molecular cytogenetic approaches. A combination of multitude multicolor banding and metaphase microdissection-based comparative genomic hybridization revealed deletions of the subtelomeric regions in 6% of the studied cases. According to these results, locus-specific probes for the subtelomeric regions of chromosomes 5, 9, 11, 12 and 13 were applied on 22 of the studied 31 NK-AML cases. Surprisingly, 50% of them showed deletions or duplications. These aberrations occurred in the in vitro proliferating as well as in the non-proliferating cells. Meta-analysis of the aberrant regions revealed that they often include genes known to be associated with tumors, e.g. RASA3 on chromosome 13. These results implicate that aberrations in the subtelomeric regions of NK-AML occur quite often and may be considered as primary genetic changes, and should not be neglected in future diagnostic approaches.

MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis

PubMed Central

Shen, Li-Jing; Chen, Fang-Yuan; Zhang, Yong; Cao, Lan-Fang; Kuang, Ying; Zhong, Min; Wang, Ting; Zhong, Hua

2013-01-01

Background Amplification of MYCN (N-Myc) oncogene has been reported as a frequent event and a poor prognostic marker in human acute myeloid leukemia (AML). The molecular mechanisms and transcriptional networks by which MYCN exerts its influence in AML are largely unknown. Methodology/Principal Findings We introduced murine MYCN gene into embryonic zebrafish through a heat-shock promoter and established the stable germline Tg(MYCN:HSE:EGFP) zebrafish. N-Myc downstream regulated gene 1 (NDRG1), negatively controlled by MYCN in human and functionally involved in neutrophil maturation, was significantly under-expressed in this model. Using peripheral blood smear detection, histological section and flow cytometric analysis of single cell suspension from kidney and spleen, we found that MYCN overexpression promoted cell proliferation, enhanced the repopulating activity of myeloid cells and the accumulation of immature hematopoietic blast cells. MYCN enhanced primitive hematopoiesis by upregulating scl and lmo2 expression and promoted myelopoiesis by inhibiting gata1 expression and inducing pu.1, mpo expression. Microarray analysis identified that cell cycle, glycolysis/gluconeogenesis, MAPK/Ras, and p53-mediated apoptosis pathways were upregulated. In addition, mismatch repair, transforming and growth factor β (TGFβ) were downregulated in MYCN-overexpressing blood cells (p<0.01). All of these signaling pathways are critical in the proliferation and malignant transformation of blood cells. Conclusion/Significance The above results induced by overexpression of MYCN closely resemble the main aspects of human AML, suggesting that MYCN plays a role in the etiology of AML. MYCN reprograms hematopoietic cell fate by regulating NDRG1 and several lineage-specific hematopoietic transcription factors. Therefore, this MYCN transgenic zebrafish model facilitates dissection of MYCN-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets. PMID:23554972

Development of a biochip-based assay integrated in a global strategy for identification of fusion transcripts in acute myeloid leukemia: a work flow for acute myeloid leukemia diagnosis.

PubMed

Giusiano, S; Formisano-Tréziny, C; Benziane, A; Maroc, N; Picard, C; Hermitte, F; Taranger-Charpin, C; Gabert, J

2010-08-01

Three major types of rearrangements are involved in acute myeloid leukemias (AML): t(8;21)(q22;q22), inv(16)(p13q22), and 11q23/MLL abnormalities. Their precise identification becomes essential for diagnosis, prognosis, and therapeutic choices. Resulting fusion transcripts (FT) are also powerful markers for monitoring the efficacy of treatment, the minimal residual disease (MRD) and could become therapeutic targets. Today, the challenge is to propose an individual follow-up for each patient even for those with a rare fusion event. In this study, we propose a biochip-based assay integrated in a global strategy for identification of rare FT in AML, after fluorescence in situ hybridization detection, as described by the World Health Organization classification. Using cell lines, we developed and validated a biochip-based assay called the AMLFusionChip that identifies every FT of AML1-ETO, CBFbeta-MYH11 as well as MLL-AF9, MLL-ENL, MLL-AF6, and MLL-AF10. The original design of our AMLFusionChip.v01 enables the identification of these FT wherever the breakpoint on the partner gene may be. In case of biochip negative result, our 3'RACE amplification strategy enables to clone and then sequence the new translocation partner. This AMLFusionChip strategy fits into the concept of personalized medicine for the largest number of patients.

Accelerate Genomic Aging in Congenital Neutropenia

DTIC Science & Technology

2015-08-01

for the markedly increased risk of transformation to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) in patients with congenital...Hematopoietic stem cells Granulocyte colony-stimulating factor Granulocyte colony-stimulating factor receptor Acute myeloid leukemia Myelodysplastic... myeloid leukemia (AML) is perhaps the major clinical concern in patients with severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS

Expression and prognostic value of hemopoietic cytokine receptors in acute myeloid leukemia (AML): implications for future therapeutical strategies.

PubMed

Graf, Michaela; Hecht, Karin; Reif, Susanne; Pelka-Fleischer, Renate; Pfister, Karin; Schmetzer, Helga

2004-02-01

Hemopoietic cytokines regulate hemopoietic cell functions via specific cell surface receptors. There is evidence to suggest, that those receptors (R) could play a role in leukemia with respect to cell differentiations and its regulation, prognosis, and pathobiology. Knowledge of individual cytokine receptor (CKR) profiles could provide new discoveries about CKR-supported therapeutic considerations. We have studied the expression of CKR on mononuclear bone marrow (BM) cells of 89 patients with acute myeloid leukemia (AML) at first diagnosis, three patients at relapse or with persisting AML and eight healthy probands by fluorescence-activated cell sorting (FACS) analysis using directly fluorescein-conjugated antibodies: CD114 (hG-CSF-R), CD116 (hGM-CSF-R), CD117 (hSCF-R), CD123 (hIL-3-R), CD130 (gp130subunit), CD135 (hFL-R). A case was defined as positive, if more than 20% of the cells expressed the regarding CKR. All investigated CKR were more frequently expressed in AML-samples than in healthy BM-samples, except CD130, which was only expressed on 5-6% of AML-blasts in all and with only one healthy BM-sample being CD130(+). Within the French-American-British (FAB) types we observed a maturation- and lineage (granulocytic/monocytic)-committed expression profile. Monocytic subtypes (FAB-type M4/M5) showed significantly more GM-CSF-R(+) (P = 0.001) and FL-R(+) (P = 0.001) and significantly less stem cell factor-R (SCF-R(+)) (P = 0.02) cases. Highest proportions of G-CSF-R(+) blasts were observed in FAB-type M3. In undifferentiated leukemias (FAB-type M1, M2) high amounts of SCF-R(+), IL-3-R(+), and FL-R(+) blasts could be detected. FL-R was the only CKR, which was positive in FAB-type M0 (n = 2). No differences in CKR-expression were detected between primary (p) and secondary (s). Separating our patient cohorts in cytogenetic risk groups we could detect a significant higher proportion of G-CSF-R(+) blasts in the cytogenetic good risk group than in the bad risk group (P = 0.027), but G-CSF-R-expression did not correlate with remission-rate or relapse-free survival probability of the patients. For clinical evaluation only patients treated by the AML-CG-protocol, were included (n = 53). There were no differences of CKR-expression in the responder and non-responder group, however, significant lower relapse-free survival probabilities for patients with more than 85.5% FL-R(+) (P = 0.001) and more than 45.5% SCF-R(+) blasts were found (P = 0.02). Patients with more than 32.5% IL-3-R(+) cells also showed a tendency to a lower relapse-free survival probability (P = 0.26), whereas patients with more than 33% GM-CSF-R(+) (P = 0.06) and patients with more than 52% G-CSF-R(+) (P = 0.175) blasts tended to have a higher relapse-free survival probability. We can conclude, that CKR-expression in AML is maturation- and lineage-committed and the proportions of especially early acting CKR have influence on relapse-free survival probability of AML-patients, independently of the karyotype. With respect to the individual CKR status the benefit of cytokines as priming agents, as agents to treat neutropenia or to influence the metabolism of chemotherapy can be discussed under new points of view.

Epigenetic inactivation of the MIR129-2 in hematological malignancies

PubMed Central

2013-01-01

Background MIR129-2 has been shown to be a tumor suppressor microRNA hypermethylated in epithelial cancers. Patients and methods Epigenetic inactivation of MIR129-2 was studied by methylation-specific PCR (MSP) in 13 cell lines (eight myeloma and five lymphoma), 15 normal controls and 344 primary samples including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM) at diagnosis, MM at relapse/progression, and monoclonal gammopathy of undetermined significance (MGUS). Expression of MIR129 and its target, SOX4, in cell lines was measured before and after hypomethylating treatment and MIR129 overexpression. MIR129 expression was correlated with MIR129-2 methylation status in primary lymphoma samples. Tumor suppressor function of MIR129 was demonstrated by MTT and trypan blue exclusion assay after MIR129 overexpression. Results The sensitivity of the methylated-MSP was one in 103. Different MSP statuses, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. All five lymphoma and seven of eight myeloma cell lines showed complete and partial MIR129-2 methylation. In primary samples, MIR129-2 methylation was absent in AML and CML, but detected in 5% ALL, 45.9% CLL, 49.5% MM at diagnosis, and 59.1% NHL. In CLL, MIR129-2 methylation adversely impacted on survival (p=0.004). In MM, MIR129-2 methylation increased from 27.5% MGUS to 49.5% MM at diagnosis and 41.5% at relapse/progression (p=0.023). In NHL, MIR129-2 methylation was associated with MIR124-1 and MIR203 methylation (p<0.001), and lower MIR129 expression (p=0.009). Hypomethylation treatment of JEKO-1, homozygously methylated for MIR129-2, led to MIR129-2 demethylation and MIR129 re-expression, with downregulation of SOX4 mRNA. Moreover, MIR129 overexpression in both mantle cell lines, JEKO-1 and GRANTA-519, inhibited cellular proliferation and enhanced cell death, with concomitant SOX4 mRNA downregulation. Conclusions MIR129-2 is a tumor suppressive microRNA frequently methylated in lymphoid but not myeloid malignancies, leading to reversible MIR129-2 silencing. In CLL, MIR129-2 methylation was associated with an inferior survival. In MM, MIR129-2 methylation might be acquired during progression from MGUS to symptomatic MM. In NHL, MIR129-2 methylation might collaborate with MIR124-1 and MIR203 methylation in lymphomagenesis. PMID:23406679

Risk of therapy-related secondary leukemia in Hodgkin lymphoma: the Stanford University experience over three generations of clinical trials.

PubMed

Koontz, Michael Zach; Horning, Sandra J; Balise, Raymond; Greenberg, Peter L; Rosenberg, Saul A; Hoppe, Richard T; Advani, Ranjana H

2013-02-10

To assess therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS) risk in patients treated for Hodgkin lymphoma (HL) on successive generations of Stanford clinical trials. Patients with HL treated at Stanford with at least 5 years of follow-up after completing therapy were identified from our database. Records were reviewed for outcome and development of t-AML/MDS. Seven hundred fifty-four patients treated from 1974 to 2003 were identified. Therapy varied across studies. Radiotherapy evolved from extended fields (S and C studies) to involved fields (G studies). Primary chemotherapy was mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) or procarbazine, mechlorethamine, and vinblastine (PAVe) in S studies; MOPP, PAVe, vinblastine, bleomycin, and methotrexate (VBM), or doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) in C studies; and VbM (reduced dose of bleomycin compared with VBM) or mechlorethamine, doxorubicin, vinblastine, vincristine, bleomycin, etoposide, and prednisone (Stanford V) in G studies. Cumulative exposure to alkylating agent (AA) was notably lower in the G studies compared with the S and C studies, with a 75% to 83% lower dose of nitrogen mustard in addition to omission of procarbazine and melphalan. Twenty-four (3.2%) of 754 patients developed t-AML/MDS, 15 after primary chemotherapy and nine after salvage chemotherapy for relapsed HL. The incidence of t-AML/MDS was significantly lower in the G studies (0.3%) compared with the S (5.7%) or C (5.2%) studies (P < .001). Additionally, in the G studies, no t-AML/MDS was noted after primary therapy, and the only patient who developed t-AML/MDS did so after second-line therapy. Our data demonstrate the relationship between the cumulative AA dose and t-AML/MDS. Limiting the dose of AA and decreased need for secondary treatments have significantly reduced the incidence of t-AML/MDS, which was extremely rare in the G studies (Stanford V era).

Potential clinical applications of rhGM-CSF in acute myeloid leukemia based on its biologic activity and receptor interaction.

PubMed

Lanza, F; Rigolin, G M; Castagnari, B; Moretti, S; Castoldi, G

1997-01-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multilineage hemopoietic growth factor that stimulates proliferation, differentiation, and survival of progenitor cells, enhances the functional activities of mature myeloid effector cells, and plays a key role in host defense and the inflammatory process. Although the clinical use of rhGM-CSF in patients affected by lymphoid malignancies is widely accepted, its utility and safety in the management of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) is still controversial. The three main schedules adopted for clinical application of GM-CSF in AML are as follows: A) post-chemotherapy, in order to shorten the duration of neutropenia and/or monocytopenia; B) prechemotherapy to recruit blast cells into active cell cycle phases, and to increase their sensitivity to cell cycle-dependent cytotoxic drugs; C) as a mobilizing agent to induce the release of progenitor cells from bone marrow into circulation (peripheral blood progenitor cell transplantation-PBPC). The objective of this paper is to analyze the potential clinical applications of rhGM-CSF in AML. The material examined in the present review includes several personal papers in this field and articles and abstracts published in journals covered by the Science Citation Index. Based on current knowledge, it may be argued that rhGM-CSF should be used only in a subset of AML patients at high risk of infection mortality, including elderly subjects, and/or in those AML patients who relapse or are resistant to induction treatment. However, the risk of stimulating the leukemic clone following GM-CSF therapy should be kept in mind when using this growth factor in the clinical setting, even though the great majority of the reported papers on this subject have shown that GM-CSF therapy does not affect relapse rates, frequency of remissions or patient life expectancy. It is likely that new data from controlled clinical trials will clarify the therapeutic role of GM-CSF in myeloid-derived malignancies, allowing the establishment of consensus guidelines for its use.

Systematic review of health state utility values for acute myeloid leukemia.

PubMed

Forsythe, Anna; Brandt, Patricia S; Dolph, Mike; Patel, Sachin; Rabe, Adrian Paul J; Tremblay, Gabriel

2018-01-01

Cost-utility analyses for acute myeloid leukemia (AML) require health state utility values (HSUVs) in order to calculate quality-adjusted life-years (QALYs) for each health state. This study reviewed AML-related HSUVs that could be used in economic evaluation studies. EMBASE, MEDLINE, and Cochrane databases were searched from January 2000 to November 2016 for relevant studies that reported quality of life (QoL) and HSUVs in AML. Identified relevant European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 values were mapped to HSUVs. HSUVs for each health state in the AML treatment pathway were then collated. Ten relevant studies were identified. Six were cost-effectiveness analyses utilizing HSUVs for calculation of QALYs, one was an effectiveness analysis (incremental QALY), and two were QoL studies reporting AML-specific utilities. An additional study reported QoL for patients undergoing stem cell transplantation (SCT). Since no study reported HSUVs for relapse, values from a study of secondary AML patients who failed prior treatment for myelodysplastic syndrome were used. Where multiple HSUVs were available, collected values were given priority over assumed values. AML treatment (induction, consolidation, or SCT) was associated with decreased HSUV, while post-treatment complete remission led to increased HSUV. There are some methodologically robust HSUVs that can be directly used in economic evaluations for AML. Careful interpretation is advised considering significant differences in methodologies and patient population (inclusion, size). We need to develop HSUVs with larger-sized studies, making greater use of condition-specific data.

Prognostic factors for acute myeloid leukaemia in adults--biological significance and clinical use.

PubMed

Liersch, Ruediger; Müller-Tidow, Carsten; Berdel, Wolfgang E; Krug, Utz

2014-04-01

Acute myeloid leukaemia (AML) is a heterogeneous disease. Prognosis of AML is influenced both by patient-specific as well as disease-specific factors. Age is the most prominent patient-specific risk factor, while chromosomal aberrations are the strongest disease-specific risk factors. For patients with cytogenetically normal AML, prognosis can be specified by mutational status of the genes NPM1, FLT3 and CEBPA. A growing number of recurrent mutations in additional genes have recently been identified, for which the prognostic effect yet has to be determined. Performance status, geriatric assessment, secondary leukaemia following myelodysplastic syndrome or cytotoxic treatment, common laboratory parameters, leukaemic stem cell frequency, bone marrow microenvironment, gene expression levels, epigenetic changes, micro-RNA's as well as kinetics and depth of response to treatment influence prognosis of AML patients. Despite the high number of established risk factors, only few predictive markers exist which can truly aid therapy decisions in patients with AML. © 2014 John Wiley & Sons Ltd.

MicroRNAs as New Biomarkers for Diagnosis and Prognosis, and as Potential Therapeutic Targets in Acute Myeloid Leukemia

PubMed Central

Trino, Stefania; Caivano, Antonella; Laurenzana, Ilaria; Tagliaferri, Daniela; Falco, Geppino; Del Vecchio, Luigi; Musto, Pellegrino; De Luca, Luciana

2018-01-01

Acute myeloid leukemias (AML) are clonal disorders of hematopoietic progenitor cells which are characterized by relevant heterogeneity in terms of phenotypic, genotypic, and clinical features. Among the genetic aberrations that control disease development there are microRNAs (miRNAs). miRNAs are small non-coding RNAs that regulate, at post-transcriptional level, translation and stability of mRNAs. It is now established that deregulated miRNA expression is a prominent feature in AML. Functional studies have shown that miRNAs play an important role in AML pathogenesis and miRNA expression signatures are associated with chemotherapy response and clinical outcome. In this review we summarized miRNA signature in AML with different cytogenetic, molecular and clinical characteristics. Moreover, we reviewed the miRNA regulatory network in AML pathogenesis and we discussed the potential use of cellular and circulating miRNAs as biomarkers for diagnosis and prognosis and as therapeutic targets. PMID:29401684

Genetic hierarchy and temporal variegation in the clonal history of acute myeloid leukaemia.

PubMed

Hirsch, Pierre; Zhang, Yanyan; Tang, Ruoping; Joulin, Virginie; Boutroux, Hélène; Pronier, Elodie; Moatti, Hannah; Flandrin, Pascale; Marzac, Christophe; Bories, Dominique; Fava, Fanny; Mokrani, Hayat; Betems, Aline; Lorre, Florence; Favier, Rémi; Féger, Frédéric; Mohty, Mohamad; Douay, Luc; Legrand, Ollivier; Bilhou-Nabera, Chrystèle; Louache, Fawzia; Delhommeau, François

2016-08-18

In acute myeloid leukaemia (AML) initiating pre-leukaemic lesions can be identified through three major hallmarks: their early occurrence in the clone, their persistence at relapse and their ability to initiate multilineage haematopoietic repopulation and leukaemia in vivo. Here we analyse the clonal composition of a series of AML through these characteristics. We find that not only DNMT3A mutations, but also TET2, ASXL1 mutations, core-binding factor and MLL translocations, as well as del(20q) mostly fulfil these criteria. When not eradicated by AML treatments, pre-leukaemic cells with these lesions can re-initiate the leukaemic process at various stages until relapse, with a time-dependent increase in clonal variegation. Based on the nature, order and association of lesions, we delineate recurrent genetic hierarchies of AML. Our data indicate that first lesions, variegation and treatment selection pressure govern the expansion and adaptive behaviour of the malignant clone, shaping AML in a time-dependent manner.

Bone marrow VEGFC expression is associated with multilineage dysplasia and several prognostic markers in adult acute myeloid leukemia, but not with survival.

PubMed

Guillem, Vicent; Calabuig, Marisa; Brunet, Salut; Esteve, Jordi; Escoda, Lourdes; Gallardo, David; Ribera, Josep-Maria; Queipo de Llano, María Paz; Arnan, Montserrat; Pedro, Carme; Amigo, María Luz; Martí-Tutusaus, Josep M; García-Guiñón, Antoni; Bargay, Joan; Sampol, Antonia; Salamero, Olga; Font, Llorenç; Talarn, Carme; Hoyos, Montserrat; Díaz-Beyá, Marina; Garrido, Ana; Navarro, Blanca; Nomdédeu, Josep; Sierra, Jordi; Tormo, Mar

2018-01-18

Vascular endothelial growth factor C (VEGFC) stimulates leukemia cell proliferation and survival, and promotes angiogenesis. We studied VEGFC expression in bone marrow samples from 353 adult acute myeloid leukemia (AML) patients and its relationship with several clinical, cytogenetic, and molecular variables. We also studied the expression of 84 genes involved in VEGF signaling in 24 patients. We found that VEGFC expression was higher in AML patients with myelodysplasia-related changes (AML-MRC) than in patients with non-AML-MRC. We also found an association between VEGFC expression and the patient cytogenetic risk group, with those with a worse prognosis having higher VEGFC expression levels. No correlation was observed between VEGFC expression and survival or complete remission. VEGFC expression strongly correlated with expression of the VEGF receptors FLT1, KDR, and NRP1. Thus, in this series, VEGFC expression was increased in AML-MRC and in subgroups with a poorer prognosis, but has no impact on survival.

What happened to anti-CD33 therapy for acute myeloid leukemia?

PubMed

Jurcic, Joseph G

2012-03-01

CD33, a 67-kDa glycoprotein expressed on the majority of myeloid leukemia cells as well as on normal myeloid and monocytic precursors, has been an attractive target for monoclonal antibody (mAb)-based therapy of acute myeloid leukemia (AML). Lintuzumab, an unconjugated, humanized anti-CD33 mAb, has modest single-agent activity against AML but failed to improve patient outcomes in two randomized trials when combined with conventional chemotherapy. Gemtuzumab ozogamicin, an anti-CD33 mAb conjugated to the antitumor antibiotic calicheamicin, improved survival in a subset of AML patients when combined with standard chemotherapy, but safety concerns led to US marketing withdrawal. The activity of these agents confirms that CD33 remains a viable therapeutic target for AML. Strategies to improve the results of mAb-based therapies for AML include antibody engineering to enhance effector function, use of alternative drugs and chemical linkers to develop safer and more effective drug conjugates, and radioimmunotherapeutic approaches.

Biology and Clinical Relevance of Acute Myeloid Leukemia Stem Cells.

PubMed

Reinisch, Andreas; Chan, Steven M; Thomas, Daniel; Majeti, Ravindra

2015-07-01

Evidence for the cancer stem cell model was first demonstrated in xenotransplanted blood and bone marrow samples from patients with acute myeloid leukemia (AML) almost two decades ago, supporting the concept that a rare clonal and mutated leukemic stem cell (LSC) population is sufficient to drive leukemic growth. The inability to eliminate LSCs with conventional therapies is thought to be the primary cause of disease relapse in AML patients, and as such, novel therapies with the ability to target this population are required to improve patient outcomes. An important step towards this goal is the identification of common immunophenotypic surface markers and biological properties that distinguish LSCs from normal hematopoietic stem and progenitor cells (HSPCs) across AML patients. This work has resulted in the development of a large number of potential LSC-selective therapies that target cell surface molecules, intracellular signaling pathways, and the bone marrow microenvironment. Here, we will review the basic biology, immunophenotypic detection, and clinical relevance of LSCs, as well as emerging biological and small-molecule strategies that either directly target LSCs or indirectly target these cells through modulation of their microenvironment. Copyright © 2015 Elsevier Inc. All rights reserved.

20(S)-Ginsenoside Rh2 Induce the Apoptosis and Autophagy in U937 and K562 Cells.

PubMed

Zhuang, Jianjian; Yin, Juxin; Xu, Chaojian; Mu, Ying; Lv, Shaowu

2018-03-08

Acute myeloid leukemia (AML) and Chronic myelogenous leukemia (CML) are common leukemia in adults. 20(S)-GRh2 is an important bioactive substance that is present in Panax ginseng. However, there are no investigations that deal with the comparison of apoptosis, the occurrence of autophagy, and the relationship between apoptosis and autophagy after being treated with 20(S)-GRh2 in AML and CML. In this study, we explored the effect of 20(S)-GRh2 on the AML and CML (U937 and K562). Fluorescence microscopy, CCK-8, Quantitative realtime PCR, Western blot, transmission electron microscopy (TEM), and flow cytometric analysis were used to detect the occurrence of cell proliferation inhibition, apoptosis, and autophagy. By using the above methods, it was determined that apoptosis induced by 20(S)-GRh2 was more obvious in K562 than U937 cells and 20(S)-GRh2 could generate autophagy in K562 and U937 cells. When pretreated by a specific inhibitor of autophagy, (3-methyladenine), the 20(S)-GRh2-induced apoptosis was enhanced, which indicated that 20(S)-GRh2-induced autophagy may protect U937 and K562 cells from undergoing apoptotic cell death. On the other hand, pretreated by an apoptosis suppressor (Z-VAD-FMK), it greatly induced the autophagy and partially prevented 20(S)-GRh2 induced apoptosis. This phenomenon indicated that 20(S)-GRh2-induced autophagy may serve as a survival mechanism and apoptosis and autophagy could act as partners to induce cell death in a cooperative manner. These findings may provide a rationale for future clinical application by using 20(S)-GRh2 combined autophagy inhibitors for AML and CML.

Perturbed hematopoietic stem and progenitor cell hierarchy in myelodysplastic syndromes patients with monosomy 7 as the sole cytogenetic abnormality.

PubMed

Dimitriou, Marios; Woll, Petter S; Mortera-Blanco, Teresa; Karimi, Mohsen; Wedge, David C; Doolittle, Helen; Douagi, Iyadh; Papaemmanuil, Elli; Jacobsen, Sten Eirik W; Hellström-Lindberg, Eva

2016-11-08

The stem and progenitor cell compartments in low- and intermediate-risk myelodysplastic syndromes (MDS) have recently been described, and shown to be highly conserved when compared to those in acute myeloid leukemia (AML). Much less is known about the characteristics of the hematopoietic hierarchy of subgroups of MDS with a high risk of transforming to AML. Immunophenotypic analysis of immature stem and progenitor cell compartments from patients with an isolated loss of the entire chromosome 7 (isolated -7), an independent high-risk genetic event in MDS, showed expansion and dominance of the malignant -7 clone in the granulocyte and macrophage progenitors (GMP), and other CD45RA+ progenitor compartments, and a significant reduction of the LIN-CD34+CD38low/-CD90+CD45RA- hematopoietic stem cell (HSC) compartment, highly reminiscent of what is typically seen in AML, and distinct from low-risk MDS. Established functional in vitro and in vivo stem cell assays showed a poor readout for -7 MDS patients irrespective of marrow blast counts. Moreover, while the -7 clone dominated at all stages of GM differentiation, the -7 clone had a competitive disadvantage in erythroid differentiation. In azacitidine-treated -7 MDS patients with a clinical response, the decreased clonal involvement in mononuclear bone marrow cells was not accompanied by a parallel reduced clonal involvement in the dominant CD45RA+ progenitor populations, suggesting a selective azacitidine-resistance of these distinct -7 progenitor compartments. Our data demonstrate, in a subgroup of high risk MDS with monosomy 7, that the perturbed stem and progenitor cell compartments resemble more that of AML than low-risk MDS.

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity.

PubMed

Jayachandran, Aparna; Shrestha, Ritu; Dhungel, Bijay; Huang, I-Tao; Vasconcelos, Marianna Yumi Kawashima; Morrison, Brian J; Ramlogan-Steel, Charmaine A; Steel, Jason C

2017-09-26

To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai , Zeb and Twist family of EMT transcription factors. Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Rational Combinations of Targeted Agents in AML

PubMed Central

Bose, Prithviraj; Grant, Steven

2015-01-01

Despite modest improvements in survival over the last several decades, the treatment of AML continues to present a formidable challenge. Most patients are elderly, and these individuals, as well as those with secondary, therapy-related, or relapsed/refractory AML, are particularly difficult to treat, owing to both aggressive disease biology and the high toxicity of current chemotherapeutic regimens. It has become increasingly apparent in recent years that coordinated interruption of cooperative survival signaling pathways in malignant cells is necessary for optimal therapeutic results. The modest efficacy of monotherapy with both cytotoxic and targeted agents in AML testifies to this. As the complex biology of AML continues to be elucidated, many “synthetic lethal” strategies involving rational combinations of targeted agents have been developed. Unfortunately, relatively few of these have been tested clinically, although there is growing interest in this area. In this article, the preclinical and, where available, clinical data on some of the most promising rational combinations of targeted agents in AML are summarized. While new molecules should continue to be combined with conventional genotoxic drugs of proven efficacy, there is perhaps a need to rethink traditional philosophies of clinical trial development and regulatory approval with a focus on mechanism-based, synergistic strategies. PMID:26113989

[Successful treatment with reduced-intensity cord blood transplantation for acute myeloid leukemia with complete tetraploidy (92, XXXX)].

PubMed

Iwasaki, Junko; Onozawa, Masahiro; Takahashi, Shojiro; Okada, Kohei; Takahata, Mutsumi; Shigematsu, Akio; Kahata, Kaoru; Kondo, Takeshi; Hashino, Satoshi; Imamura, Masahiro; Asaka, Masahiro

2011-03-01

A 56-year-old female was diagnosed with acute myeloid leukemia (FAB: AML-M1). G-banding karyotype of her bone marrow showed complete tetraploidy (92, XXXX [24/24]). Although she achieved complete remission (CR) after induction therapy and maintained CR during consolidation therapy, relapse occurred only 2 months after discharge. When the relapse occurred, bone marrow karyotypic analysis showed complete tetraploidy again. The patient received reduced-intensity cord blood transplantation (RI-CBT), which induced CR for the second time. The patient is currently alive 24 months after transplantation and there have not been any signs of recurrence to date. There have been a few reports of AML with near-tetraploidy, but cases of AML with complete tetraploidy are extremely rare. Tetraploid AML has been reported to have a poor prognosis and there have been very few cases maintaining CR over the long term after chemotherapy alone. This is the first case of complete tetraploid AML successfully treated by RI-CBT. The clinical course of this case suggests that hematopoietic stem cell transplantation during the first CR phase should be considered a treatment option for tetraploid AML.

FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions.

PubMed

Larrosa-Garcia, Maria; Baer, Maria R

2017-06-01

The receptor tyrosine kinase fms -like tyrosine kinase 3 (FLT3), involved in regulating survival, proliferation, and differentiation of hematopoietic stem/progenitor cells, is expressed on acute myeloid leukemia (AML) cells in most patients. Mutations of FLT3 resulting in constitutive signaling are common in AML, including internal tandem duplication (ITD) in the juxtamembrane domain in 25% of patients and point mutations in the tyrosine kinase domain in 5%. Patients with AML with FLT3-ITD have a high relapse rate and short relapse-free and overall survival after chemotherapy and after transplant. A number of inhibitors of FLT3 signaling have been identified and are in clinical trials, both alone and with chemotherapy, with the goal of improving clinical outcomes in patients with AML with FLT3 mutations. While inhibitor monotherapy produces clinical responses, they are usually incomplete and transient, and resistance develops rapidly. Diverse combination therapies have been suggested to potentiate the efficacy of FLT3 inhibitors and to prevent development of resistance or overcome resistance. Combinations with epigenetic therapies, proteasome inhibitors, downstream kinase inhibitors, phosphatase activators, and other drugs that alter signaling are being explored. This review summarizes the current status of translational and clinical research on FLT3 inhibitors in AML, and discusses novel combination approaches. Mol Cancer Ther; 16(6); 991-1001. ©2017 AACR . ©2017 American Association for Cancer Research.

Iron overload may promote alteration of NK cells and hematopoietic stem/progenitor cells by JNK and P38 pathway in myelodysplastic syndromes.

PubMed

Hua, Yanni; Wang, Chaomeng; Jiang, Huijuan; Wang, Yihao; Liu, Chunyan; Li, Lijuan; Liu, Hui; Shao, Zonghong; Fu, Rong

2017-08-01

The objective of the study was to examine levels of intracellular iron, reactive oxygen species (ROS) and the expression of JNK and p38MAPK in NK cells and hematopoietic stem/progenitor cells (HSPCs) in MDS patients, and explore potential mechanisms by which iron overload (IOL) promotes MDS progression. Thirty-four cases of MDS and six cases of AML transformed from MDS (MDS/AML) were included. HSPCs and NK cells were isolated by magnetic absorption cell sorting. We used flow cytometry to detect the levels of ROS and intracellular JNK and P38 in NK cells and HSPCs. Total RNA and protein were extracted from NK cells and CD34 + cells to examine the expression of JNK and p38MAPK using RT-PCR and Western blotting. Intracellular iron concentration was detected. Data were analyzed by SPSS 21 statistical software. Intracellular iron concentration and ROS were increased in both NK cells and HSPCs in MDS patients with iron overload (P < 0.05). MDS patients with iron overload had higher JNK expression and lower p38 expression in NK cells, and higher p38 expression in HSPCs compared with non-iron overload group. IOL may cause alterations in NK cells and HSPCs through the JNK and p38 pathways, and play a role in the transformation to AML from MDS.

Gene stage-specific expression in the microenvironment of pediatric myelodysplastic syndromes.

PubMed

Roela, Rosimeire A; Carraro, Dirce M; Brentani, Helena P; Kaiano, Jane H L; Simão, Daniel F; Guarnieiro, Roberto; Lopes, Luiz Fernando; Borojevic, Radovan; Brentani, M Mitzi

2007-05-01

Using cDNA microarray assays we have observed a clear difference in the gene expression pattern between bone marrow stromal cells obtained from healthy children (CT) and from pediatric patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) associated with MDS (MDS-AML). The global gene function profiling analysis indicated that in the pediatric MDS microenvironment the disease stages may be characterized mainly by underexpression of genes associated with biological processes such as transport. Furthermore, a subset of downregulated genes related to endocytosis and protein secretion was able to discriminate MDS from MDS-AML.

Comparison of matched sibling donors versus unrelated donors in allogeneic stem cell transplantation for primary refractory acute myeloid leukemia: a study on behalf of the Acute Leukemia Working Party of the EBMT.

PubMed

Brissot, Eolia; Labopin, Myriam; Stelljes, Matthias; Ehninger, Gerhard; Schwerdtfeger, Rainer; Finke, Jürgen; Kolb, Hans-Jochem; Ganser, Arnold; Schäfer-Eckart, Kerstin; Zander, Axel R; Bunjes, Donald; Mielke, Stephan; Bethge, Wolfgang A; Milpied, Noël; Kalhs, Peter; Blau, Igor-Woflgang; Kröger, Nicolaus; Vitek, Antonin; Gramatzki, Martin; Holler, Ernst; Schmid, Christoph; Esteve, Jordi; Mohty, Mohamad; Nagler, Arnon

2017-06-24

Primary refractory acute myeloid leukemia (PRF-AML) is associated with a dismal prognosis. Allogeneic stem cell transplantation (HSCT) in active disease is an alternative therapeutic strategy. The increased availability of unrelated donors together with the significant reduction in transplant-related mortality in recent years have opened the possibility for transplantation to a larger number of patients with PRF-AML. Moreover, transplant from unrelated donors may be associated with stronger graft-mediated anti-leukemic effect in comparison to transplantations from HLA-matched sibling donor, which may be of importance in the setting of PRF-AML. The current study aimed to address the issue of HSCT for PRF-AML and to compare the outcomes of HSCT from matched sibling donors (n = 660) versus unrelated donors (n = 381), for patients with PRF-AML between 2000 and 2013. The Kaplan-Meier estimator, the cumulative incidence function, and Cox proportional hazards regression models were used where appropriate. HSCT provide patients with PRF-AML a 2-year leukemia-free survival and overall survival of about 25 and 30%, respectively. In multivariate analysis, two predictive factors, cytogenetics and time from diagnosis to transplant, were associated with lower leukemia-free survival, whereas Karnofsky performance status at transplant ≥90% was associated with better leukemia-free survival (LFS). Concerning relapse incidence, cytogenetics and time from diagnosis to transplant were associated with increased relapse. Reduced intensity conditioning regimen was the only factor associated with lower non-relapse mortality. HSCT was able to rescue about one quarter of the patients with PRF-AML. The donor type did not have any impact on PRF patients' outcomes. In contrast, time to transplant was a major prognostic factor for LFS. For patients with PRF-AML who do not have a matched sibling donor, HSCT from an unrelated donor is a suitable option, and therefore, initiation of an early search for allocating a suitable donor is indicated.

Acute myeloblastic leukemia with minimal myeloid differentiation (FAB AML-M0): a study of eleven cases.

PubMed

Sempere, A; Jarque, I; Guinot, M; Palau, J; García, R; Sanz, G F; Gomis, F; Pérez-Sirvent, M L; Senent, L; Sanz, M A

1993-12-01

The main clinical, morphological, cytochemical, immunological features and therapy results of eleven patients diagnosed as acute myeloblastic leukemia M0 (AML-M0) are reported here. There were no clinical characteristics, abnormalities on physical examination or initial laboratory parameters that distinguished these eleven patients. Bone marrow aspirates were hypocellular in four patients. The leukemic cells were undifferentiated by light microscopy and myeloperoxidase (MPO) and/or Sudan Black B (SBB) stains were negative in all cases. Myeloid differentiation antigens were present on the leukemic cells of all eleven patients, whereas B and T cell markers were clearly negative except for CD4 and CD7 antigens. Whatever the treatment employed survival was very short. Eight of the eleven patients were treated and two achieved complete remission (CR) but only one of them is alive in continuous CR. Our results like those previously reported, suggest that AML-M0 patients have a very poor prognosis with standard induction therapies and should perhaps be considered for experimental therapeutic approaches.

miR-133 regulates Evi1 expression in AML cells as a potential therapeutic target.

PubMed

Yamamoto, Haruna; Lu, Jun; Oba, Shigeyoshi; Kawamata, Toyotaka; Yoshimi, Akihide; Kurosaki, Natsumi; Yokoyama, Kazuaki; Matsushita, Hiromichi; Kurokawa, Mineo; Tojo, Arinobu; Ando, Kiyoshi; Morishita, Kazuhiro; Katagiri, Koko; Kotani, Ai

2016-01-12

The Ecotropic viral integration site 1 (Evi1) is a zinc finger transcription factor, which is located on chromosome 3q26, over-expression in some acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Elevated Evi1 expression in AML is associated with unfavorable prognosis. Therefore, Evi1 is one of the strong candidate in molecular target therapy for the leukemia. MicroRNAs (miRNAs) are small non-coding RNAs, vital to many cell functions that negatively regulate gene expression by translation or inducing sequence-specific degradation of target mRNAs. As a novel biologics, miRNAs is a promising therapeutic target due to its low toxicity and low cost. We screened miRNAs which down-regulate Evi1. miR-133 was identified to directly bind to Evi1 to regulate it. miR-133 increases drug sensitivity specifically in Evi1 expressing leukemic cells, but not in Evi1-non-expressing cells The results suggest that miR-133 can be promising therapeutic target for the Evi1 dysregulated poor prognostic leukemia.

Outcome of children with acute myeloid leukaemia (AML) experiencing primary induction failure in the AIEOP AML 2002/01 clinical trial.

PubMed

Quarello, Paola; Fagioli, Franca; Basso, Giuseppe; Putti, Maria C; Berger, Massimo; Luciani, Matteo; Rizzari, Carmelo; Menna, Giuseppe; Masetti, Riccardo; Locatelli, Franco

2015-11-01

Paediatric patients with acute myeloid leukaemia (AML) who fail induction due to primary resistance to chemotherapy account for a significant proportion of cases and have a particularly dismal prognosis. We report the clinical and biological data, and final outcome of 48 paediatric patients with primary-resistant AML enrolled in the Associazione Italiana di Ematologia e Oncologia Pediatrica AML 2002/01 clinical trial. These patients had a significantly higher white blood cell count at diagnosis compared to other AML patients. Cytogenetic and molecular features did not differ between patients with primary induction failure and patients allocated to the high-risk group. For the whole patient population, the probability of overall survival, event-free survival (EFS) and disease-free survival (DFS) was 21·8% ± 6·2, 20·4% ± 5·9, and 49·5% ± 11·3, respectively. Twenty-eight (58%) patients received haematopoietic stem cell transplantation (HSCT); 3 were autologous and 25 were allogeneic. Patients who underwent HSCT had improved EFS (31·2% vs. 5%, P < 0·0001). Only one of the 20 patients who did not receive HSCT is alive and disease free. The 19 patients in complete remission at time of HSCT showed significantly better DFS than the 9 with active disease (46% vs. 0%, P = 0·02). This study represents one of the largest series with long-term follow up of paediatric AML patients with primary refractory disease. Children who underwent transplantation had an encouraging long-term outcome. Disease recurrence remains the major cause of treatment failure; a better understanding of the disease biology is desirable to develop more effective treatment strategies. © 2015 John Wiley & Sons Ltd.

Musashi-2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia

PubMed Central

Kharas, Michael G; Lengner, Christopher J; Al-Shahrour, Fatima; Bullinger, Lars; Ball, Brian; Zaidi, Samir; Morgan, Kelly; Tam, Winnie; Paktinat, Mahnaz; Okabe, Rachel; Gozo, Maricel; Einhorn, William; Lane, Steven W; Scholl, Claudia; Fröhling, Stefan; Fleming, Mark; Ebert, Benjamin L; Gilliland, D Gary; Jaenisch, Rudolf; Daley, George Q

2011-01-01

RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia–associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML). PMID:20616797

Lower frequency of NPM1 and FLT3-ITD mutations in a South African adult de novo AML cohort

PubMed Central

Marshall, R. C.; Tlagadi, A.; Bronze, M.; Kana, V.; Naidoo, S.; Wiggill, T. M.; Carmona, S. C.

2014-01-01

Introduction Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of haemopoietic progenitor cells diagnosed in individuals of any age, but with a median age of 67 years at presentation in adults. Assessment of the mutation status of Nucleophosmin protein-1 (NPM1) and FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) are essential for the diagnosis, prognosis and treatment of AML. Methods A total of 160 de novo AML cases, both cytogenetically normal and abnormal, were analyzed for the presence of NPM1 and FLT3-ITD mutations and the results assessed in conjunction with epidemiological, clinical and laboratory findings. Results NPM1 mutations were found in 7.5%, while FLT3-ITD was present in 12% of these cases. Both of these were lower than expected. The median age at diagnosis of AML was 41 years and for the FLT3-ITD only cases, median age was 33 years; these ages were younger than expected. Conclusion The lower reported frequencies and younger median age at diagnosis of AML and these specific mutations may be contributed to by a number of factors including; effects of race on age of presentation, inclusion of patients diagnosed with de novo AML only and a generally younger median age of the South African population. PMID:24666762

Aberrant expression of CKLF-like MARVEL transmembrane member 5 (CMTM5) by promoter methylation in myeloid leukemia.

PubMed

Niu, Jihong; Li, Henan; Zhang, Yao; Li, Jinlan; Xie, Min; Li, Lingdi; Qin, Xiaoying; Qin, Yazhen; Guo, Xiaohuan; Jiang, Qian; Liu, Yanrong; Chen, Shanshan; Huang, Xiaojun; Han, Wenling; Ruan, Guorui

2011-06-01

CMTM5 has been shown to exhibit tumor suppressor activities, however, its role in leukemia is unclear. Herein we firstly reported the expression and function of CMTM5 in myeloid leukemia. CMTM5 was down-regulated, or undetectable, in leukemia cell lines and bone marrow cells from leukemia patients with promoter methylation. Ectopic expression of CMTM5-v1 strongly inhibited the proliferation of K562 and MEG-01 cells. In addition, significant negative correlations were observed between CMTM5 and three leukemia-specific fusion genes (AML1-ETO, PML-RARα and BCR/ABL1). CMTM5 expression was up-regulated in patients who had undergone treatment. Therefore, CMTM5 may be involved in the pathomechanism of myeloid leukemias. Copyright © 2010 Elsevier Ltd. All rights reserved.

1:100,000-scale Counties of the United States

USGS Publications Warehouse

,

1993-01-01

This is a coverage of the county boundaries of the conterminous United States (AK, HI and Puerto Rico are available separately). The lines were extracted from U.S. Census TIGER/line files using an AML program (see 4.), written by Doug Nebert and Mark Negri, running on two Data General 6220 servers.

Antileukaemic effect of PI3K-mTOR inhibitors in acute myeloid leukaemia-gene expression profiles reveal CDC25B expression as determinate of pharmacological effect.

PubMed

Reikvam, Håkon; Tamburini, Jerome; Skrede, Silje; Holdhus, Rita; Poulain, Laury; Ersvaer, Elisabeth; Hatfield, Kimberley J; Bruserud, Øystein

2014-01-01

Acute myeloid leukaemia (AML) is a heterogeneous malignancy. Intracellular signalling through the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway is important for regulation of cellular growth and metabolism, and inhibitors of this pathway is considered for AML treatment. Primary human AML cells, derived from 96 consecutive adult patients, were examined. The effects of two mTOR inhibitors (rapamycin, temsirolimus) and two PI3K inhibitors (GDC-0941, 3-methyladenine) were studied, and we investigated cytokine-dependent proliferation, regulation of apoptosis and global gene expression profiles. Only a subset of patients demonstrated strong antiproliferative effects of PI3K-mTOR inhibitors. Unsupervised hierarchical clustering analysis identified two main clusters of patients; one subset showing weak or absent antiproliferative effects (59%) and another group showing a strong growth inhibition for all drugs and concentrations examined (41%). Global gene expression analyses showed that patients with AML cell resistance against PI3K-mTOR inhibitors showed increased mRNA expression of the CDC25B gene that encodes the cell cycle regulator Cell Division Cycle 25B. The antileukaemic effect of PI3K-Akt-mTOR inhibition varies between patients, and resistance to these inhibitors is associated with the expression of the cell cycle regulator CDC25B, which is known to crosstalk with the PI3K-Akt-mTOR pathway and mediate rapamycin resistance in experimental models. © 2013 John Wiley & Sons Ltd.

Evolution of acute myelogenous leukemia stem cell properties after treatment and progression.

PubMed

Ho, Tzu-Chieh; LaMere, Mark; Stevens, Brett M; Ashton, John M; Myers, Jason R; O'Dwyer, Kristen M; Liesveld, Jane L; Mendler, Jason H; Guzman, Monica; Morrissette, Jennifer D; Zhao, Jianhua; Wang, Eunice S; Wetzler, Meir; Jordan, Craig T; Becker, Michael W

2016-09-29

Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered. © 2016 by The American Society of Hematology.

Evolution of acute myelogenous leukemia stem cell properties after treatment and progression

PubMed Central

Ho, Tzu-Chieh; LaMere, Mark; Stevens, Brett M.; Ashton, John M.; Myers, Jason R.; O’Dwyer, Kristen M.; Liesveld, Jane L.; Mendler, Jason H.; Guzman, Monica; Morrissette, Jennifer D.; Zhao, Jianhua; Wang, Eunice S.; Wetzler, Meir; Jordan, Craig T.

2016-01-01

Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered. PMID:27421961

Preleukemic and second-hit mutational events in an acute myeloid leukemia patient with a novel germline RUNX1 mutation.

PubMed

Ng, Isaac Ks; Lee, Joanne; Ng, Christopher; Kosmo, Bustamin; Chiu, Lily; Seah, Elaine; Mok, Michelle Meng Huang; Tan, Karen; Osato, Motomi; Chng, Wee-Joo; Yan, Benedict; Tan, Lip Kun

2018-01-01

Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations ( RUNX1 , GATA2 , DNMT3A , BCORL1 , BCOR , 2 PHF6 and CDKN2A ) were identified in the pre-induction sample, of which 5 remained ( RUNX1 , DNMT3A , BCORL1 , BCOR and 1 out of 2 PHF6 ) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1 -mutated state. On the contrary, the 5 mutations that persisted post-treatment, including the germline RUNX1 mutation, were likely to be part of the preleukemic clone. Further studies are necessary to assess the prevalence of these preleukemic and secondary mutations in the larger FPD/AML patient cohort and establish their prognostic significance. Given the molecular heterogeneity of FPD/AML and other AML subtypes, a better understanding of mutational classes and their involvement in AML pathogenesis can improve risk stratification of patients for more effective and targeted therapy.

Heterogeneous effects of M-CSF isoforms on the progression of MLL-AF9 leukemia.

PubMed

Wang, Rong; Feng, Wenli; Yang, Feifei; Yang, Xiao; Wang, Lina; Chen, Chong; Hu, Yuting; Ren, Qian; Zheng, Guoguang

2018-02-01

Macrophage colony-stimulating factor (M-CSF) regulates both malignant cells and microenvironmental cells. Its splicing isoforms show functional heterogeneity. However, their roles on leukemia have not been well established. Here, the expression of total M-CSF in patients with hematopoietic malignancies was analyzed. The roles of M-CSF isoforms on the progression of acute myeloid leukemia (AML) were studied by establishing MLL-AF9-induced mouse AML models with high level membrane-bound M-CSF (mM-CSF) or soluble M-CSF (sM-CSF). Total M-CSF was highly expressed in myeloid leukemia patients. Furthermore, mM-CSF but not sM-CSF prolonged the survival of leukemia mice. While sM-CSF was more potent to promote proliferation and self-renew, mM-CSF was more potent to promote differentiation. Moreover, isoforms had different effects on leukemia-associated macrophages (LAMs) though they both increase monocytes/macrophages by growth-promoting and recruitment effects. In addition, mM-CSF promoted specific phagocytosis of leukemia cells by LAMs. RNA-seq analysis revealed that mM-CSF enhanced phagocytosis-associated genes and activated oxidative phosphorylation and metabolism pathway. These results highlight heterogeneous effects of M-CSF isoforms on AML progression and the mechanisms of mM-CSF, that is, intrinsically promoting AML cell differentiation and extrinsically enhancing infiltration of macrophages and phagocytosis by macrophages, which may provide potential clues for clinical diagnosis and therapy. © 2017 Australasian Society for Immunology Inc.

TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements.

PubMed

Walsh, Mary F; Ampasala, Dinakar R; Rishi, Arun K; Basson, Marc D

2009-02-01

TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.

Regression Analysis of Combined Gene Expression Regulation in Acute Myeloid Leukemia

PubMed Central

Li, Yue; Liang, Minggao; Zhang, Zhaolei

2014-01-01

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (Regression Analysis of Combined Expression Regulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level. PMID:25340776

Stem Cell Modeling of Core Binding Factor Acute Myeloid Leukemia

PubMed Central

Mosna, Federico

2016-01-01

Even though clonally originated from a single cell, acute leukemia loses its homogeneity soon and presents at clinical diagnosis as a hierarchy of cells endowed with different functions, of which only a minority possesses the ability to recapitulate the disease. Due to their analogy to hematopoietic stem cells, these cells have been named “leukemia stem cells,” and are thought to be chiefly responsible for disease relapse and ultimate survival after chemotherapy. Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) is cytogenetically characterized by either the t(8;21) or the inv(16)/t(16;16) chromosomal abnormalities, which, although being pathognomonic, are not sufficient per se to induce overt leukemia but rather determine a preclinical phase of disease when preleukemic subclones compete until the acquisition of clonal dominance by one of them. In this review we summarize the concepts regarding the application of the “leukemia stem cell” theory to the development of CBF AML; we will analyze the studies investigating the leukemogenetic role of t(8;21) and inv(16)/t(16;16), the proposed theories of its clonal evolution, and the role played by the hematopoietic niches in preserving the disease. Finally, we will discuss the clinical implications of stem cell modeling of CBF AML for the therapy of the disease. PMID:26880987

Iron overload promotes erythroid apoptosis through regulating HIF-1a/ROS signaling pathway in patients with myelodysplastic syndrome.

PubMed

Zheng, Qing-Qing; Zhao, You-Shan; Guo, Juan; Zhao, Si-da; Song, Lu-Xi; Fei, Cheng-Ming; Zhang, Zheng; Li, Xiao; Chang, Chun-Kang

2017-07-01

Erythroid apoptosis increases significantly in myelodysplastic syndrome (MDS) patients with iron overload, but the underlying mechanism is not fully clear. In this study, we aim to explore the effect of HIF-1a/ROS on erythroid apoptosis in MDS patients with iron overload. We found that iron overload injured cellular functions through up-regulating ROS levels in MDS/AML cells, including inhibited cell viability, increased cell apoptosis and blocked cell cycle at G0/G1 phase. Interestingly, overexpression of hypoxia inducible factor-1a (HIF-1a), which was under-expressed in iron overload models, reduced ROS levels and attenuated cell damage caused by iron overload in MDS/AML cells. And gene knockdown of HIF-1a got the similar results as iron overload in MDS/AML cells. Furthermore, iron overload caused high erythroid apoptosis was closely related with ROS in MDS patients. Importantly, the HIF-1a protein levels of erythrocytes elevated obviously after incubation with desferrioxamine (DFO) from MDS patients with iron overload, accompanied by ROS levels inhibited and erythroid apoptosis reduced. Taken together, our findings determine that the HIF-1a/ROS signaling pathway plays a key role in promoting erythroid apoptosis in MDS patients with iron overload. Copyright © 2017 Elsevier Ltd. All rights reserved.

An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia.

PubMed

Leong, Steven R; Sukumaran, Siddharth; Hristopoulos, Maria; Totpal, Klara; Stainton, Shannon; Lu, Elizabeth; Wong, Alfred; Tam, Lucinda; Newman, Robert; Vuillemenot, Brian R; Ellerman, Diego; Gu, Chen; Mathieu, Mary; Dennis, Mark S; Nguyen, Allen; Zheng, Bing; Zhang, Crystal; Lee, Genee; Chu, Yu-Waye; Prell, Rodney A; Lin, Kedan; Laing, Steven T; Polson, Andrew G

2017-02-02

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML. © 2017 by The American Society of Hematology.

Subtype Differentiation of Small (≤ 4 cm) Solid Renal Mass Using Volumetric Histogram Analysis of DWI at 3-T MRI.

PubMed

Li, Anqin; Xing, Wei; Li, Haojie; Hu, Yao; Hu, Daoyu; Li, Zhen; Kamel, Ihab R

2018-05-29

The purpose of this article is to evaluate the utility of volumetric histogram analysis of apparent diffusion coefficient (ADC) derived from reduced-FOV DWI for small (≤ 4 cm) solid renal mass subtypes at 3-T MRI. This retrospective study included 38 clear cell renal cell carcinomas (RCCs), 16 papillary RCCs, 18 chromophobe RCCs, 13 minimal fat angiomyolipomas (AMLs), and seven oncocytomas evaluated with preoperative MRI. Volumetric ADC maps were generated using all slices of the reduced-FOV DW images to obtain histogram parameters, including mean, median, 10th percentile, 25th percentile, 75th percentile, 90th percentile, and SD ADC values, as well as skewness, kurtosis, and entropy. Comparisons of these parameters were made by one-way ANOVA, t test, and ROC curves analysis. ADC histogram parameters differentiated eight of 10 pairs of renal tumors. Three subtype pairs (clear cell RCC vs papillary RCC, clear cell RCC vs chromophobe RCC, and clear cell RCC vs minimal fat AML) were differentiated by mean ADC. However, five other subtype pairs (clear cell RCC vs oncocytoma, papillary RCC vs minimal fat AML, papillary RCC vs oncocytoma, chromophobe RCC vs minimal fat AML, and chromophobe RCC vs oncocytoma) were differentiated by histogram distribution parameters exclusively (all p < 0.05). Mean ADC, median ADC, 75th and 90th percentile ADC, SD ADC, and entropy of malignant tumors were significantly higher than those of benign tumors (all p < 0.05). Combination of mean ADC with histogram parameters yielded the highest AUC (0.851; sensitivity, 80.0%; specificity, 86.1%). Quantitative volumetric ADC histogram analysis may help differentiate various subtypes of small solid renal tumors, including benign and malignant lesions.