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Sample records for flt3 ligand targeted

  1. Eradication of Acute Myeloid Leukemia with FLT3 Ligand-Targeted miR-150 Nanoparticles.

    PubMed

    Jiang, Xi; Bugno, Jason; Hu, Chao; Yang, Yang; Herold, Tobias; Qi, Jun; Chen, Ping; Gurbuxani, Sandeep; Arnovitz, Stephen; Strong, Jennifer; Ferchen, Kyle; Ulrich, Bryan; Weng, Hengyou; Wang, Yungui; Huang, Hao; Li, Shenglai; Neilly, Mary Beth; Larson, Richard A; Le Beau, Michelle M; Bohlander, Stefan K; Jin, Jie; Li, Zejuan; Bradner, James E; Hong, Seungpyo; Chen, Jianjun

    2016-08-01

    Acute myeloid leukemia (AML) is a common and fatal form of hematopoietic malignancy. Overexpression and/or mutations of FLT3 have been shown to occur in the majority of cases of AML. Our analysis of a large-scale AML patient cohort (N = 562) indicates that FLT3 is particularly highly expressed in some subtypes of AML, such as AML with t(11q23)/MLL-rearrangements or FLT3-ITD. Such AML subtypes are known to be associated with unfavorable prognosis. To treat FLT3-overexpressing AML, we developed a novel targeted nanoparticle system: FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) nanosized dendriplex encapsulating miR-150, a pivotal tumor suppressor and negative regulator of FLT3 We show that the FLT3L-guided miR-150 nanoparticles selectively and efficiently target FLT3-overexpressing AML cells and significantly inhibit viability/growth and promote apoptosis of the AML cells. Our proof-of-concept animal model studies demonstrate that the FLT3L-guided miR-150 nanoparticles tend to concentrate in bone marrow, and significantly inhibit progression of FLT3-overexpressing AML in vivo, while exhibiting no obvious side effects on normal hematopoiesis. Collectively, we have developed a novel targeted therapeutic strategy, using FLT3L-guided miR-150-based nanoparticles, to treat FLT3-overexpressing AML with high efficacy and minimal side effects. Cancer Res; 76(15); 4470-80. ©2016 AACR. PMID:27280396

  2. Serum concentrations of Flt-3 ligand in rheumatic diseases.

    PubMed

    Nakamura, Kayo; Nakatsuka, Noriko; Jinnin, Masatoshi; Makino, Takamitsu; Kajihara, Ikko; Makino, Katsunari; Honda, Noritoshi; Inoue, Kuniko; Fukushima, Satoshi; Ihn, Hironobu

    2015-10-01

    Fms-like tyrosine kinase 3 (Flt-3) is a cytokine receptor expressed on the surface of bone-marrow progenitor of hematopoietic cells. Flt-3 ligands are produced by peripheral blood mononuclear cells, and found in various human body fluids. Flt-3 signal is involved in the regulation of vessel formation as well as B cell differentiation, suggesting that Flt-3 signal contributes to the pathogenesis of vascular abnormalities and immune dysregulation in rheumatic diseases. The aim of the present study is to examine serum Flt-3 ligand levels in patients with various rheumatic diseases, and to evaluate the possibility that serum Flt-3 ligand levels can be a useful disease marker. Sera were obtained from 20 dermatomyositis (DM) patients, 36 systemic sclerosis (SSc) patients, 10 systemic lupus erythematosus (SLE) patients, 10 scleroderma spectrum disorder (SSD) patients, 4 mixed connective tissue disease (MCTD) patients, and 12 normal subjects. Flt-3 ligand levels were determined with ELISA. Serum Flt-3 ligand levels were significantly elevated in patients with DM, SSc, SSD and MCTD compared to those in normal subjects. DM patients with elevated Flt-3 ligand levels were accompanied with significantly increased CRP levels and increased frequency of heliotrope rash than those with normal levels. In addition, SSc patients with elevated Flt-3 ligand levels showed significantly reduced frequency of nailfold bleeding. Serum Flt-3 ligand levels can be a marker of cutaneous manifestation in DM and a marker of microangiopathy in SSc. Clarifying the role of Flt-3 ligand in rheumatic diseases may lead to further understanding of these diseases and new therapeutic approaches. PMID:26559027

  3. Computer aided drug discovery of highly ligand efficient, low molecular weight imidazopyridine analogs as FLT3 inhibitors

    PubMed Central

    Frett, Brendan; McConnell, Nick; Smith, Catherine C.; Wang, Yuanxiang; Shah, Neil P.; Li, Hong-yu

    2015-01-01

    The FLT3 kinase represents an attractive target to effectively treat AML. Unfortunately, no FLT3 targeted therapeutic is currently approved. In line with our continued interests in treating kinase related disease for anti-FLT3 mutant activity, we utilized pioneering synthetic methodology in combination with computer aided drug discovery and identified low molecular weight, highly ligand efficient, FLT3 kinase inhibitors. Compounds were analyzed for biochemical inhibition, their ability to selectively inhibit cell proliferation, for FLT3 mutant activity, and preliminary aqueous solubility. Validated hits were discovered that can serve as starting platforms for lead candidates. PMID:25765758

  4. Computer aided drug discovery of highly ligand efficient, low molecular weight imidazopyridine analogs as FLT3 inhibitors.

    PubMed

    Frett, Brendan; McConnell, Nick; Smith, Catherine C; Wang, Yuanxiang; Shah, Neil P; Li, Hong-yu

    2015-04-13

    The FLT3 kinase represents an attractive target to effectively treat AML. Unfortunately, no FLT3 targeted therapeutic is currently approved. In line with our continued interests in treating kinase related disease for anti-FLT3 mutant activity, we utilized pioneering synthetic methodology in combination with computer aided drug discovery and identified low molecular weight, highly ligand efficient, FLT3 kinase inhibitors. Compounds were analyzed for biochemical inhibition, their ability to selectively inhibit cell proliferation, for FLT3 mutant activity, and preliminary aqueous solubility. Validated hits were discovered that can serve as starting platforms for lead candidates. PMID:25765758

  5. Flt3 Ligand Regulates the Development of Innate Lymphoid Cells in Fetal and Adult Mice.

    PubMed

    Baerenwaldt, Anne; von Burg, Nicole; Kreuzaler, Matthias; Sitte, Selina; Horvath, Edit; Peter, Annick; Voehringer, David; Rolink, Antonius G; Finke, Daniela

    2016-03-15

    Flt3 ligand (Flt3L) promotes survival of lymphoid progenitors in the bone marrow and differentiation of dendritic cells (DCs), but its role in regulating innate lymphoid cells (ILCs) during fetal and adult life is not understood. By using Flt3L knockout and transgenic mice, we demonstrate that Flt3L controls ILC numbers by regulating the pool of α4β7(-) and α4β7(+) lymphoid tissue inducer cell progenitors in the fetal liver and common lymphoid progenitors in the bone marrow. Deletion of flt3l severely reduced the number of fetal liver progenitors and lymphoid tissue inducer cells in the neonatal intestine, resulting in impaired development of Peyer's patches. In the adult intestine, NK cells and group 2 and 3 ILCs were severely reduced. This effect occurred independently of DCs as ILC numbers were normal in mice in which DCs were constitutively deleted. Finally, we could show that administration of Flt3L increased the number of NKp46(-) group 3 ILCs in wild-type and even in Il7(-/-) mice, which generally have reduced numbers of ILCs. Taken together, Flt3L significantly contributes to ILC and Peyer's patches development by targeting lymphoid progenitor cells during fetal and adult life. PMID:26851220

  6. Flt3 Ligand Is Essential for Survival and Protective Immune Responses during Toxoplasmosis.

    PubMed

    Dupont, Christopher D; Harms Pritchard, Gretchen; Hidano, Shinya; Christian, David A; Wagage, Sagie; Muallem, Gaia; Tait Wojno, Elia D; Hunter, Christopher A

    2015-11-01

    Dendritic cells (DCs) are critical for resistance to Toxoplasma gondii, and infection with this pathogen leads to increased numbers of DCs at local sites of parasite replication and in secondary lymphoid organs, but the factors that regulate this expansion are poorly understood. The cytokine Flt3 ligand (Flt3L) is critical for the generation and maintenance of DCs, and Flt3L(-/-) mice were found to be highly susceptible to acute toxoplasmosis. This phenotype correlated with decreased production of IL-12 and IFN-γ, as well as impaired NK cell responses. Surprisingly, despite low basal numbers of DCs, Flt3L(-/-) mice infected with T. gondii displayed an expansion of CD8α(+) and CD11b(lo)CD8α(-) DCs. Infection also induced an expansion of parasite-specific CD4(+) and CD8(+) T cells in Flt3L(-/-) mice; however, these cells were reduced in number and displayed impaired ability to produce IFN-γ relative to wild-type controls. Exogenous IL-12 treatment partially restored NK and T cell responses in Flt3L(-/-) mice, as well as acute resistance; however, these mice eventually succumbed to toxoplasmic encephalitis, despite the presence of large numbers of DCs and T cells in the brain. These results highlight the importance of Flt3L for resistance to toxoplasmosis and demonstrate the existence of Flt3L-independent pathways that can mediate infection-induced expansion of DCs and T cell priming. PMID:26385522

  7. Effect of FLT3 Ligand on Survival and Disease Phenotype in Murine Models Harboring a FLT3 Internal Tandem Duplication Mutation

    PubMed Central

    Bailey, Emily J; Duffield, Amy S; Greenblatt, Sarah M; Aplan, Peter D; Small, Donald

    2013-01-01

    Many of the mutations contributing to leukemogenesis in acute myeloid leukemia have been identified. A common activating mutation is an internal tandem duplication (ITD) mutation in the FLT3 gene that is found in approximately 25% of patients and confers a poor prognosis. FLT3 inhibitors have been developed and have some efficacy, but patients often relapse. Levels of FLT3 ligand (FL) are significantly elevated in patients during chemotherapy and may be an important component contributing to relapse. We used a mouse model to investigate the possible effect of FL expression on leukemogenesis involving FLT3-ITD mutations in an in vivo system. FLT3ITD/ITD FL−/− (knockout) mice had a statistically significant increase in survival compared with FLT3ITD/ITD FL+/+ (wildtype) mice, most of which developed a fatal myeloproliferative neoplasm. These findings suggest that FL levels may have prognostic significance in human patients. We also studied the effect of FL expression on survival in a FLT3-ITD NUP98–HOX13 (NHD13) fusion mouse model. These mice develop an aggressive leukemia with short latency. We asked whether FL expression played a similar role in this context. The NUP98-HOX13 FLT3ITD/wt FL−/− mice did not have a survival advantage, compared with NUP98-HOX13 FLT3ITD/wt FL+/+ mice (normal FL levels). The loss of the survival advantage of the FL knockout group in the NUP98–HOX13 model suggests that adding a second mutation changes the effect of FL expression in the context of more aggressive disease. PMID:23759524

  8. Barley as a green factory for the production of functional Flt3 ligand.

    PubMed

    Erlendsson, Lýdur S; Muench, Marcus O; Hellman, Ulf; Hrafnkelsdóttir, Soffía M; Jonsson, Anders; Balmer, Yves; Mäntylä, Einar; Orvar, Björn L

    2010-02-01

    Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors. PMID:19844912

  9. Lineage-specific STAT5 target gene activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype.

    PubMed

    Müller, T A; Grundler, R; Istvanffy, R; Rudelius, M; Hennighausen, L; Illert, A L; Duyster, J

    2016-08-01

    Mutations that activate FMS-like tyrosine kinase 3 (FLT3) are frequent occurrences in acute myeloid leukemia. Two distinct types of mutations have been described: internal duplication of the juxtamembranous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both mutations lead to constitutive FLT3 signaling, only FLT3-ITD strongly activates signal transducer and activator of transcription 5 (STAT5). In a murine transplantation model, FLT3-ITD induces a myeloproliferative neoplasm, whereas FLT3-TKD leads to a lymphoid malignancy with significantly longer latency. Here we report that the presence of STAT5 is critical for the development of a myeloproliferative disease by FLT3-ITD in mice. Deletion of Stat5 in FLT3-ITD-induced leukemogenesis leads not only to a significantly longer survival (82 vs 27 days) of the diseased mice, but also to an immunophenotype switch with expansion of the lymphoid cell compartment. Interestingly, we were able to show differential STAT5 activation in FLT3-ITD(+) myeloid and lymphoid murine progenitors. STAT5 target genes such as Oncostatin M were highly expressed in FLT3-ITD(+) myeloid but not in FLT3-ITD(+) lymphoid progenitor cells. Strikingly, FLT3-TKD expression in combination with Oncostatin M is sufficient to reverse the phenotype to a myeloproliferative disease in FLT3-TKD mice. Thus, lineage-specific STAT5 activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype in mice. PMID:27046463

  10. Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell–dependent induction of CD4+CD25+FoxP3+ Treg

    PubMed Central

    Biswas, Moanaro; Sarkar, Debalina; Kumar, Sandeep R. P.; Nayak, Sushrusha; Rogers, Geoffrey L.; Markusic, David M.; Liao, Gongxian; Terhorst, Cox

    2015-01-01

    CD4+CD25+FoxP3+ regulatory T cells (Treg) are critical elements for maintaining immune tolerance, for instance to exogenous antigens that are introduced during therapeutic interventions such as cell/organ transplant or gene/protein replacement therapy. Coadministration of antigen with rapamycin simultaneously promotes deletion of conventional CD4+ T cells and induction of Treg. Here, we report that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin. This occurs via selective expansion of plasmacytoid dendritic cells (pDCs), which further augments the number of Treg. Whereas in conventional DCs, rapamycin effectively blocks mammalian target of rapamycin (mTOR) 1 signaling induced by Flt3L, increased mTOR1 activity renders pDCs more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDCs. This concept is supported by the finding that Treg induction is abrogated upon pDC depletion. The combination with pDCs and rapamycin is requisite for Flt3L/antigen-induced Treg induction because Flt3L/antigen by itself fails to induce Treg. As coadministering Flt3L, rapamycin, and antigen blocked CD8+ T-cell and antibody responses in models of gene and protein therapy, we conclude that the differential effect of rapamycin on DC subsets can be exploited for improved tolerance induction. PMID:25833958

  11. Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy

    PubMed Central

    Warkentin, Alexander A; Lopez, Michael S; Lasater, Elisabeth A; Lin, Kimberly; He, Bai-Liang; Leung, Anskar YH; Smith, Catherine C; Shah, Neil P; Shokat, Kevan M

    2014-01-01

    Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a ‘synthetic lethal toxicity’ arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity. DOI: http://dx.doi.org/10.7554/eLife.03445.001 PMID:25531068

  12. FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.

    PubMed

    Kreiter, Sebastian; Diken, Mustafa; Selmi, Abderraouf; Petschenka, Jutta; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects. PMID:27236799

  13. Fms-like tyrosine kinase 3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse.

    PubMed

    Jacobsen, Rebecca N; Nowlan, Bianca; Brunck, Marion E; Barbier, Valerie; Winkler, Ingrid G; Levesque, Jean-Pierre

    2016-03-01

    The cytokines granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (Flt3-L) mobilize hematopoietic stem and progenitor cells into the peripheral blood of primates, humans, and mice. We recently reported that G-CSF administration causes a transient blockade of medullar erythropoiesis by suppressing erythroblastic island (EI) macrophages in the bone marrow. In the study described here, we investigated the effect of mobilizing doses of Flt3-L on erythropoiesis in mice in vivo. Similar to G-CSF, Flt3-L caused whitening of the bone marrow with significant reduction in the numbers of EI macrophages and erythroblasts. This was compensated by an increase in the numbers of EI macrophages and erythroblasts in the spleen. However, unlike G-CSF, Flt3-L had an indirect effect on EI macrophages, as it was not detected at the surface of EI macrophages or erythroid progenitors. PMID:26607596

  14. Flt3/Flk-2 ligand in combination with thrombopoietin decreases apoptosis in megakaryocyte development.

    PubMed

    Sigurjonsson, Olafur Eysteinn; Gudmundsson, Kristbjorn Orri; Haraldsdottir, Vilhelmina; Rafnar, Thorunn; Agnarsson, Bjarni A; Gudmundsson, Sveinn

    2004-04-01

    The growth factors thrombopoietin (TPO) and Flt3/Flk-2-ligand (FL), either independently or in combination, modulate megakaryocyte development. Our results show that bone marrow CD34+ cells cultured with TPO and FL differentiate at a slower rate into CD41+ cells and are delayed in apoptosis at the later stages of the cultures compared to cells cultured with TPO alone. Our data also show that FL in synergy with TPO may inhibit apoptosis in megakaryocyte development by up-regulating bcl-2 and inducing conformational changes of p53, in MK progenitors. FL in combination with TPO slows down maturation and consequently delays apoptosis of MK progenitor cells. PMID:15186734

  15. Flt3 is a target of coumestrol in protecting against UVB-induced skin photoaging.

    PubMed

    Park, Gaeun; Baek, Sohee; Kim, Jong-Eun; Lim, Tae-gyu; Lee, Charles C; Yang, Hee; Kang, Young-Gyu; Park, Jun Seong; Augustin, Martin; Mrosek, Michael; Lee, Chang Yong; Dong, Zigang; Huber, Robert; Lee, Ki Won

    2015-12-01

    While skin aging is a naturally occurring process by senescence, exposure to ultraviolet (UV) radiation accelerates wrinkle formation and sagging of skin. UV induces skin aging by degrading collagen via activating matrix metalloproteinases (MMPs). In this study, we show that coumestrol, a metabolite of the soybean isoflavone daidzein, has a preventive effect on skin photoaging in three-dimensional human skin equivalent model. Coumestrol inhibited UVB-induced MMP-1 expression and activity. Whole human kinase profiling assay identified FLT3 kinase as a novel target protein of coumestrol in UVB-induced signaling pathway in skin. Coumestrol suppresses FLT3 kinase activity, and subsequently, Ras/MEK/ERK and Akt/p70 ribosomal S6 kinase pathway. This suppresses AP-1 activity and in turn, diminishes MMP-1 gene transcription. Using X-ray crystallography, the binding of coumestrol to FLT3 was defined and implied ATP-competitive inhibition. Residues Lys644 and Phe830 showed local changes to accommodate coumestrol in the ATP-binding pocket. 4-APIA, a pharmacological inhibitor of FLT3, inhibited MMP-1 expression and induced signal transduction changes similar to coumestrol. Taken together, coumestrol inhibits UVB-induced MMP-1 expression by suppressing FLT3 kinase activity. These findings suggest that coumestrol is a novel dietary compound with potential application in preventing and improving UVB-associated skin aging. PMID:26341390

  16. In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development.

    PubMed

    Tsapogas, Panagiotis; Swee, Lee Kim; Nusser, Anja; Nuber, Natko; Kreuzaler, Matthias; Capoferri, Giuseppina; Rolink, Hannie; Ceredig, Rhodri; Rolink, Antonius

    2014-04-01

    Cytokines are essential regulators of hematopoiesis, acting in an instructive or permissive way. Fms-like tyrosine kinase 3 ligand (FLT3L) is an important cytokine for the development of several hematopoietic populations. Its receptor (FLT3) is expressed on both myeloid and lymphoid progenitors and deletion of either the receptor or its ligand leads to defective developmental potential of hematopoietic progenitors. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of this cytokine in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. These transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, the transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce the earliest erythroid progenitors when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential. PMID:24463214

  17. In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development

    PubMed Central

    Tsapogas, Panagiotis; Swee, Lee Kim; Nusser, Anja; Nuber, Natko; Kreuzaler, Matthias; Capoferri, Giuseppina; Rolink, Hannie; Ceredig, Rhodri; Rolink, Antonius

    2014-01-01

    Cytokines are essential regulators of hematopoiesis, acting in an instructive or permissive way. Fms-like tyrosine kinase 3 ligand (FLT3L) is an important cytokine for the development of several hematopoietic populations. Its receptor (FLT3) is expressed on both myeloid and lymphoid progenitors and deletion of either the receptor or its ligand leads to defective developmental potential of hematopoietic progenitors. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of this cytokine in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. These transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, the transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce the earliest erythroid progenitors when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential. PMID:24463214

  18. Thrombopoietin cooperates with FLT3-ligand in the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors

    PubMed Central

    Chen, Wei; Antonenko, Svetlana; Sederstrom, Joel M.; Liang, Xueqing; Chan, Anissa S. H.; Kanzler, Holger; Blom, Bianca; Blazar, Bruce R.; Liu, Yong-Jun

    2005-01-01

    Type 1 interferon–producing cells (IPCs), also known as plasmacytoid dendritic cell (DC) precursors, represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection, autoimmune SLE, and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3), IL-7, stem cell factor (SCF), macrophage–colony-stimulating factor (M-CSF), and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture, they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L, inducing CD34+HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 × 106 IPCs per 106 CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-α (IFN-α) and to differentiate into mature DCs. This culture system, combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF–mobilized donors, permits the generation of more than 109 IPCs from a single blood donor. PMID:14670916

  19. Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system.

    PubMed

    Zhang, Yan-Li; Chen, Song-Sen; Yang, Ke-Gong; Su, Lin; Deng, Yan-Chun; Liu, Chang-Zheng

    2005-08-01

    Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg/L. rhFL was purified to about 95% purity with overnight dialysis, filtration and an anion-exchange step. Further purification steps employing Sephacryl S-200 and reverse-phase HPLC raised the purity to over 99%. The purified rhFL possessed correct N-terminal amino acid sequence and positive Western blotting bands. SDS-PAGE and mass spectrometry analysis showed molecular weight of rhFL was about 21 and 34 kDa, suggesting that rhFL was glycosylated. The result of capillary electrophoresis showed that its pI is 3.12-4.72. Endo H deglycosylation analysis indicated that there was O-glycosylation besides N-glycosylation in rhFL secreted from P. pastoris. Bioactivity assay showed that the purified rhFL had dose-dependent expansion activity on bone marrow nucleated cells. PMID:15914030

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

    PubMed Central

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

    2015-01-01

    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. PMID:26292723

  1. Expression of GADS enhances FLT3-induced mitogenic signaling.

    PubMed

    Chougule, Rohit A; Cordero, Eugenia; Moharram, Sausan A; Pietras, Kristian; Rönnstrand, Lars; Kazi, Julhash U

    2016-03-22

    GADS is a member of a family of SH2 and SH3 domain-containing adaptors that functions in tyrosine kinase-mediated signaling cascades. Its expression is largely restricted to hematopoietic tissues and cell lines. Therefore, GADS is mainly involved in leukocyte-specific protein tyrosine kinase signaling. GADS is known to interact with tyrosine-phosphorylated SHC, BCR-ABL and KIT. The SH2 domain of GADS has a similar binding specificity to that of GRB2 but its SH3 domain displays a different binding specificity, and thus it is involved in other downstream signaling pathways than GRB2. In the present study, we examined the role of GADS in FLT3 signaling. FLT3 is a type III receptor tyrosine kinase, which is mutated in more than 30% of acute myeloid leukemia (AML) and the most common mutations is the internal tandem duplication (ITD) mutations. We observed that expression of GADS enhanced oncogenic FLT3-ITD-induced cell proliferation and colony formation in vitro. In a mouse xenograft model, GADS accelerated FLT3-ITD-dependent tumor formation. Furthermore, expression of GADS induced a transcriptional program leading to upregulation of MYC and mTORC1 target genes. GADS localizes to the cell membrane and strongly binds to ligand-stimulated wild-type FLT3 or is constitutively associated with the oncogenic mutant FLT3-ITD. We mapped the binding sites in FLT3 to pY955 and pY969 which overlaps with the GRB2 binding sites. Expression of GADS enhanced FLT3-mediated phosphorylation of AKT, ERK1/2, p38 and STAT5. Taken together, our data suggests that GADS is an important downstream component of FLT3 signaling and expression of GADS potentiates FLT3-mediated mitogenic signaling. PMID:26895103

  2. Expression of GADS enhances FLT3-induced mitogenic signaling

    PubMed Central

    Chougule, Rohit A.; Cordero, Eugenia; Moharram, Sausan A.; Pietras, Kristian; Rönnstrand, Lars; Kazi, Julhash U.

    2016-01-01

    GADS is a member of a family of SH2 and SH3 domain-containing adaptors that functions in tyrosine kinase-mediated signaling cascades. Its expression is largely restricted to hematopoietic tissues and cell lines. Therefore, GADS is mainly involved in leukocyte-specific protein tyrosine kinase signaling. GADS is known to interact with tyrosine-phosphorylated SHC, BCR-ABL and KIT. The SH2 domain of GADS has a similar binding specificity to that of GRB2 but its SH3 domain displays a different binding specificity, and thus it is involved in other downstream signaling pathways than GRB2. In the present study, we examined the role of GADS in FLT3 signaling. FLT3 is a type III receptor tyrosine kinase, which is mutated in more than 30% of acute myeloid leukemia (AML) and the most common mutations is the internal tandem duplication (ITD) mutations. We observed that expression of GADS enhanced oncogenic FLT3-ITD-induced cell proliferation and colony formation in vitro. In a mouse xenograft model, GADS accelerated FLT3-ITD-dependent tumor formation. Furthermore, expression of GADS induced a transcriptional program leading to upregulation of MYC and mTORC1 target genes. GADS localizes to the cell membrane and strongly binds to ligand-stimulated wild-type FLT3 or is constitutively associated with the oncogenic mutant FLT3-ITD. We mapped the binding sites in FLT3 to pY955 and pY969 which overlaps with the GRB2 binding sites. Expression of GADS enhanced FLT3-mediated phosphorylation of AKT, ERK1/2, p38 and STAT5. Taken together, our data suggests that GADS is an important downstream component of FLT3 signaling and expression of GADS potentiates FLT3-mediated mitogenic signaling. PMID:26895103

  3. 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

  4. Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand–receptor complexes

    PubMed Central

    Verstraete, Kenneth; Remmerie, Bert; Elegheert, Jonathan; Lintermans, Beatrice; Haegeman, Guy; Vanhoenacker, Peter; Van Craenenbroeck, Kathleen; Savvides, Savvas N.

    2011-01-01

    The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3D1–D5 and Flt3D1–D4, for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI−/−) that can secrete the target proteins with limited and homogeneous N-­linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor–ligand complexes yielded monodisperse complex prep­arations that were amenable to crystallization. Crystals of the Flt3D1–D4–FL and Flt3D1–D5–FL complexes diffracted to 4.3 and 7.8 Å resolution, respectively, and exhibited variable diffraction quality even within the same crystal. The resulting data led to the successful structure determination of Flt3D1–D4–FL via a combination of molecular-replacement and density-modification protocols exploiting the noncrystallographic symmetry and high solvent content of the crystals. PMID:21393836

  5. Discovery and Rational Design of Pteridin-7(8H)-one-Based Inhibitors Targeting FMS-like Tyrosine Kinase 3 (FLT3) and Its Mutants.

    PubMed

    Sun, Deheng; Yang, Yu; Lyu, Jiankun; Zhou, Wei; Song, Wenlin; Zhao, Zhenjiang; Chen, Zhuo; Xu, Yufang; Li, Honglin

    2016-07-14

    FLT3 has been validated as a therapeutic target for the treatment of acute myeloid leukemia (AML). In this paper, we describe for the first time, pteridin-7(8H)-one as a scaffold for potent FLT3 inhibitors derived from structural optimizations on irreversible EGFR inhibitors. The representative inhibitor (31) demonstrates single-digit nanomolar inhibition against FLT3 and subnanomolar KD for drug-resistance FLT3 mutants. In profiling of the in vitro tumor cell lines, it shows good selectivity against AML cells harboring FLT3-ITD mutations over other leukemia and solid tumor cell lines. The mechanism of action study illustrates that pteridin-7(8H)-one derivatives suppress the phosphorylation of FLT3 and its downstream pathways, thereby inducing G0/G1 cell cycle arrest and apoptosis in AML cells. In in vivo studies, 31 significantly suppresses the tumor growth in MV4-11 xenograft model. Overall, we provide a structurally distinct chemical scaffold with which to develop FLT3 mutants-selective inhibitors for AML treatment. PMID:27266526

  6. FMS-like tyrosine kinase 3 (FLT3) inhibitors: Molecular docking and experimental studies.

    PubMed

    Mashkani, Baratali; Tanipour, Mohammad Hossein; Saadatmandzadeh, Mohammad; Ashman, Leonie K; Griffith, Renate

    2016-04-01

    Activating mutations in FMS-like tyrosine kinase 3 (FLT3) occur in 25% of acute lymphoid and 30% of acute myeloid leukaemia cases. Therefore, FLT3 is a potential therapeutic target for small molecule kinase inhibitors. In this study, protein-ligand interactions between FLT3 and kinase inhibitors (CEP701, PKC412, sunitinib, imatinib and dasatinib) were obtained through homology modelling and molecular docking. A cellular system for experimental testing of the inhibitors was also established by expressing wildtype and internal tandem duplication mutant FLT3 (FLT3-WT and FLT3-ITD) in FDC-P1 cells. Imatinib and dasatinib could not be docked into any of the FLT3 models, consistent with their lack of activity in the experimental assays. CEP701, PKC412 and sunitinib interacted with the ATP-binding pocket of FLT3, forming H-bonds with Cys694 and Glu692. Based on the EC50 values in the cell proliferation assay, CEP701 was the most potent inhibitor; sunitinib and PKC412 were ranked second and third, respectively. Sunitinib was the most selective inhibitor, followed by PKC421 and CEP701. The potency of sunitinib and to a lesser extent CEP701 in inhibition of FLT3 autophosphorylation was lower than the cell proliferation inhibition, indicating that inhibition of FLT3 downstream proteins may contribute to the cellular effects. It was shown in this study that the docking procedure was able to differentiate FLT3 inhibitors from ineffective compounds. Additionally, interaction with the phosphate binding region in the ATP-binding pocket increased potency at the cost of selectivity. These findings can be applied in designing highly effective and selective inhibitors for FLT3 and other related kinases. PMID:26896780

  7. Flt3 ligand improves the innate response to respiratory syncytial virus and limits lung disease upon RSV reexposure in neonate mice.

    PubMed

    Remot, Aude; Descamps, Delphyne; Jouneau, Luc; Laubreton, Daphné; Dubuquoy, Catherine; Bouet, Stephan; Lecardonnel, Jérôme; Rebours, Emmanuelle; Petit-Camurdan, Agnès; Riffault, Sabine

    2016-04-01

    Respiratory syncytial virus (RSV) causes severe bronchiolitis in infants worldwide. The immunological factors responsible for RSV susceptibility in infants are poorly understood. Here, we used the BALB/c mouse model of neonatal RSV infection to study the mechanisms leading to severe disease upon reexposure to the virus when adults. Two major deficiencies in neonatal lung innate responses were found: a poor DCs mobilization, and a weak engagement of the IFNI pathway. The administration of Flt3 ligand (Flt3-L), a growth factor that stimulates the proliferation of hematopoietic cells, to neonates before RSV-infection, resulted in increased lung DC number, and reconditioned the IFNI pathway upon RSV neonatal infection. Besides, neonates treated with Flt3-L were protected against exacerbated airway disease upon adult reexposure to RSV. This was associated with a reorientation of RSV-specific responses toward Th1-mediated immunity. Thus, the poor lung DCs and IFNI responses to RSV in neonates may be partly responsible for the deleterious long-term consequences revealed upon adult reexposure to RSV, which could be prevented by Flt3-L treatment. These results open new perspectives for developing neonatal immuno-modulating strategies to reduce the burden of bronchiolitis. PMID:26681580

  8. FLT3 ligand administration after hematopoietic cell transplantation increases circulating dendritic cell precursors that can be activated by CpG oligodeoxynucleotides to enhance T-cell and natural killer cell function.

    PubMed

    Chen, Wei; Chan, Anissa S H; Dawson, Amanda J; Liang, Xueqing; Blazar, Bruce R; Miller, Jeffrey S

    2005-01-01

    Dendritic cells (DCs) are key effectors in innate immunity and play critical roles in triggering adaptive immune responses. FLT3 ligand (FLT3-L) is essential for DC development from hematopoietic progenitors. In a phase I clinical trial, we demonstrated that immunotherapy with subcutaneous injection of FLT3-L is safe and well tolerated in cancer patients recovering from autologous hematopoietic cell transplantation (HCT). FLT3-L administration significantly increased the frequency and absolute number of blood DC precursors without affecting other mature cell lineages during the 6-week course of FLT3-L therapy. After 14 days of FLT3-L administration, the number of blood CD11c + DCs, plasmacytoid DCs (PDCs), and CD14 + monocytes increased by 5.3-, 2.9-, 3.8-fold, respectively, and was maintained at increased levels throughout FLT3-L therapy. FLT3-L-increased blood DCs in HCT patients were immature and had modest enhancing effects on in vitro T-cell proliferation to antigens and natural killer (NK) cell function. The addition of type B CpG oligodeoxynucleotides (ODNs) to peripheral blood mononuclear cells obtained from HCT patients receiving FLT3-L therapy induced rapid maturation of both CD11c + DCs and PDCs and enhanced T-cell proliferative responses. In addition, CpG ODN induced potent activation of NK cells from FLT3-L-treated patients with increased surface CD69 expression and augmented cytotoxicity. CpG ODN-induced activation of NK cells was primarily via an indirect mechanism through PDCs. These findings suggest that FLT3-L mobilization of DC precursors followed by a specific DC stimulus such as CpG ODN may provide a novel strategy to manipulate antitumor immunity in patients after HCT. PMID:15625541

  9. Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus.

    PubMed

    Kwissa, Marcin; Amara, Rama R; Robinson, Harriet L; Moss, Bernard; Alkan, Sefik; Jabbar, Abdul; Villinger, Francois; Pulendran, Bali

    2007-10-29

    DNA vaccines offer promising strategies for immunization against infections. However, their clinical use requires improvements in immunogenicity. We explored the efficacy of Toll-like receptor (TLR) ligands (TLR-Ls) on augmenting the immunogenicity of a DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine against SIV. Rhesus macaques were injected with Fms-like tyrosine kinase 3 (Flt3)-ligand (FL) to expand dendritic cells (DCs) and were primed with a DNA vaccine encoding immunodeficiency virus antigens mixed with ligands for TLR9 or TLR7/8. Subsequently, the animals were boosted with DNA and twice with recombinant MVA expressing the same antigens. TLR9-L (CpG DNA) mediated activation of DCs in vivo and enhanced the magnitude of antigen-specific CD8(+) interferon (IFN) gamma(+) T cells and polyfunctional CD8(+) T cells producing IFN-gamma, tumor necrosis factor alpha, and interleukin 2. Although this trial was designed primarily as an immunogenicity study, we challenged the animals with pathogenic SIVmac(251) and observed a reduction in peak viremia and cumulative viral loads in the TLR9-L plus FL-adjuvanted group relative to the unvaccinated group; however, the study design precluded comparisons between the adjuvanted groups and the group vaccinated with DNA/MVA alone. Viral loads were inversely correlated with the magnitude and quality of the immune response. Thus, the immunogenicity of DNA vaccines can be augmented with TLR9-L plus FL. PMID:17954572

  10. The combination of FLT3 and DNA methyltransferase inhibition is synergistically cytotoxic to FLT3/ITD acute myeloid leukemia cells.

    PubMed

    Chang, E; Ganguly, S; Rajkhowa, T; Gocke, C D; Levis, M; Konig, H

    2016-05-01

    Effective treatment regimens for elderly acute myeloid leukemia (AML) patients harboring internal tandem duplication mutations in the FMS-like tyrosine kinase-3 (FLT3) gene (FLT3/ITD) are lacking and represent a significant unmet need. Recent data on the effects of FLT3 tyrosine kinase inhibitors on FLT3/ITD(+) AML showed promising clinical activity, including in elderly patients. DNA methyltransferase (DNMT) inhibitors such as decitabine (5-aza-2-deoxycytidine, DEC) and 5-azacitidine (AZA) demonstrated clinical benefit in AML, are well tolerated and are associated with minimal increases in FLT3 ligand, which can represent a potential resistance mechanism to FLT3 inhibitors. In addition, both FLT3 and DNMT inhibition are associated with the induction of terminal differentiation of myeloid blasts. Consequently, there is a strong theoretical rationale for combining FLT3 and DNMT inhibition for FLT3/ITD(+) AML. We therefore sought to study the anti-leukemic effects of DEC, AZA and FLT3 inhibitors, either as single agents or in combination, on AML cell lines and primary cells derived from newly diagnosed and relapsed AML patients. Our studies indicate that combined treatment using FLT3 inhibition and hypomethylation confers synergistic anti-leukemic effects, including apoptosis, growth inhibition and differentiation. The simultaneous administration of AZA and FLT3 inhibition appears to be the most efficacious combination in this regard. These drugs may provide a novel therapeutic approach for FLT3/ITD(+) AML, in particular for older patients. PMID:26686245

  11. Flt3/Flk-2-ligand in synergy with thrombopoietin delays megakaryocyte development and increases the numbers of megakaryocyte progenitor cells in serum-free cultures initiated with CD34+ cells.

    PubMed

    Sigurjónsson, Olafur Eysteinn; Gudmundsson, Kristbjörn Orri; Haraldsdóttir, Vilhelmína; Rafnar, Thorunn; Gudmundsson, Sveinn; Guomundsson, Kristbjörn Orri; Guomundsson, Sveinn

    2002-04-01

    Megakaryocytopoiesis involves proliferation and maturation of committed precursors that increase their size by polyploidy, a process that is believed to be critical for the efficient production and release of platelets. Thrombopoietin has been shown to act on proliferation, maturation, and survival pathways in megakaryocytopoiesis. Less is known about the role of Flt3/Flk-2-ligand in this development. Apoptosis has an important role in hematopoiesis in general. It has been shown to have an effect on senescent megakaryocytes but not megakaryocyte progenitor cells. In this study, a serum-free culture model was developed, differentiating bone marrow CD34(+) hematopoietic stem cells into megakaryocytes, using thrombopoietin and Flt3/Flk-2-ligand. The model was used to study the effect of these growth factors on expansion of megakaryocyte progenitor cells, differentiation of megakaryocytes, and ploidy. Our results demonstrate that bone marrow CD34(+) cells cultured with thrombopoietin and Flt3/Flk-2-ligand show a lower developmental rate into MK cells compared to cells cultured with thrombopoietin alone. Cells cultured with thrombopoietin and Flt3/Flk-2-ligand expressed less CD41, the ploidy level was lower, and they appeared less mature. On the other hand, the cells showed up to 10-fold increase in cell numbers compared to five-fold increase when cultured with thrombopoietin alone. These results suggest that Flt3/Flk-2-ligand in synergy with thrombopoietin may slow down megakaryocyte development by causing increased proliferation of megakaryocyte progenitor cells. PMID:11983110

  12. Priming of qualitatively superior human effector CD8+ T cells using TLR8 ligand combined with FLT3L1

    PubMed Central

    Afonso, Georgia; Larsen, Martin; Gostick, Emma; Price, David A; Mallone, Roberto; Appay, Victor

    2015-01-01

    The quality of antigen specific CD8+ T cell responses is central to immune efficacy in infectious and malignant settings. Inducing effector CD8+ T cells with potent functional properties is therefore a priority of immunotherapy. However, the optimal assessment of new therapeutic molecules or strategies in humans is limited by the models currently used. Here, we introduce an original model of in vitro CD8+ T cell priming, based on an accelerated dendritic cell coculture system, which uses unfractionated human PBMCs as the starting material. This approach allows for a rapid study of adjuvant effects on the functional properties of human CD8+ T cells, primed from antigen-specific naïve precursors. We demonstrate that a selective TLR8 agonist, in combination with FLT3L, primes high-quality CD8+ T cell responses. TLR8L/FLT3L primed CD8+ T cells displayed enhanced cytolytic activity, polyfunctionality and antigen sensitivity. The acquisition of this superior functional profile was associated with increased T-bet expression in T cells induced via an IL-12-dependent mechanism. Collectively, these data validate an expedited route to vaccine delivery or optimal T cell expansion for adoptive cell transfer. PMID:26608912

  13. The Development of Murine Plasmacytoid Dendritic Cell Precursors Is Differentially Regulated by FLT3-ligand and Granulocyte/Macrophage Colony-Stimulating Factor

    PubMed Central

    Gilliet, Michel; Boonstra, Andre; Paturel, Carine; Antonenko, Svetlana; Xu, Xiu-Ling; Trinchieri, Giorgio; O'Garra, Anne; Liu, Yong-Jun

    2002-01-01

    Plasmacytoid predendritic cells or type 1 interferon (IFN)-producing cells (IPCs) have recently been identified in mice. Although culture systems giving rise to different murine dendritic cell subsets have been established, the developmental regulation of murine plasmacytoid IPCs and the culture conditions leading to their generation remain unknown. Here we show that large numbers of over 40% pure CD11c+CD11b−B220+Gr-1+ IPCs can be generated from mouse bone marrow cultures with FLT3-ligand. By contrast GM-CSF or TNF-α, which promote the generation of CD11c+CD11b+B220− myeloid DCs, block completely the development of IPCs. IPCs generated display similar features to human IPCs, such as the plasmacytoid morphology, the ability to produce large amounts of IFN-α in responses to herpes simplex virus, and the capacity to respond to ligands for Toll-like receptor 9 (TLR-9; CpG ODN 1668), but not to ligands for TLR-4 (lipopolysaccharide [LPS]). Unlike human IPCs which produce little IL-12p70, mouse IPCs produce IL-12p70 in response to CpG ODN 1668 and herpes simplex virus. This study demonstrates that the development of murine CD11c+CD11b−B220+Gr-1+ IPCs and CD11c+CD11b+B220− myeloid DCs is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. Human IPCs and mouse IPCs display different ability to produce IL-12p70. Large numbers of mouse IPCs can now be obtained from total bone marrow culture. PMID:11927638

  14. A novel Flt3-deficient HIS mouse model with selective enhancement of human DC development.

    PubMed

    Li, Yan; Mention, Jean-Jacques; Court, Nathalie; Masse-Ranson, Guillemette; Toubert, Antoine; Spits, Hergen; Legrand, Nicolas; Corcuff, Erwan; Strick-Marchand, Helene; Di Santo, James P

    2016-05-01

    Humanized mice harboring human immune systems (HIS) represent a platform to study immune responses against pathogens and to screen vaccine candidates and novel immunotherapeutics. Innate and adaptive immune responses are suboptimal in HIS mice, possibly due to poor reconstitution of human antigen-presenting cells, including dendritic cells (DCs). DC homeostasis is regulated by cytokine availability, and Flt3-ligand (Flt3L) is one factor that conditions this process. Mouse myelopoiesis is essentially normal in most current HIS models. As such, developing mouse myeloid cells may limit human DC reconstitution by reducing available Flt3L and by cellular competition for specific "niches." To address these issues, we created a novel HIS model that compromises host myeloid cell development via deficiency in the receptor tyrosine kinase Flk2/Flt3. In Balb/c Rag2(-/-) Il2rg(-/-) Flt3(-/-) (BRGF) recipients, human conventional DCs and plasmacytoid DCs develop from hCD34(+) precursors and can be specifically boosted with exogenous Flt3L. Human DCs that develop in this context normally respond to TLR stimulation, and improved human DC homeostasis is associated with increased numbers of human NK and T cells. This new HIS-DC model should provide a means to dissect human DC differentiation and represents a novel platform to screen immune adjuvants and DC targeting therapies. PMID:26865269

  15. Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia

    PubMed Central

    Green, Alexa S.; Maciel, Thiago T.; Hospital, Marie-Anne; Yin, Chae; Mazed, Fetta; Townsend, Elizabeth C.; Pilorge, Sylvain; Lambert, Mireille; Paubelle, Etienne; Jacquel, Arnaud; Zylbersztejn, Florence; Decroocq, Justine; Poulain, Laury; Sujobert, Pierre; Jacque, Nathalie; Adam, Kevin; So, Jason C. C.; Kosmider, Olivier; Auberger, Patrick; Hermine, Olivier; Weinstock, David M.; Lacombe, Catherine; Mayeux, Patrick; Vanasse, Gary J.; Leung, Anskar Y.; Moura, Ivan C.; Bouscary, Didier; Tamburini, Jerome

    2015-01-01

    ABSTRACT Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD–induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD+ cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy. PMID:26601252

  16. An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

    PubMed

    Kawashita, Yujo; Deb, Niloy J; Garg, Madhur K; Kabarriti, Rafi; Fan, Zuoheng; Alfieri, Alan A; Roy-Chowdhury, Jayanta; Guha, Chandan

    2014-08-01

    Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and

  17. An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 1. Flt3 ligand gene transfer increases antitumor effects of a radio-inducible suicide gene therapy in an ectopic tumor model.

    PubMed

    Kawashita, Yujo; Deb, Niloy J; Garg, Madhur; Kabarriti, Rafi; Alfieri, Alan; Takahashi, Masahiko; Roy-Chowdhury, Jayanta; Guha, Chandan

    2014-08-01

    Hepatocellular carcinoma (HCC) often presents as a diffuse or multifocal tumor making it difficult to control by surgery or radiation. Radio-inducible herpes simplex virus thymidine kinase (HSV-TK) gene therapy has been shown to enhance local tumor control after radiation therapy (RT), while limiting the expression of the transgene in the irradiated tumor tissues. To prevent liver tumor recurrence and control systemic disease while limiting the potential bystander toxicity of HSV-TK therapy, we proposed to stimulate endogenous dendritic cell (DC) proliferation with systemic adenovirus Flt3 ligand (Adeno-Flt3L) gene therapy, followed by primary tumor radiation therapy combined with a radio-inducible HSV-TK gene therapy. We hypothesized that adenovirus-expressing Flt3L gene therapy will stimulate DC proliferation, allowing the upregulated DCs to locally harness tumor antigens released from HSV-TK/RT-treated HCC cells, thereby converting irradiated tumors to an autologous in situ tumor vaccine in mice with primary liver tumors. To test this hypothesis, an expression vector of HSV-TK was constructed under the control of a radio-inducible promoter early-growth response (Egr-TK) and a recombinant adenovirus-expressing human Flt3L was constructed. The Adeno-Flt3L [10(9) plaque forming units (pfu)] was administered intravenously on days 1 and 8 after radiation therapy. The murine hepatoma cell line (BNL1ME) was stably transfected by Egr-TK or Egr-Null (encoding no therapeutic gene). Palpable tumors in BALB/c mice were treated with a localized dose of 25 Gy of radiation followed by ganciclovir (GCV, 100 mg/kg, 14 days). Four treatment cohorts were compared: Egr-Null/GCV + RT + Adeno-LacZ; Egr-Null/GCV + RT + Adeno-Flt3L; Egr-TK/GCV + RT + Adeno-LacZ; and Egr-TK/GCV + RT + Adeno-Flt3L. There was no primary tumor regression in the Egr-Null tumors after radiation therapy alone. In contrast, Egr-TK tumors had nearly complete tumor regression for 3 weeks after radiation therapy

  18. Identification of an orally available compound with potent and broad FLT3 inhibition activity.

    PubMed

    Chen, Y; Guo, Y; Zhao, W; Tina Ho, W-T; Fu, X; Zhao, Z J

    2016-06-01

    FLT3 internal tandem duplication (FLT3-ITD) is an activating mutation found in 20-30% of patients with acute myeloid leukemia (AML), which makes FLT3 an attractive target for the treatment of AML. Although FLT3-mutant patients respond to current FLT3 inhibitors, relapse usually happens because of the acquisition of resistant secondary mutations at the FLT3 catalytic domain, which is mainly on D835. In the search for compounds with broad FLT3 inhibition activities, we screened a kinase inhibitor library by using our unique FLT3 substrate and identified JAK3 inhibitor VI (designated JI6 hereafter) as a novel FLT3 inhibitor, which selectively targets FLT3 D835 mutants as well as FLT3-ITD. JI6 effectively inhibited FLT3-ITD-containing MV4-11 cells and HCD-57 cells transformed with FLT3-ITD and D835 mutants. Furthermore, administration of JI6 effectively targeted FLT3 signaling in vivo and suppressed the myeloproliferative phenotypes in FLT3-ITD knock-in mice, and significantly prolonged the survival of immunodeficient mice implanted with the transformed HCD-57 cells. Therefore, JI6 is a promising candidate for the development of next-generation anti-AML drugs. PMID:26411368

  19. Identification of an orally available compound with potent and broad FLT3 inhibition activity

    PubMed Central

    Chen, Yun; Guo, Yao; Zhao, Wanke; Tina Ho, Wan-Ting; Fu, Xueqi; Joe Zhao, Zhizhuang

    2015-01-01

    FLT3 internal tandem duplication (FLT3-ITD) is an activating mutation found in 20%-30% of patients with acute myeloid leukemia (AML), which makes FLT3 an attractive target for the treatment of AML. Although FLT3-mutant patients respond to current FLT3 inhibitors, relapse usually happens due to the acquisition of resistant secondary mutations at the FLT3 catalytic domain, which is mainly on D835. In the search for compounds with broad FLT3 inhibition activities, we screened a kinase inhibitor library by using our unique FLT3 substrate and identified JAK3 inhibitor VI (designated JI6 hereafter) as a novel FLT3 inhibitor, which selectively targets FLT3 D835 mutants as well as FLT3-ITD. JI6 effectively inhibited FLT3-ITD-containing MV4-11 cells and HCD-57 cells transformed with FLT3-ITD and D835 mutants. Furthermore, administration of JI6 effectively targeted FLT3 signaling in vivo and suppressed the myeloproliferative phenotypes in FLT3-ITD knock-in mice and significantly prolonged the survival of immunodeficient mice implanted with the transformed HCD-57 cells. Therefore, JI6 is a promising candidate for development of next generation anti-AML drugs. PMID:26411368

  20. LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling

    PubMed Central

    Scheffler, Julia M.; Sparber, Florian; Tripp, Christoph H.; Herrmann, Caroline; Humenberger, Alexandra; Blitz, Johanna; Romani, Nikolaus; Stoitzner, Patrizia; Huber, Lukas A.

    2014-01-01

    The receptor tyrosine kinase Flt3 and its ligand are crucial for dendritic cell (DC) homeostasis by activating downstream effectors including mammalian target of Rapamycin (mTOR) signalling. LAMTOR2 is a member of the Ragulator/LAMTOR complex known to regulate mTOR and extracellular signal-regulated kinase activation on the late endosome as well as endosomal biogenesis. Here we show in mice that conditional ablation of LAMTOR2 in DCs results in a severe disturbance of the DC compartment caused by accumulation of Flt3 on the cell surface. This results in an increased downstream activation of the AKT/mTOR signalling pathway and subsequently to a massive expansion of conventional DCs and plasmacytoid DCs in ageing mice. Finally, we can revert the symptoms in vivo by inhibiting the activation of Flt3 and its downstream target mTOR. PMID:25336251

  1. Leukemia-associated activating mutation of Flt3 expands dendritic cells and alters T cell responses.

    PubMed

    Lau, Colleen M; Nish, Simone A; Yogev, Nir; Waisman, Ari; Reiner, Steven L; Reizis, Boris

    2016-03-01

    A common genetic alteration in acute myeloid leukemia is the internal tandem duplication (ITD) in FLT3, the receptor for cytokine FLT3 ligand (FLT3L). Constitutively active FLT3-ITD promotes the expansion of transformed progenitors, but also has pleiotropic effects on hematopoiesis. We analyzed the effect of FLT3-ITD on dendritic cells (DCs), which express FLT3 and can be expanded by FLT3L administration. Pre-leukemic mice with the Flt3(ITD) knock-in allele manifested an expansion of classical DCs (cDCs) and plasmacytoid DCs. The expansion originated in DC progenitors, was cell intrinsic, and was further enhanced in Flt3(ITD/ITD) mice. The mutation caused the down-regulation of Flt3 on the surface of DCs and reduced their responsiveness to Flt3L. Both canonical Batf3-dependent CD8(+) cDCs and noncanonical CD8(+) cDCs were expanded and showed specific alterations in their expression profiles. Flt3(ITD) mice showed enhanced capacity to support T cell proliferation, including a cell-extrinsic expansion of regulatory T (T reg) cells. Accordingly, these mice restricted alloreactive T cell responses during graft-versus-host reaction, but failed to control autoimmunity without T reg cells. Thus, the FLT3-ITD mutation directly affects DC development, indirectly modulating T cell homeostasis and supporting T reg cell expansion. We hypothesize that this effect of FLT3-ITD might subvert immunosurveillance and promote leukemogenesis in a cell-extrinsic manner. PMID:26903243

  2. MUC1-C oncoprotein promotes FLT3 receptor activation in acute myeloid leukemia cells

    PubMed Central

    Liu, Suiyang; Yin, Li; Stroopinsky, Dina; Rajabi, Hasan; Puissant, Alexandre; Stegmaier, Kimberly; Avigan, David; Kharbanda, Surender; Kufe, Donald

    2014-01-01

    Blasts from approximately one-third of patients with acute myeloid leukemia (AML) harbor activating mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase that confer a poor prognosis. The Mucin 1-C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in AML blasts and stem cells; however, there is no known interaction between MUC1-C and FLT3. The present studies demonstrate that MUC1-C associates with wild-type and mutant FLT3 in AML cells. Targeting MUC1-C with the cell-penetrating peptide inhibitor GO-203 disrupts MUC1-C/FLT3 complexes and downregulates FLT3 activation. GO-203 treatment of AML cells was also associated with inhibition of the FLT3 downstream effectors AKT, extracellular signal-regulated kinase, and STAT5. The results further show that AML cells with FLT3-activating mutations and resistant to the FLT3 inhibitor midostaurin/PKC412 are sensitive to GO-203–induced growth arrest and death. Moreover, GO-203 increases sensitivity of mutant FLT3 AML cells to FLT3 inhibitor treatment. These results indicate that MUC1-C contributes to FLT3 activation in AML cells and that targeting MUC1-C inhibits the FLT3 signaling pathway. Our findings support the development of MUC1-C inhibitors alone and in combination with agents that target FLT3 for the treatment of wild-type and mutant FLT3 AML. PMID:24282218

  3. The Dual MEK/FLT3 Inhibitor E6201 Exerts Cytotoxic Activity against Acute Myeloid Leukemia Cells Harboring Resistance-Conferring FLT3 Mutations.

    PubMed

    Zhang, Weiguo; Borthakur, Gautam; Gao, Chen; Chen, Ye; Mu, Hong; Ruvolo, Vivian R; Nomoto, Kenichi; Zhao, Nanding; Konopleva, Marina; Andreeff, Michael

    2016-03-15

    Fms-like tyrosine kinase 3 (FLT3) inhibition has elicited encouraging responses in acute myeloid leukemia (AML) therapy. Unfortunately, unless combined with a bone marrow transplant, disease relapse is frequent. In addition to the acquired point mutations in the FLT3 kinase domain that contribute to FLT3 inhibitor resistance, MEK/ERK signaling is persistently activated in AML cells even when FLT3 phosphorylation is continually suppressed. Thus, concomitant targeting of FLT3 and MAPK may potentially exert synergistic activity to counteract the resistance of AML cells to FLT3-targeted therapy. In this study, we investigated the antileukemia activity of a MEK1 and FLT3 dual inhibitor, E6201, in AML cells resistant to FLT3 inhibition. We found that E6201 exerted profound apoptogenic effects on AML cells harboring resistance-conferring FLT3 mutations. This activity appeared to be p53 dependent, and E6201-induced cytotoxicity was retained under hypoxic culture conditions and during coculture with mesenchymal stem cells that mimic the AML microenvironment. Furthermore, E6201 markedly reduced leukemia burden and improved the survival of mice in a human FLT3-mutated AML model. Collectively, our data provide a preclinical basis for the clinical evaluation of E6201 in AML patients harboring FLT3 mutations, including those who relapse following FLT3-targeted monotherapy. PMID:26822154

  4. Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice.

    PubMed

    Manfra, Denise J; Chen, Shu-Cheng; Jensen, Kristian K; Fine, Jay S; Wiekowski, Maria T; Lira, Sergio A

    2003-03-15

    The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease. PMID:12626534

  5. CDC25A governs proliferation and differentiation of FLT3-ITD acute myeloid leukemia

    PubMed Central

    Bertoli, Sarah; Boutzen, Helena; David, Laure; Larrue, Clément; Vergez, François; Fernandez-Vidal, Anne; Yuan, Lingli; Hospital, Marie-Anne; Tamburini, Jérôme; Demur, Cécile; Delabesse, Eric; Saland, Estelle; Sarry, Jean-Emmanuel; Galcera, Marie-Odile; Mas, Véronique Mansat-De; Didier, Christine; Dozier, Christine; Récher, Christian; Manenti, Stéphane

    2015-01-01

    We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of higher clonogenic potential in FLT3-ITD+ samples, not in FLT3-wt ones. Importantly, pharmacological inhibition as well as RNA interference-mediated knock-down of CDC25A also induced monocytic differentiation of FLT3-ITD positive cells, as judged by cell surface markers expression, morphological modifications, and C/EBPα phosphorylation. CDC25 inhibition also re-induced monocytic differentiation in primary AML blasts carrying the FLT3-ITD mutation, but not in blasts expressing wild type FLT3. Altogether, these data identify CDC25A as an early cell cycle transducer of FLT3-ITD oncogenic signaling, and as a promising target to inhibit proliferation and re-induce differentiation of FLT3-ITD AML cells. PMID:26515730

  6. 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

    2016-06-01

    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. PMID:27103744

  7. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor

    PubMed Central

    Sandhöfer, Nadine; Bauer, Julia; Reiter, Katrin; Dufour, Annika; Rothenberg, Maja; Konstandin, Nikola P.; Zellmeier, Evelyn; Tizazu, Belay; Greif, Philipp A.; Metzeler, Klaus H.; Hiddemann, Wolfgang; Polzer, Harald; Spiekermann, Karsten

    2016-01-01

    In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor. PMID:27346558

  8. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor.

    PubMed

    Sandhöfer, Nadine; Bauer, Julia; Reiter, Katrin; Dufour, Annika; Rothenberg, Maja; Konstandin, Nikola P; Zellmeier, Evelyn; Tizazu, Belay; Greif, Philipp A; Metzeler, Klaus H; Hiddemann, Wolfgang; Polzer, Harald; Spiekermann, Karsten

    2016-01-01

    In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor. PMID:27346558

  9. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia

    PubMed Central

    Minson, Katherine A.; Smith, Catherine C.; DeRyckere, Deborah; Libbrecht, Clara; Lee-Sherick, Alisa B.; Huey, Madeline G.; Lasater, Elisabeth A.; Kirkpatrick, Gregory D.; Stashko, Michael A.; Zhang, Weihe; Jordan, Craig T.; Kireev, Dmitri; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton; Shah, Neil P.; Graham, Douglas K.

    2016-01-01

    FMS-like tyrosine kinase 3–targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD–mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML. PMID:27158668

  10. The evolving role of FLT3 inhibitors in acute myeloid leukemia: quizartinib and beyond

    PubMed Central

    Wander, Seth A.; Levis, Mark J.

    2014-01-01

    Acute myeloid leukemia remains associated with poor outcomes despite advances in our understanding of the complicated molecular events driving leukemogenesis and malignant progression. Those patients harboring mutations in the FLT3 receptor tyrosine kinase have a particularly poor prognosis; however, significant excitement has been generated by the emergence of a variety of targeted inhibitors capable of suppressing FLT3 signaling in vivo. Here we will review results from preclinical studies and early clinical trials evaluating both first- and second-generation FLT3 inhibitors. Early FLT3 inhibitors (including sunitinib, midostaurin, and lestaurtinib) demonstrated significant promise in preclinical models of FLT3 mutant AML. Unfortunately, many of these compounds failed to achieve robust and sustained FLT3 inhibition in early clinical trials, at best resulting in only transient decreases in peripheral blast counts. These results have prompted the development of second-generation FLT3 inhibitors, epitomized by the novel agent quizartinib. These second-generation inhibitors have demonstrated enhanced FLT3 specificity and have been generally well tolerated in early clinical trials. Several FLT3 inhibitors have reached phase III clinical trials, and a variety of phase I/II trials exploring a role for these novel compounds in conjunction with conventional chemotherapy or hematopoietic stem cell transplantation are ongoing. Finally, molecular insights provided by FLT3 inhibitors have shed light upon the variety of mechanisms underlying the acquisition of resistance and have provided a rationale supporting the use of combinatorial regimens with other emerging targeted therapies. PMID:24883179

  11. Molecular Dynamics Studies on D835N Mutation in FLT3-Its Impact on FLT3 Protein Structure.

    PubMed

    Swetha, Rayapadi G; Ramaiah, Sudha; Anbarasu, Anand

    2016-06-01

    Mutations in Fetal Liver Tyrosine Kinase 3 (FLT3) genes are implicated in the constitutive activation and development of Acute Myeloid Leukaemia (AML). They are involved in signalling pathway of autonomous proliferation and block differentiation in leukaemia cells. FLT3 is considered as a promising target for the therapeutic intervention of AML. There are a few missense mutations associated with FLT3 that are found in AML patients. The D835N mutation is the most frequently observed and the aspartic acid in this position acts as a key residue for the receptor activation. The present study aims to understand the structural effect of D835N mutation in FLT3. We carried out the molecular dynamics (MD) simulation for a period of 120 ns at 300 K. Root-mean square deviation, root-mean square fluctuations, surface accessibility, radius of gyration, hydrogen bond, eigenvector projection analysis, trace of covariance matrix, and density analysis revealed the instability of mutant (D835N) protein. Our study provides new insights on the conformational changes in the mutant (D835N) structure of FLT3 protein. Our observations will be useful for researchers exploring AML and for the development of FLT3 inhibitors. PMID:26566084

  12. Crenolanib is a potent inhibitor of FLT3 with activity against resistance-conferring point mutants

    PubMed Central

    Galanis, Allison; Ma, Hayley; Rajkhowa, Trivikram; Ramachandran, Abhijit; Small, Donald; Cortes, Jorge

    2014-01-01

    Mutations of the type III receptor tyrosine kinase FLT3 occur in approximately 30% of acute myeloid leukemia patients and lead to constitutive activation. This has made FLT3-activating mutations an attractive drug target because they are probable driver mutations of this disease. As more potent FLT3 inhibitors are developed, a predictable development of resistance-conferring point mutations, commonly at residue D835, has been observed. Crenolanib is a highly selective and potent FLT3 tyrosine kinase inhibitor (TKI) with activity against the internal tandem duplication (FLT3/ITD) mutants and the FLT3/D835 point mutants. We tested crenolanib against a panel of D835 mutant cell lines and primary patient blasts and observed superior cytotoxic effects when compared with other available FLT3 TKIs such as quizartinib and sorafenib. Another potential advantage of crenolanib is its reduced inhibition of c-Kit compared with quizartinib. In progenitor cell assays, crenolanib was less disruptive of erythroid colony growth, which may result in relatively less myelosuppression than quizartinib. Finally, correlative data from an ongoing clinical trial demonstrate that acute myeloid leukemia patients can achieve sufficient levels of crenolanib to inhibit both FLT3/ITD and resistance-conferring FLT3/D835 mutants in vivo. Crenolanib is thus an important next-generation FLT3 TKI. This study is registered at clinicaltrials.gov (ID: NCT01657682). PMID:24227820

  13. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6.

    PubMed

    Uras, Iris Z; Walter, Gina J; Scheicher, Ruth; Bellutti, Florian; Prchal-Murphy, Michaela; Tigan, Anca S; Valent, Peter; Heidel, Florian H; Kubicek, Stefan; Scholl, Claudia; Fröhling, Stefan; Sexl, Veronika

    2016-06-01

    Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD(+) acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration-approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors. PMID:27099147

  14. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

    PubMed Central

    Uras, Iris Z.; Walter, Gina J.; Scheicher, Ruth; Bellutti, Florian; Prchal-Murphy, Michaela; Tigan, Anca S.; Valent, Peter; Heidel, Florian H.; Kubicek, Stefan; Scholl, Claudia; Fröhling, Stefan

    2016-01-01

    Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD+ acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration–approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors. PMID:27099147

  15. FLT3-ITD drives Ara-C resistance in leukemic cells via the induction of RUNX3.

    PubMed

    Damdinsuren, Anar; Matsushita, Hiromichi; Ito, Masatoshi; Tanaka, Masayuki; Jin, Guilan; Tsukamoto, Hideo; Asai, Satomi; Ando, Kiyoshi; Miyachi, Hayato

    2015-12-01

    Internal tandem duplication (ITD) mutations of the FLT3 gene (FLT3-ITD) are well known to correlate with a poor prognosis in acute myeloid leukemia (AML). We previously reported that FLT3-ITD confers resistance to cytosine arabinoside (Ara-C), a key cytotoxic agent in AML treatments. In order to elucidate the detailed molecular mechanisms underlying the Ara-C resistance induced by FLT3-ITD, we performed a microarray gene expression analysis of the human leukemic cell line K562 transduced with FLT3-ITD (K562/FLT3-ITD) and identified RUNX3 as a downstream target of FLT3-ITD. The transcriptional induction of the RUNX3 expression by FLT3-ITD was noted on a Luciferase assay. The knockdown of the RUNX3 expression in the K562/FLT3-ITD cells increased the sensitivity to Ara-C, and the exogenous expression of RUNX3 per se resulted in the enhancement of Ara-C resistance in the K562 cells. A relationship between the FLT3-ITD-induced RUNX3 expression and Ara-C resistance was also observed in AML cells with an endogenous FLT3-ITD expression. Collectively, these findings demonstrate that RUNX3 is a prerequisite for Ara-C resistance via FLT3-ITD signaling. PMID:26475207

  16. 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

  17. Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of acquired drug resistance to PTK inhibitors in FLT3-ITD-transformed hematopoietic cells.

    PubMed

    Bagrintseva, Ksenia; Schwab, Ruth; Kohl, Tobias M; Schnittger, Susanne; Eichenlaub, Sabine; Ellwart, Joachim W; Hiddemann, Wolfgang; Spiekermann, Karsten

    2004-03-15

    Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML. PMID:14604974

  18. Inhibition of MEK5 by BIX02188 induces apoptosis in cells expressing the oncogenic mutant FLT3-ITD

    SciTech Connect

    Razumovskaya, Elena; Sun, Jianmin; Roennstrand, Lars

    2011-08-26

    Highlights: {yields} In this study we have demonstrated that FLT3 activation leads to activation of ERK5. {yields} We have demonstrated that ERK5 is involved in activation of AKT downstream of FLT3. {yields} (BIX02188) blocks activation of ERK5 and induces apoptosis in FLT3 Ba/F3 cells. {yields} (BIX02188) induce apoptosis in the two leukemic cell lines MV4-11 and MOLM-13. -- Abstract: Fms-like tyrosine kinase-3 (FLT3) is a growth factor receptor normally expressed on hematopoietic progenitor cells. Approximately one third of all patients with AML carry an activating mutation in FLT3 that drives proliferation and survival of the leukemic cells. The most common activating mutation is the so-called internal tandem duplication (ITD), which involves an in-frame duplication of a segment of varying length in the region of the FLT3 gene that encodes the juxtamembrane domain. The pathways downstream of FLT3-ITD are partially known but further knowledge regarding the downstream signal transduction molecules is important in order to develop alternative strategies for pharmacological intervention. In this paper we have studied the role of MEK/ERK5 in FLT3-ITD mediated transformation. We have found that both wild-type FLT3 and FLT3-ITD activate MEK5 leading to the activation of ERK5. By use of the selective inhibitor of MEK5, (BIX02188), we have shown that activation of AKT downstream of FLT3 is partially dependent on ERK5. Furthermore, inhibition of MEK5/ERK5 induces apoptosis of both FLT3-ITD transfected Ba/F3 cells as well as the FLT3-ITD carrying leukemic cell lines MV4-11 and MOLM-13. These results suggest that MEK5/ERK5 is important for FLT3-ITD induced hematopoietic transformation and may thus represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia.

  19. Enzyme-free and sensitive electrochemical determination of the FLT3 gene based on a dual signal amplified strategy: Controlled nanomaterial multilayers and a target-catalyzed hairpin assembly.

    PubMed

    Sun, Yingying; Ren, Qunxiang; Liu, Bo; Qin, Yan; Zhao, Shuang

    2016-04-15

    An isothermal, enzyme-free and sensitive electrochemical DNA sensor was developed for the detection of the FLT3 gene in acute myeloid leukemia (AML). First, aminated multi-walled carbon nanotubes (AMWNTs) and gold nanoparticles (AuNPs) were alternately self-assembled on a gold electrode using a layer-by-layer strategy. Then, the hairpin DNA probe 1 (H1), with a thiol group at the 3' end and a ferrocenyl moiety (Fc) at the 5' end, was immobilized on the AMWNTs/AuNPs multilayer films through Au-S bonding. When the target DNA (TD) appeared, it hybridized with and opened the hairpin structure of H1, and Fc was forced away from the electrode surface, leading to a significant decrease in the current peak of square wave voltammetry. Subsequently, the hairpin DNA probe 2 (H2) bound to H1, freeing the TD to trigger another reaction cycle. The combination of this target-catalyzed hairpin assembly and the LBL assembly of nanomaterials achieved a detection limit of 0.1 pM with a wide linear range of 0.1-1000 pM. The sensor discriminated between mismatched DNA and the target DNA with high selectivity. This dual signal amplification strategy is relatively simple and inexpensive because it does not need any enzymes or sophisticated equipment and successfully assayed the FLT3 gene from real samples. PMID:26584077

  20. Flt3 permits survival during infection by rendering dendritic cells competent to activate NK cells.

    PubMed

    Eidenschenk, Céline; Crozat, Karine; Krebs, Philippe; Arens, Ramon; Popkin, Daniel; Arnold, Carrie N; Blasius, Amanda L; Benedict, Chris A; Moresco, Eva Marie Y; Xia, Yu; Beutler, Bruce

    2010-05-25

    A previously unappreciated signal necessary for dendritic cell (DC)-mediated activation of natural killer (NK) cells during viral infection was revealed by a recessive N-ethyl-N-nitrosourea-induced mutation called warmflash (wmfl). Wmfl homozygotes displayed increased susceptibility to mouse cytomegalovirus (MCMV) infection. In response to MCMV infection in vivo, delayed NK cell activation was observed, but no intrinsic defects in NK cell activation or function were identified. Rather, coculture experiments demonstrated that NK cells are suboptimally activated by wmfl DCs, which showed impaired cytokine production in response to MCMV or synthetic TLR7 and TLR9 ligands. The wmfl mutation was identified in the gene encoding the Fms-like tyrosine kinase 3 (Flt3). Flt3 ligand (Flt3L) is transiently induced in the serum upon infection or TLR activation. However, antibody blockade reveals no acute requirement for Flt3L, suggesting that the Flt3L --> Flt3 axis programs the development of DCs, making them competent to support NK effector function. In the absence of Flt3 signaling, NK cell activation is delayed and survival during MCMV infection is markedly compromised. PMID:20457904

  1. Flt3 permits survival during infection by rendering dendritic cells competent to activate NK cells

    PubMed Central

    Eidenschenk, Céline; Crozat, Karine; Krebs, Philippe; Arens, Ramon; Popkin, Daniel; Arnold, Carrie N.; Blasius, Amanda L.; Benedict, Chris A.; Moresco, Eva Marie Y.; Xia, Yu; Beutler, Bruce

    2010-01-01

    A previously unappreciated signal necessary for dendritic cell (DC)-mediated activation of natural killer (NK) cells during viral infection was revealed by a recessive N-ethyl-N-nitrosourea-induced mutation called warmflash (wmfl). Wmfl homozygotes displayed increased susceptibility to mouse cytomegalovirus (MCMV) infection. In response to MCMV infection in vivo, delayed NK cell activation was observed, but no intrinsic defects in NK cell activation or function were identified. Rather, coculture experiments demonstrated that NK cells are suboptimally activated by wmfl DCs, which showed impaired cytokine production in response to MCMV or synthetic TLR7 and TLR9 ligands. The wmfl mutation was identified in the gene encoding the Fms-like tyrosine kinase 3 (Flt3). Flt3 ligand (Flt3L) is transiently induced in the serum upon infection or TLR activation. However, antibody blockade reveals no acute requirement for Flt3L, suggesting that the Flt3L → Flt3 axis programs the development of DCs, making them competent to support NK effector function. In the absence of Flt3 signaling, NK cell activation is delayed and survival during MCMV infection is markedly compromised. PMID:20457904

  2. FYN expression potentiates FLT3-ITD induced STAT5 signaling in acute myeloid leukemia

    PubMed Central

    Chougule, Rohit A.; Kazi, Julhash U.; Rönnstrand, Lars

    2016-01-01

    FYN is a non-receptor tyrosine kinase belonging to the SRC family of kinases, which are frequently over-expressed in human cancers, and play key roles in cancer biology. SRC has long been recognized as an important oncogene, but little attention has been given to its other family members. In this report, we have studied the role of FYN in FLT3 signaling in respect to acute myeloid leukemia (AML). We observed that FYN displays a strong association with wild-type FLT3 as well as oncogenic FLT3-ITD and is dependent on the kinase activity of FLT3 and the SH2 domain of FYN. We identified multiple FYN binding sites in FLT3, which partially overlapped with SRC binding sites. To understand the role of FYN in FLT3 signaling, we generated FYN overexpressing cells. We observed that expression of FYN resulted in slightly enhanced phosphorylation of AKT, ERK1/2 and p38 in response to ligand stimulation. Furthermore, FYN expression led to a slight increase in FLT3-ITD-dependent cell proliferation, but potent enhancement of STAT5 phosphorylation as well as colony formation. We also observed that FYN expression is deregulated in AML patient samples and that higher expression of FYN, in combination with FLT3-ITD mutation, resulted in enrichment of the STAT5 signaling pathway and correlated with poor prognosis in AML. Taken together our data suggest that FYN cooperates with oncogenic FLT3-ITD in cellular transformation by selective activation of the STAT5 pathway. Therefore, inhibition of FYN, in combination with FLT3 inhibition, will most likely be beneficial for this group of AML patients. PMID:26848862

  3. FYN expression potentiates FLT3-ITD induced STAT5 signaling in acute myeloid leukemia.

    PubMed

    Chougule, Rohit A; Kazi, Julhash U; Rönnstrand, Lars

    2016-03-01

    FYN is a non-receptor tyrosine kinase belonging to the SRC family of kinases, which are frequently over-expressed in human cancers, and play key roles in cancer biology. SRC has long been recognized as an important oncogene, but little attention has been given to its other family members. In this report, we have studied the role of FYN in FLT3 signaling in respect to acute myeloid leukemia (AML). We observed that FYN displays a strong association with wild-type FLT3 as well as oncogenic FLT3-ITD and is dependent on the kinase activity of FLT3 and the SH2 domain of FYN. We identified multiple FYN binding sites in FLT3, which partially overlapped with SRC binding sites. To understand the role of FYN in FLT3 signaling, we generated FYN overexpressing cells. We observed that expression of FYN resulted in slightly enhanced phosphorylation of AKT, ERK1/2 and p38 in response to ligand stimulation. Furthermore, FYN expression led to a slight increase in FLT3-ITD-dependent cell proliferation, but potent enhancement of STAT5 phosphorylation as well as colony formation. We also observed that FYN expression is deregulated in AML patient samples and that higher expression of FYN, in combination with FLT3-ITD mutation, resulted in enrichment of the STAT5 signaling pathway and correlated with poor prognosis in AML. Taken together our data suggest that FYN cooperates with oncogenic FLT3-ITD in cellular transformation by selective activation of the STAT5 pathway. Therefore, inhibition of FYN, in combination with FLT3 inhibition, will most likely be beneficial for this group of AML patients. PMID:26848862

  4. Discovery of LY2457546: a multi-targeted anti-angiogenic kinase inhibitor with a novel spectrum of activity and exquisite potency in the acute myelogenous leukemia-Flt-3-internal tandem duplication mutant human tumor xenograft model.

    PubMed

    Burkholder, Timothy P; Clayton, Joshua R; Rempala, Mark E; Henry, James R; Knobeloch, John M; Mendel, David; McLean, Johnathan A; Hao, Yan; Barda, David A; Considine, Eileen L; Uhlik, Mark T; Chen, Yuefeng; Ma, Liandong; Bloem, Laura J; Akunda, Jacqueline K; McCann, Denis J; Sanchez-Felix, Manuel; Clawson, David K; Lahn, Michael M; Starling, James J

    2012-06-01

    LY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non

  5. FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors

    PubMed Central

    Mead, Adam J.; Kharazi, Shabnam; Atkinson, Deborah; Macaulay, Iain; Pecquet, Christian; Loughran, Stephen; Lutteropp, Michael; Woll, Petter; Chowdhury, Onima; Luc, Sidinh; Buza-Vidas, Natalija; Ferry, Helen; Clark, Sally-Ann; Goardon, Nicolas; Vyas, Paresh; Constantinescu, Stefan N.; Sitnicka, Ewa; Nerlov, Claus; Jacobsen, Sten Eirik W.

    2013-01-01

    Summary Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies. PMID:23727242

  6. Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia.

    PubMed

    Lim, Yiting; Gondek, Lukasz; Li, Li; Wang, Qiuju; Ma, Hayley; Ma, Haley; Chang, Emily; Huso, David L; Foerster, Sarah; Marchionni, Luigi; McGovern, Karen; Watkins, David Neil; Peacock, Craig D; Levis, Mark; Smith, Bruce Douglas; Merchant, Akil A; Small, Donald; Matsui, William

    2015-06-10

    FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML. PMID:26062848

  7. Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia

    PubMed Central

    Lim, Yiting; Gondek, Lukasz; Li, Li; Wang, Qiuju; Ma, Haley; Chang, Emily; Huso, David L.; Foerster, Sarah; Marchionni, Luigi; McGovern, Karen; Watkins, D. Neil; Peacock, Craig D.; Levis, Mark; Smith, B. Douglas; Merchant, Akil A.; Small, Donald; Matsui, William

    2015-01-01

    FLT3 internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing STAT5 signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML. PMID:26062848

  8. A genome-wide RNAi screen identifies proteins modulating aberrant FLT3-ITD signaling

    PubMed Central

    Caldarelli, A; Müller, J P; Paskowski-Rogacz, M; Herrmann, K; Bauer, R; Koch, S; Heninger, A K; Krastev, D; Ding, L; Kasper, S; Fischer, T; Brodhun, M; Böhmer, F-D; Buchholz, F

    2013-01-01

    Fms-like tyrosine kinase-3 is a commonly mutated gene in acute myeloid leukemia, with about one-third of patients carrying an internal-tandem duplication of the juxtamembrane domain in the receptor (FLT3-ITD). FLT3-ITD exhibits altered signaling quality, including aberrant activation of STAT5. To identify genes affecting FLT3-ITD-mediated STAT5 signaling, we performed an esiRNA-based RNAi screen utilizing a STAT5-driven reporter assay. Knockdowns that caused reduced FLT3-ITD-mediated STAT5 signaling were enriched for genes encoding proteins involved in protein secretion and intracellular protein transport, indicating that modulation of protein transport processes could potentially be used to reduce constitutive STAT5 signaling in FLT3-ITD-positive cells. The relevance of KDELR1, a component involved in the Golgi-ER retrograde transport, was further analyzed. In FLT3-ITD-expressing leukemic MV4-11 cells, downregulation of KDELR1 resulted in reduced STAT5 activation, proliferation and colony-forming capacity. Stable shRNA-mediated depletion of KDELR1 in FLT3-ITD-expressing 32D cells likewise resulted in reduced STAT5 signaling and cell proliferation. Importantly, these cells also showed a reduced capacity to generate a leukemia-like disease in syngeneic C3H/HeJ mice. Together our data suggest intracellular protein transport as a potential target for FLT3-ITD driven leukemias, with KDELR1 emerging as a positive modulator of oncogenic FLT3-ITD activity. PMID:23508117

  9. Knock-in of a FLT3/ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model

    PubMed Central

    Greenblatt, Sarah; Li, Li; Slape, Christopher; Nguyen, Bao; Novak, Rachel; Duffield, Amy; Huso, David; Desiderio, Stephen; Borowitz, Michael J.; Aplan, Peter

    2012-01-01

    Constitutive activation of FLT3 by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML). FLT3/ITD mutations have also been observed in myelodysplastic syndrome patients both before and during progression to AML. Previous work has shown that insertion of an FLT3/ITD mutation into the murine Flt3 gene induces a myeloproliferative neoplasm, but not progression to acute leukemia, suggesting that additional cooperating events are required. We therefore combined the FLT3/ITD mutation with a model of myelodysplastic syndrome involving transgenic expression of the Nup98-HoxD13 (NHD13) fusion gene. Mice expressing both the FLT3/ITD and NHD13 transgene developed AML with 100% penetrance and short latency. These leukemias were driven by mutant FLT3 expression and were susceptible to treatment with FLT3 tyrosine kinase inhibitors. We also observed a spontaneous loss of the wild-type Flt3 allele in these AMLs, further modeling the loss of the heterozygosity phenomenon that is seen in human AML with FLT3-activating mutations. Because resistance to FLT3 inhibitors remains an important clinical issue, this model may help identify new molecular targets in collaborative signaling pathways. PMID:22323452

  10. Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse.

    PubMed

    Garg, Manoj; Nagata, Yasunobu; Kanojia, Deepika; Mayakonda, Anand; Yoshida, Kenichi; Haridas Keloth, Sreya; Zang, Zhi Jiang; Okuno, Yusuke; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Ding, Ling-Wen; Alpermann, Tamara; Sun, Qiao-Yang; Lin, De-Chen; Chien, Wenwen; Madan, Vikas; Liu, Li-Zhen; Tan, Kar-Tong; Sampath, Abhishek; Venkatesan, Subhashree; Inokuchi, Koiti; Wakita, Satoshi; Yamaguchi, Hiroki; Chng, Wee Joo; Kham, Shirley-Kow Yin; Yeoh, Allen Eng-Juh; Sanada, Masashi; Schiller, Joanna; Kreuzer, Karl-Anton; Kornblau, Steven M; Kantarjian, Hagop M; Haferlach, Torsten; Lill, Michael; Kuo, Ming-Chung; Shih, Lee-Yung; Blau, Igor-Wolfgang; Blau, Olga; Yang, Henry; Ogawa, Seishi; Koeffler, H Phillip

    2015-11-26

    Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone. PMID:26438511

  11. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia

    PubMed Central

    Lee, Benjamin H.; Tothova, Zuzana; Levine, Ross L.; Anderson, Kristina; Buza-Vidas, Natalija; Cullen, Dana E.; McDowell, Elizabeth P.; Adelsperger, Jennifer; Fröhling, Stefan; Huntly, Brian J.P.; Beran, Miloslav; Jacobsen, Sten Eirik; Gilliland, D. Gary

    2007-01-01

    SUMMARY Despite their known transforming properties, the effects of leukemogenic FLT3-ITD mutations on hematopoietic stem and multipotent progenitor cells and on hematopoietic differentiation are not well understood. We report a mouse model harboring an ITD in the murine Flt3 locus that develops myeloproliferative disease resembling CMML and further identified FLT3-ITD mutations in a subset of human CMML. These findings correlated with an increase in number, cell cycling and survival of multipotent stem and progenitor cells in an ITD dose-dependent manner in animals that exhibited alterations within their myeloid progenitor compartments and a block in normal B-cell development. This model provides insights into the consequences of constitutive signaling by an oncogenic tyrosine kinase on hematopoietic progenitor quiescence, function, and cell fate. SIGNIFICANCE Activating FLT3 mutations are among the most common genetic events in AML and confer a poor clinical prognosis. Essential to our understanding of how these lesions contribute to myeloid leukemia is the development of a Flt3-ITD ‘knock-in’ murine model that has allowed examination of the consequences of constitutive FLT3 signaling on primitive hematopoietic progenitors when expressed at appropriate physiologic levels. These animals informed us to the existence of FLT3-ITD-positive human CMML, which has clinical importance given the availability of FLT3 small molecule inhibitors. This model will not only serve as a powerful biological tool to identify mutations that cooperate with FLT3 in leukemogenesis, but also to assess molecular therapies that target either FLT3 or components of its signaling pathways. PMID:17936561

  12. Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

    PubMed Central

    Feder, Rachel; Mollah, Shamim; Tse, Sze-Wah; Longhi, Maria Paula; Mehandru, Saurabh; Matos, Ines; Cheong, Cheolho; Ruane, Darren; Brane, Lucas; Teixeira, Angela; Dobrin, Joseph; Mizenina, Olga; Park, Chae Gyu; Meredith, Matthew; Clausen, Björn E.; Nussenzweig, Michel C.; Steinman, Ralph M.

    2014-01-01

    DCs are critical for initiating immunity. The current paradigm in vaccine biology is that DCs migrating from peripheral tissue and classical lymphoid-resident DCs (cDCs) cooperate in the draining LNs to initiate priming and proliferation of T cells. Here, we observe subcutaneous immunity is Fms-like tyrosine kinase 3 ligand (Flt3L) dependent. Flt3L is rapidly secreted after immunization; Flt3 deletion reduces T cell responses by 50%. Flt3L enhances global T cell and humoral immunity as well as both the numbers and antigen capture capacity of migratory DCs (migDCs) and LN-resident cDCs. Surprisingly, however, we find immunity is controlled by cDCs and actively tempered in vivo by migDCs. Deletion of Langerin+ DC or blockade of DC migration improves immunity. Consistent with an immune-regulatory role, transcriptomic analyses reveals different skin migDC subsets in both mouse and human cluster together, and share immune-suppressing gene expression and regulatory pathways. These data reveal that protective immunity to protein vaccines is controlled by Flt3L-dependent, LN-resident cDCs. PMID:25135299

  13. FLT3 kinase inhibitor TTT-3002 overcomes both activating and drug resistance mutations in FLT3 in acute myeloid leukemia

    PubMed Central

    Ma, Hayley S.; Nguyen, Bao; Duffield, Amy S.; Li, Li; Galanis, Allison; Williams, Allen B.; Brown, Patrick A.; Levis, Mark J.; Leahy, Daniel J.; Small, Donald

    2014-01-01

    There have been a number of clinical trials testing the efficacy of FLT3 tyrosine kinase inhibitors (TKIs) in acute myeloid leukemia (AML). patients harboring a constitutively activating mutation in FLT3 However, there has been limited efficacy, most often due to inadequate achievement of FLT3 inhibition through a variety of mechanisms In a previous study, TTT-3002 was identified as a novel FLT3 inhibitor with the most potent activity to date against FLT3 internal tandem duplication (FLT3/ITD) mutations Here the activity of TTT-3002 is demonstrated against a broad spectrum of FLT3 activating point mutations (FLT3/PMs), including the most frequently occurring D835 mutations The compound is also active against a number of point mutations selected for in FLT3/ITD alleles that confer resistance to other TKIs, including the F691L gatekeeper mutation TTT-3002 maintains activity against relapsed AML patient samples that are resistant to sorafenib and AC220 Studies utilizing human plasma samples from healthy donors and AML patients indicate that TTT-3002 is only moderately protein bound compared to several other TKIs currently in clinical trials Tumor burden of mice in a FLT3 TKI-resistant transplant model is significantly improved by oral dosing of TTT-3002 Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI that may overcome some of the limitations of other TKIs in the treatment of FLT3-mutant AML PMID:25060518

  14. Development of a potent and selective FLT3 kinase inhibitor by systematic expansion of a non-selective fragment-screening hit.

    PubMed

    Nakano, Hirofumi; Hasegawa, Tsukasa; Imamura, Riyo; Saito, Nae; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo

    2016-05-01

    A non-selective inhibitor (1) of FMS-like tyrosine kinase-3 (FLT3) was identified by fragment screening and systematically modified to afford a potent and selective inhibitor 26. We confirmed that 26 inhibited the growth of FLT-3-activated human acute myeloid leukemia cell line MV4-11. Our design strategy enabled rapid development of a novel type of FLT3 inhibitor from the hit fragment in the absence of target-structural information. PMID:26995531

  15. Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia.

    PubMed

    Khalife, J; Radomska, H S; Santhanam, R; Huang, X; Neviani, P; Saultz, J; Wang, H; Wu, Y-Z; Alachkar, H; Anghelina, M; Dorrance, A; Curfman, J; Bloomfield, C D; Medeiros, B C; Perrotti, D; Lee, L J; Lee, R J; Caligiuri, M A; Pichiorri, F; Croce, C M; Garzon, R; Guzman, M L; Mendler, J H; Marcucci, G

    2015-10-01

    High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients. PMID:25971362

  16. Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia

    PubMed Central

    Khalife, J; Radomska, HS; Santhanam, R; Huang, X; Neviani, P; Saultz, J; Wang, H; Wu, Y-Z; Alachkar, H; Anghelina, M; Dorrance, A; Curfman, J; Bloomfield, CD; Medeiros, BC; Perrotti, D; Lee, LJ; Lee, RJ; Caligiuri, MA; Pichiorri, F; Croce, CM; Garzon, R; Guzman, ML; Mendler, JH; Marcucci, G

    2016-01-01

    High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluatedin clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients. PMID:25971362

  17. Recent advances and novel agents for FLT3 mutated acute myeloid leukemia

    PubMed Central

    Pawar, Rahul; Bali, Omar Preet Singh; Malhotra, Bharat Kumar

    2014-01-01

    Acute myeloid leukemia (AML) is a devastating hematologic malignancy that affects both older adults as well as children. Treatments available for AML largely depend on cytotoxic agents and often the only curative option is an allogeneic bone marrow transplant, an option limited to young persons and associated with high morbidity and mortality. There is an urgent need for the identification of new myeloid targets and an understanding of the key genetic mutations involved in disease progression and prognosis. One such mutation is the internal tandem duplication (ITD) in the FMS-like tyrosine kinase receptor-3 (FLT3) gene which confers an inferior outcome that is attributed to a higher relapse rate. In this review, we evaluate the FLT3-ITD mutation and discuss the recent data regarding emerging approaches using FLT3 inhibitors for the treatment of AML.

  18. Discovery of (R)-1-(3-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-(dimethylamino)ethanone (CHMFL-FLT3-122) as a Potent and Orally Available FLT3 Kinase Inhibitor for FLT3-ITD Positive Acute Myeloid Leukemia.

    PubMed

    Li, Xixiang; Wang, Aoli; Yu, Kailin; Qi, Ziping; Chen, Cheng; Wang, Wenchao; Hu, Chen; Wu, Hong; Wu, Jiaxin; Zhao, Zheng; Liu, Juan; Zou, Fengming; Wang, Li; Wang, Beilei; Wang, Wei; Zhang, Shanchun; Liu, Jing; Liu, Qingsong

    2015-12-24

    FLT3-ITD mutant has been observed in about 30% of AML patients and extensively studied as a drug discovery target. On the basis of the structure of PCI-32765 (ibrutinib), a BTK kinase inhibitor that was recently reported to bear FLT3 kinase activity through a structure-guided drug design approach, we have discovered compound 18 (CHMFL-FLT3-122), which displayed an IC50 of 40 nM against FLT3 kinase and achieved selectivity over BTK kinase (over 10-fold). It significantly inhibited the proliferation of FLT3-ITD positive AML cancer cell lines MV4-11 (GI50 = 22 nM), MOLM13/14 (GI50 = 21 nM/42 nM). More importantly, 18 demonstrated 170-fold selectivity between FLT3 kinase and c-KIT kinase (GI50 = 11 nM versus 1900 nM) in the TEL-fusion isogenic BaF3 cells indicating a potential to avoid the FLT3/c-KIT dual inhibition induced myelosuppression toxicity. In the cellular context it strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting the cell cycle into the G0/G1 phase. In the in vivo studies 18 demonstrated a good bioavailability (30%) and significantly suppressed the tumor growth in MV4-11 cell inoculated xenograft model (50 mg/kg) without exhibiting obvious toxicity. Compound 18 might be a potential drug candidate for FLT3-ITD positive AML. PMID:26630553

  19. MuLV-related endogenous retroviral elements and Flt3 participate in aberrant end-joining events that promote B-cell leukemogenesis

    PubMed Central

    Johnson, Radia M.; Papp, Eniko; Grandal, Ildiko; Kowalski, Paul E.; Nutter, Lauryl; Wong, Raymond C.C.; Joseph-George, Ann M.; Danska, Jayne S.; Guidos, Cynthia J.

    2014-01-01

    During V(D)J recombination of immunoglobulin genes, p53 and nonhomologous end-joining (NHEJ) suppress aberrant rejoining of DNA double-strand breaks induced by recombinase-activating genes (Rags)-1/2, thus maintaining genomic stability and limiting malignant transformation during B-cell development. However, Rag deficiency does not prevent B-cell leukemogenesis in p53/NHEJ mutant mice, revealing that p53 and NHEJ also suppress Rag-independent mechanisms of B-cell leukemogenesis. Using several cytogenomic approaches, we identified a novel class of activating mutations in Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase important for normal hematopoiesis in Rag/p53/NHEJ triple-mutant (TM) B-cell leukemias. These mutant Flt3 alleles were created by complex genomic rearrangements with Moloney leukemia virus (MuLV)-related endogenous retroviral (ERV) elements, generating ERV-Flt3 fusion genes encoding an N-terminally truncated mutant form of Flt3 (trFlt3) that was transcribed from ERV long terminal repeats. trFlt3 protein lacked most of the Flt3 extracellular domain and induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, expression of trFlt3 in p53/NHEJ mutant hematopoietic progenitor cells promoted development of clinically aggressive B-cell leukemia. Thus, repetitive MuLV-related ERV sequences can participate in aberrant end-joining events that promote development of aggressive B-cell leukemia. PMID:24888589

  20. Sorafenib plus all-trans retinoic acid for AML patients with FLT3-ITD and NPM1 mutations.

    PubMed

    Guenounou, Sarah; Delabesse, Eric; Récher, Christian

    2014-12-01

    Knowledge of the molecular basis of acute myeloid leukaemia has increased considerably in the past few years, and therapies targeting specific molecular defects of this disease are intensively investigated. Patients with both NPM1 and FLT3-ITD mutations encompass 20% of cytogenetically normal AML. The multikinase and FLT3 inhibitor, sorafenib, has shown some efficacy in patients with relapsed FLT3-ITD(+) AML. In addition, it is suggested that all-trans retinoic acid (ATRA) used in combination with chemotherapy has shown to improve outcome of patients harbouring NPM1 mutations. We report here the clinical course of three patients with refractory or relapsed FLT3-ITD(+) /NPM1(+) AML who achieved significant response upon sorafenib and ATRA combination. PMID:24689895

  1. Evolution of a FLT3-TKD mutated subclone at meningeal relapse in acute promyelocytic leukemia

    PubMed Central

    Bochtler, Tilmann; Fröhling, Stefan; Weichert, Wilko; Endris, Volker; Thiede, Christian; Hutter, Barbara; Hundemer, Michael; Ho, Anthony D.; Krämer, Alwin

    2016-01-01

    Here, we report the case of an acute promyelocytic leukemia (APL) patient who—although negative for FLT3 mutations at diagnosis—developed isolated FLT3 tyrosine kinase II domain (FLT3-TKD)-positive meningeal relapse, which, in retrospect, could be traced back to a minute bone marrow subclone present at first diagnosis. Initially, the 48-yr-old female diagnosed with high-risk APL had achieved complete molecular remission after standard treatment with all-trans retinoic acid (ATRA) and chemotherapy according to the AIDA (ATRA plus idarubicin) protocol. Thirteen months after the start of ATRA maintenance, the patient suffered clinically overt meningeal relapse along with minute molecular traces of PML/RARA (promyelocytic leukemia/retinoic acid receptor alpha) in the bone marrow. Following treatment with arsenic trioxide and ATRA in combination with intrathecal cytarabine and methotrexate, the patient achieved a complete molecular remission in both cerebrospinal fluid (CSF) and bone marrow, which currently lasts for 2 yr after completion of therapy. Whole-exome sequencing and subsequent ultradeep targeted resequencing revealed a heterozygous FLT3-TKD mutation in CSF leukemic cells (p.D835Y, c.2503G>T, 1000/1961 reads [51%]), which was undetectable in the concurrent bone marrow sample. Interestingly, the FLT3-TKD mutated meningeal clone originated from a small bone marrow subclone present in a variant allele frequency of 0.4% (6/1553 reads) at initial diagnosis. This case highlights the concept of clonal evolution with a subclone harboring an additional mutation being selected as the “fittest” and leading to meningeal relapse. It also further supports earlier suggestions that FLT3 mutations may play a role for migration and clonal expansion in the CSF sanctuary site. PMID:27626069

  2. Evolution of a FLT3-TKD mutated subclone at meningeal relapse in acute promyelocytic leukemia.

    PubMed

    Bochtler, Tilmann; Fröhling, Stefan; Weichert, Wilko; Endris, Volker; Thiede, Christian; Hutter, Barbara; Hundemer, Michael; Ho, Anthony D; Krämer, Alwin

    2016-09-01

    Here, we report the case of an acute promyelocytic leukemia (APL) patient who-although negative for FLT3 mutations at diagnosis-developed isolated FLT3 tyrosine kinase II domain (FLT3-TKD)-positive meningeal relapse, which, in retrospect, could be traced back to a minute bone marrow subclone present at first diagnosis. Initially, the 48-yr-old female diagnosed with high-risk APL had achieved complete molecular remission after standard treatment with all-trans retinoic acid (ATRA) and chemotherapy according to the AIDA (ATRA plus idarubicin) protocol. Thirteen months after the start of ATRA maintenance, the patient suffered clinically overt meningeal relapse along with minute molecular traces of PML/RARA (promyelocytic leukemia/retinoic acid receptor alpha) in the bone marrow. Following treatment with arsenic trioxide and ATRA in combination with intrathecal cytarabine and methotrexate, the patient achieved a complete molecular remission in both cerebrospinal fluid (CSF) and bone marrow, which currently lasts for 2 yr after completion of therapy. Whole-exome sequencing and subsequent ultradeep targeted resequencing revealed a heterozygous FLT3-TKD mutation in CSF leukemic cells (p.D835Y, c.2503G>T, 1000/1961 reads [51%]), which was undetectable in the concurrent bone marrow sample. Interestingly, the FLT3-TKD mutated meningeal clone originated from a small bone marrow subclone present in a variant allele frequency of 0.4% (6/1553 reads) at initial diagnosis. This case highlights the concept of clonal evolution with a subclone harboring an additional mutation being selected as the "fittest" and leading to meningeal relapse. It also further supports earlier suggestions that FLT3 mutations may play a role for migration and clonal expansion in the CSF sanctuary site. PMID:27626069

  3. Association of the EGF-TM7 receptor CD97 expression with FLT3-ITD in acute myeloid leukemia

    PubMed Central

    Wobus, Manja; Bornhäuser, Martin; Jacobi, Angela; Kräter, Martin; Otto, Oliver; Ortlepp, Claudia; Guck, Jochen; Ehninger, Gerhard; Thiede, Christian; Oelschlägel, Uta

    2015-01-01

    Internal tandem duplications within the juxtamembrane region of the FMS-like tyrosine kinase receptor FLT3 (FLT3-ITD) represents one of the most common mutations in patients with acute myeloid leukemia (AML) which results in constitutive aberrant activation, increased proliferation of leukemic progenitors and is associated with an aggressive clinical phenotype. The expression of CD97, an EGF-TM7 receptor, has been linked to invasive behavior in thyroid and colorectal cancer. Here, we have investigated the association of CD97 with FLT3-ITD and its functional consequences in AML. Higher CD97 expression levels have been detected in 208 out of 385 primary AML samples. This was accompanied by a significantly increased bone marrow blast count as well as by mutations in the FLT3 gene. FLT3-ITD expressing cell lines as MV4-11 and MOLM-13 revealed significantly higher CD97 levels than FLT3 wildtype EOL-1, OCI-AML3 and HL-60 cells which were clearly decreased by the tyrosine kinase inhibitors PKC412 and SU5614. CD97 knock down by short hairpin RNA in MV4-11 cells resulted in inhibited trans-well migration towards fetal calf serum (FCS) and lysophosphatidic acid (LPA) being at least in part Rho-A dependent. Moreover, knock down of CD97 led to an altered mechanical phenotype, reduced adhesion to a stromal layer and lower wildtype FLT3 expression. Our results, thus, constitute the first evidence for the functional relevance of CD97 expression in FLT3-ITD AML cells rendering it a potential new theragnostic target. PMID:26462154

  4. Small molecules ATP-competitive inhibitors of FLT3: a chemical overview.

    PubMed

    Schenone, S; Brullo, C; Botta, M

    2008-01-01

    FLT3 is a tyrosine kinase (TK), member of the class III TK receptor family, normally expressed in hematopoietic, immune and neural systems, also playing an important role in the pathogenesis of acute leukemias, particularly acute myeloid leukemia (AML), where it is present in constitutively activated mutated forms, correlated with poor prognosis, in a notable percentage of patients. For these reasons FLT3 soon appeared as a promising target for the therapeutic intervention for this severe and aggressive malignancy; the recent determination of the crystal structure of the autoinhibited form of FLT3 gave new trend for the design and the synthesis of potent inhibitors. Small molecules tyrosine kinase inhibitors represent one of the largest drug family currently targeted by pharmaceutical companies for the treatment of cancer. Exciting examples of such molecules have reached advanced clinical trials and have been recently approved by FDA for the treatment of different solid or haematological tumors. Usually TK inhibitors share common features, namely two hydrophobic/aromatic regions bearing one or more hydrogen bonding substituents. These two regions can be connected by different spacers and almost all the molecules contain a component resembling the ATP purine structure. This review will deal with FLT3 synthetic inhibitors, reporting not only the most important molecules that are in clinical trials, but also the new compounds that have appeared in literature in the last few years. Our attention will be focused on chemical structures, mechanisms of action and structure-activity relationships. PMID:19075657

  5. TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality

    PubMed Central

    Wang, Jue; Li, Tongjuan; Zhou, Mi; Hu, Zheng; Zhou, Xiaoxi; Zhou, Shiqiu; Wang, Na; Huang, Liang; Zhao, Lei; Cao, Yang; Xiao, Min; Ma, Ding; Zhou, Pengfei; Shang, Zhen; Zhou, Jianfeng

    2015-01-01

    Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations. PMID:26669855

  6. BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor–induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3

    PubMed Central

    Nordigården, Amanda; Kraft, Maria; Eliasson, Pernilla; Labi, Verena; Lam, Eric W.-F.; Villunger, Andreas; Jönsson, Jan-Ingvar

    2012-01-01

    Constitutively activating internal tandem duplications (ITD) of FLT3 (FMS-like tyrosine kinase 3) are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD–mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapototic mechanisms of 2 small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras–mitogen-activated protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition. PMID:19064725

  7. Tumor Targeting via Integrin Ligands

    PubMed Central

    Marelli, Udaya Kiran; Rechenmacher, Florian; Sobahi, Tariq Rashad Ali; Mas-Moruno, Carlos; Kessler, Horst

    2013-01-01

    Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side-effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability, and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor-specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug-delivery systems, and discuss the prospects of such therapies to specifically target tumor cells. PMID:24010121

  8. The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice

    PubMed Central

    Schessl, Christina; Rawat, Vijay P.S.; Cusan, Monica; Deshpande, Aniruddha; Kohl, Tobias M.; Rosten, Patricia M.; Spiekermann, Karsten; Humphries, R. Keith; Schnittger, Susanne; Kern, Wolfgang; Hiddemann, Wolfgang; Quintanilla-Martinez, Leticia; Bohlander, Stefan K.; Feuring-Buske, Michaela; Buske, Christian

    2005-01-01

    The molecular characterization of leukemia has demonstrated that genetic alterations in the leukemic clone frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-ETO) and mutations affecting genes involved in signal transduction (e.g., activating mutations of FLT3 and KIT). This finding has favored a model of leukemogenesis in which the collaboration of these 2 classes of genetic alterations is necessary for the malignant transformation of hematopoietic progenitor cells. The model is supported by experimental data indicating that AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model. Moreover, in a series of 135 patients with AML1-ETO–positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS. These data support the concept of oncogenic cooperation between AML1-ETO and a class of activating mutations, recurrently found in patients with t(8;21), and provide a rationale for therapies targeting signal transduction pathways in AML1-ETO–positive leukemias. PMID:16025155

  9. Ligand-targeted liposomes for cancer treatment.

    PubMed

    Sapra, Puja; Tyagi, Pradeep; Allen, Theresa M

    2005-10-01

    Selective targeting of ligand-targeted liposomes containing anticancer drugs or therapeutic genes to cell surface receptors expressed on cancer cells is a recognized strategy for improving the therapeutic effectiveness of conventional chemotherapeutics or gene therapeutics. Some recent advances in the field of ligand-targeted liposomes for the treatment of cancer are summarized including: selection criteria for the receptors to be targeted, choice of targeting ligands and choice of encapsulated therapeutics. Targeting of liposomes to solid tumors, versus angiogenic endothelial cells versus vascular targets is discussed. Ligand-targeted liposomes have shown considerable promise in preclinical xenograft models and are poised for clinical development. PMID:16305440

  10. Hoxa9 Regulates Flt3 in Lymphohematopoietic Progenitors

    PubMed Central

    Gwin, Kimberly; Frank, Elena; Bossou, Ayoko; Medina, Kay L.

    2014-01-01

    Early B cell factor (EBF) is a transcription factor essential for specification and commitment to the B cell fate. In this study, we show downregulation of a developmentally regulated cluster of hoxa genes, notably hoxa9, coincides with induction of EBF at the Pro-B cell stage of B cell differentiation. Analysis of the hematopoietic progenitor compartment in Hoxa9−/− mice revealed significantly reduced frequencies and expression levels of Flt3, a cytokine receptor important for lymphoid priming and the generation of B cell precursors (BCPs). We show that Hoxa9 directly regulates the flt3 gene. Chromatin immunoprecipitation analysis revealed binding of Hoxa9 to the flt3 promoter in a lymphoid progenitor cell line. Knockdown of Hoxa9 significantly reduced Flt3 transcription and expression. Conversely, forced expression of Hoxa9 increased Flt3 transcription and expression in a Pro-B cell line that expressed low levels of Flt3. Hoxa9 inversely correlated with ebf1 in ex vivo-isolated bone marrow progenitors and BCPs, suggesting that EBF might function to silence a Hoxa9 transcriptional program. Restoration of EBF function in an EBF−/− cell line induced B lineage gene expression but did not directly suppress hoxa9 transcription, revealing alternate mechanisms of Hoxa9 regulation in BCPs. These data provide new insight into Hoxa9 function and regulation during lymphoid and B cell development. Furthermore, they suggest that failure to upregulate Flt3 provides a molecular basis for the lymphoid/early B cell deficiencies in Hoxa9−/− mice. PMID:20971928

  11. Leukemogenic potency of the novel FLT3-N676K mutant.

    PubMed

    Huang, Kezhi; Yang, Min; Pan, Zengkai; Heidel, Florian H; Scherr, Michaela; Eder, Matthias; Fischer, Thomas; Büsche, Guntram; Welte, Karl; von Neuhoff, Nils; Ganser, Arnold; Li, Zhixiong

    2016-04-01

    The novel FMS-like tyrosine kinase 3 (FLT3)-N676K point mutation within the FLT3 kinase domain-1 was recently identified in 6 % of de novo acute myeloid leukemia (AML) patients with inv(16). Because FLT3-N676K was encountered almost exclusively in inv(16) AML, we investigated the transforming potential of FLT3-N676K, the cooperation between FLT3-N676K and core binding factor ß-smooth muscle myosin heavy chain (CBFß-SMMHC) (encoded by the inv(16) chimeric gene CBFB-MYH11) in inducing acute leukemia, and tested the sensitivity of FLT3-N676K-positive leukemic cells to FLT3 inhibitors. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n = 11/13, median latency 58 days), with a transforming activity similar to FLT3-internal tandem duplication (ITD) (n = 8/8), FLT3-TKD D835Y (n = 8/9), and FLT3-ITD-N676K (n = 9/9) mutations. Three out of 14 (21.4 %) C57BL/6J mice transplanted with FLT3-N676K-transduced primary hematopoietic progenitor cells developed acute leukemia (latency of 68, 77, and 273 days), while no hematological malignancy was observed in the control groups including FLT3-ITD. Moreover, co-expression of FLT3-N676K/CBFß-SMMHC did not promote acute leukemia in three independent experiments (n = 16). In comparison with FLT3-ITD, FLT3-N676K induced much higher activation of FLT3 and tended to trigger stronger phosphorylation of MAPK and AKT. Importantly, leukemic cells carrying the FLT3-N676K mutant in the absence of an ITD mutation were highly sensitive to FLT3 inhibitors AC220 and crenolanib, and crenolanib even retained activity against the AC220-resistant FLT3-ITD-N676K mutant. Taken together, the FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, further experiments investigating molecular mechanisms for leukemogenesis induced by FLT3-N676

  12. Cabozantinib is selectively cytotoxic in acute myeloid leukemia cells with FLT3-internal tandem duplication (FLT3-ITD).

    PubMed

    Lu, Jeng-Wei; Wang, An-Ni; Liao, Heng-An; Chen, Chien-Yuan; Hou, Hsin-An; Hu, Chung-Yi; Tien, Hwei-Fan; Ou, Da-Liang; Lin, Liang-In

    2016-07-01

    Cabozantinib is an oral multikinase inhibitor that exhibits anti-tumor activity in several cancers. We found that cabozantinib was significantly cytotoxic to MV4-11 and Molm-13 cells that harbored FLT3-ITD, resulting in IC50 values of 2.4 nM and 2.0 nM, respectively. However, K562, OCI-AML3 and THP-1 (leukemia cell lines lacking FLT3-ITD) were resistant to cabozantinib, showing IC50 values in the micromolar range. Cabozantinib arrested MV4-11 cell growth at the G0/G1 phase within 24 h, which was associated with decreased phosphorylation of FLT3, STAT5, AKT and ERK. Additionally, cabozantinib induced MV4-11 cell apoptosis in a dose-dependent manner (as indicated by annexin V staining and high levels of cleaved caspase 3 and PARP-1), down-regulated the anti-apoptotic protein survivin and up-regulated the pro-apoptotic protein Bak. Thus, cabozantinib is selectively cytotoxic to leukemia cells with FLT3-ITD, causing cell-cycle arrest and apoptosis. In mouse xenograft model, cabozantinib significantly inhibited MV4-11 and Molm-13 tumor growth at a dosage of 10 mg/kg and showed longer survival rate. Clinical trials evaluating the efficacy of cabozantinib in acute myeloid leukemia (AML) with FLT3-ITD are warranted. PMID:27060207

  13. TTT-3002 is a novel FLT3 tyrosine kinase inhibitor with activity against FLT3-associated leukemias in vitro and in vivo

    PubMed Central

    Ma, Hayley; Nguyen, Bao; Li, Li; Greenblatt, Sarah; Williams, Allen; Zhao, Ming; Levis, Mark; Rudek, Michelle; Duffield, Amy

    2014-01-01

    More than 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FMS-like tyrosine kinase-3 (FLT3). The most common type, internal tandem duplication (ITD), confers poor prognosis. We report for the first time on TTT-3002, a tyrosine kinase inhibitor (TKI) that is one of the most potent FLT3 inhibitors discovered to date. Studies using human FLT3/ITD mutant leukemia cell lines revealed the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation is from 100 to 250 pM. The proliferation IC50 for TTT-3002 in these same cells was from 490 to 920 pM. TTT-3002 also showed potent activity when tested against the most frequently occurring FLT3-activating point mutation, FLT3/D835Y, against which many current TKIs are ineffective. These findings were validated in vivo by using mouse models of FLT3-associated AML. Survival and tumor burden of mice in several FLT3/ITD transplantation models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrated that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD-expressing AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from healthy blood and bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI in the treatment of FLT3-mutant AML. PMID:24408321

  14. FLT3/D835Y mutation knock-in mice display less aggressive disease compared with FLT3/internal tandem duplication (ITD) mice

    PubMed Central

    Bailey, Emily; Li, Li; Duffield, Amy S.; Ma, Hayley S.; Huso, David L.; Small, Don

    2013-01-01

    FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately one third of acute myeloid leukemia cases. The most common FLT3 mutations in acute myeloid leukemia are internal tandem duplication (ITD) mutations in the juxtamembrane domain (23%) and point mutations in the tyrosine kinase domain (10%). The mutation substituting the aspartic acid at position 838 (equivalent to the human aspartic acid residue at position 835) with a tyrosine (referred to as FLT3/D835Y hereafter) is the most frequent kinase domain mutation, converting aspartic acid to tyrosine. Although both of these mutations constitutively activate FLT3, patients with an ITD mutation have a significantly poorer prognosis. To elucidate the mechanisms behind this prognostic difference, we have generated a knock-in mouse model with a D838Y point mutation in FLT3 that corresponds to the FLT3/D835Y mutation described in humans. Compared with FLT3/ITD knock-in mice, the FLT3/D835Y knock-in mice survive significantly longer. The majority of these mice develop myeloproliferative neoplasms with a less-aggressive phenotype. In addition, FLT3/D835Y mice have distinct hematopoietic development patterns. Unlike the tremendous depletion of the hematopoietic stem cell compartment we have observed in FLT3/ITD mice, FLT3/D835Y mutant mice are not depleted in hematopoietic stem cells. Further comparisons of these FLT3/D835Y knock-in mice with FLT3/ITD mice should provide an ideal platform for dissecting the molecular mechanisms that underlie the prognostic differences between the two different types of FLT3 mutations. PMID:24255108

  15. Structural modifications at the 6-position of thieno[2,3-d]pyrimidines and their effects on potency at FLT3 for treatment of acute myeloid leukemia.

    PubMed

    Kim, Hyuntae; Lee, Chulho; Yang, Jee Sun; Choi, Seonghwi; Park, Chun-Ho; Kang, Jong Soon; Oh, Soo Jin; Yun, Jieun; Kim, Myung-Hwa; Han, Gyoonhee

    2016-09-14

    Fms-like tyrosine kinase 3 (FLT3) is a well-known and important target for the treatment of acute myeloid leukemia (AML). A series of thieno[2,3-d]pyrimidine derivatives from a modification at the 6-position were synthesized to identify effective FLT3 inhibitors. Although compounds 1 and 2 emerged as promising FLT3 inhibitors among the synthesized compounds, both compounds exhibited poor metabolic stability in human and rat liver microsomes. Hence, further optimization was required for the discovery of FLT3 inhibitors, with a focus on improving metabolic stability. Compound 16d, which had structural modifications of the methyl group at the 5-position and the 4-(2-methylaminoethoxy) phenyl group at the 6-position, exhibited good inhibitory activity against FLT3 and showed effective antiproliferative activity against four leukemia cell lines, including MV4-11. Moreover, compound 16d displayed enhanced metabolic stability. The results of this study indicated that 16d could be a promising compound for further optimization and development as a potent FLT3 inhibitor. PMID:27187860

  16. SIRT1 activation by a c-MYC oncogenic network promotes the maintenance and drug resistance of human FLT3-ITD acute myeloid leukemia stem cells.

    PubMed

    Li, Ling; Osdal, Tereza; Ho, Yinwei; Chun, Sookhee; McDonald, Tinisha; Agarwal, Puneet; Lin, Allen; Chu, Su; Qi, Jing; Li, Liang; Hsieh, Yao-Te; Dos Santos, Cedric; Yuan, Hongfeng; Ha, Trung-Quang; Popa, Mihaela; Hovland, Randi; Bruserud, Oystein; Gjertsen, Bjørn Tore; Kuo, Ya-Huei; Chen, Wenyong; Lain, Sonia; McCormack, Emmet; Bhatia, Ravi

    2014-10-01

    The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes. PMID:25280219

  17. SIRT1 Activation by a c-MYC Oncogenic Network Promotes the Maintenance and Drug Resistance of Human FLT3-ITD Acute Myeloid Leukemia Stem Cells

    PubMed Central

    Li, Ling; Osdal, Tereza; Ho, Yinwei; Chun, Sookhee; McDonald, Tinisha; Agarwal, Puneet; Lin, Allen; Chu, Su; Qi, Jing; Li, Liang; Hsieh, Yao-Te; Santos, Cedric Dos; Yuan, Hongfeng; Ha, Trung-Quang; Popa, Mihaela; Hovland, Randi; Bruserud, Øystein; Gjertsen, Bjørn Tore; Kuo, Ya-Huei; Chen, Wenyong; Lain, Sonia; McCormack, Emmet; Bhatia, Ravi

    2015-01-01

    SUMMARY The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 de-acetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes. PMID:25280219

  18. Sorafenib in combination with low-dose-homoharringtonine as a salvage therapy in primary refractory FLT3-ITD-positive AML: a case report and review of literature

    PubMed Central

    Xu, Gaixiang; Mao, Liping; Liu, Hui; Yang, Min; Jin, Jie; Qian, Wenbin

    2015-01-01

    The presence of internal tandem duplications (ITD) in the Fms-related tyrosine kinase 3 receptor (FLT3) has been associated with a poor prognosis in acute myeloid leukemia (AML). Over the past decade, FLT3 is a promising target in FLT3-ITD-positive AML. Sorafenib which is one of the commonly focused FLT3 inhibitors may improve outcome, but only few patients display long-term responses in previously reported cases, prompting the search for underlying resistance mechanisms and therapeutic strategies to overcome them. To the best of our knowledge, this is the first case report about sorafenib in combination with low-dose-homoharringtonine as a salvage therapy successfully administrated and got complete remission (CR) in primary refractory FLT3-ITD-positive AML. Our result demonstrates the combination of this two drugs may be a good choice for the primary refractory FLT3-ITD-positive AML patient, although cooperative studies of large numbers of these patients are needed to evaluate and optimize this combination. PMID:26884901

  19. NKG2D ligands as therapeutic targets

    PubMed Central

    Spear, Paul; Wu, Ming-Ru; Sentman, Marie-Louise; Sentman, Charles L.

    2013-01-01

    The Natural Killer Group 2D (NKG2D) receptor plays an important role in protecting the host from infections and cancer. By recognizing ligands induced on infected or tumor cells, NKG2D modulates lymphocyte activation and promotes immunity to eliminate ligand-expressing cells. Because these ligands are not widely expressed on healthy adult tissue, NKG2D ligands may present a useful target for immunotherapeutic approaches in cancer. Novel therapies targeting NKG2D ligands for the treatment of cancer have shown preclinical success and are poised to enter into clinical trials. In this review, the NKG2D receptor and its ligands are discussed in the context of cancer, infection, and autoimmunity. In addition, therapies targeting NKG2D ligands in cancer are also reviewed. PMID:23833565

  20. Prognostic impact of FLT3-ITD load in NPM1 mutated acute myeloid leukemia.

    PubMed

    Schnittger, S; Bacher, U; Kern, W; Alpermann, T; Haferlach, C; Haferlach, T

    2011-08-01

    High FLT3-ITD/wildtype (wt) load in FLT3-ITD-mutated AML has been associated with adverse impact on outcome in several studies. To clarify whether FLT3-ITD load as expressed as FLT3-ITD/wt ratio is also relevant in patients with NPM1 mutated AML, we assessed the FLT3-ITD mutation status and FLT3-ITD/wt ratio by fragment analysis in 638 NPM1mut AML (339 females; 299 males; 17.8-88.0 years), and analyzed its prognostic relevance in 355 patients. FLT3-ITD of various length and load were detected in 243/638 cases (38.1%). Median EFS (19.3 vs 9.7 months, P<0.001) and median 2-year survival rate (72.0 vs 52.7%, P=0.006) was better in FLT3wt (n=212 with available follow-up data) than FLT3-ITD (n=143). A higher FLT3-ITD/wt ratio as continuous variable was correlated with a shorter EFS (P=0.028). When patients were separated into subgroups according to the FLT3-ITD mutation load, only a FLT3-ITD/wt ratio 0.5 conferred an independent adverse impact on EFS and OS, and retained its prognostic significance also in multivariate analysis (P=0.009 for EFS, P=0.008 for OS). In conclusion, for risk estimation in NPM1 mutated AML not only the FLT3-ITD status, but also the FLT3-ITD load has to be taken into account. These data might contribute to clinical decision making in AML. PMID:21537333

  1. FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

    PubMed Central

    Neumann, Martin; Coskun, Ebru; Fransecky, Lars; Mochmann, Liliana H.; Bartram, Isabelle; Farhadi Sartangi, Nasrin; Heesch, Sandra; Gökbuget, Nicola; Schwartz, Stefan; Brandts, Christian; Schlee, Cornelia; Haas, Rainer; Dührsen, Ulrich; Griesshammer, Martin; Döhner, Hartmut; Ehninger, Gerhard; Burmeister, Thomas; Blau, Olga; Thiel, Eckhard; Hoelzer, Dieter; Hofmann, Wolf-Karsten; Baldus, Claudia D.

    2013-01-01

    Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup. PMID:23359050

  2. Expression profile of heat shock proteins in acute myeloid leukaemia patients reveals a distinct signature strongly associated with FLT3 mutation status--consequences and potentials for pharmacological intervention.

    PubMed

    Reikvam, Håkon; Hatfield, Kimberley J; Ersvaer, Elisabeth; Hovland, Randi; Skavland, Jørn; Gjertsen, Bjørn T; Petersen, Kjell; Bruserud, Oystein

    2012-02-01

    Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSPs are regarded as possible therapeutic targets in acute myeloid leukaemia (AML). We used bioinformatical approaches to characterize the HSP profile in AML cells from 75 consecutive patients, in addition to the effect of the HSP90 inhibitor 17-DMAG. Patients harbouring a FLT3-internal tandem duplication (FLT3-ITD) were extensively overrepresented in the cluster with high HSP levels, indicating a strong dependence of HSPs in stabilizing FLT3-ITD encoded oncoproteins. FLT3 ligation further increased the levels of HSP90 and its co-chaperone HSP70. HSP90 inhibition had a stronger pro-apoptotic effect for AML cells with FLT3-ITD than for cells with wild-type FLT3, whereas the anti-proliferative effect of HSP90 inhibition was similar for the two patient subsets. HSP90 inhibition altered the constitutive cytokine release profile in an anti-angiogenic direction independent of FLT3 mutational status: (i) pro-angiogenic CXCL8, MMP-2 and MMP-9 showed a stronger decrease than anti-angiogenic CXCL9-11, (ii) the Tie-2 agonist Ang-1 showed a stronger decrease than the potentially antagonistic Ang-2, and (iii) VEGF and HGF levels were decreased. Finally, HSP90 inhibition counteracted the leukaemia-stimulating effect of endothelial cells. Our studies demonstrate that HSP90 inhibition mediates anti-leukaemic effects through both direct and indirect activity. PMID:22150087

  3. NPMc+ cooperates with Flt3/ITD mutations to cause acute leukemia recapitulating human disease

    PubMed Central

    Rau, Rachel; Magoon, Daniel; Greenblatt, Sarah; Li, Li; Annesley, Colleen; Duffield, Amy S.; Huso, David; McIntyre, Emily; Clohessy, John G.; Reschke, Markus; Pandolfi, Pier Paolo; Small, Donald; Brown, Patrick

    2013-01-01

    Cytoplasmic nucleophosmin (NPMc+) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML), and they frequently occur together suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity. PMID:24184354

  4. Predicting target-ligand interactions using protein ligand-binding site and ligand substructures

    PubMed Central

    2015-01-01

    Background Cell proliferation, differentiation, Gene expression, metabolism, immunization and signal transduction require the participation of ligands and targets. It is a great challenge to identify rules governing molecular recognition between chemical topological substructures of ligands and the binding sites of the targets. Methods We suppose that the ligand-target interactions are determined by ligand substructures as well as the physical-chemical properties of the binding sites. Therefore, we propose a fragment interaction model (FIM) to describe the interactions between ligands and targets, with the purpose of facilitating the chemical interpretation of ligand-target binding. First we extract target-ligand complexes from sc-PDB database, based on which, we get the target binding sites and the ligands. Then we represent each binding site as a fragment vector based on a target fragment dictionary that is composed of 199 clusters (denoted as fragements in this work) obtained by clustering 4200 trimers according to their physical-chemical properties. And then, we represent each ligand as a substructure vector based on a dictionary containing 747 substructures. Finally, we build the FIM by generating the interaction matrix M (representing the fragment interaction network), and the FIM can later be used for predicting unknown ligand-target interactions as well as providing the binding details of the interactions. Results The five-fold cross validation results show that the proposed model can get higher AUC score (92%) than three prevalence algorithms CS-PD (80%), BLM-NII (85%) and RF (85%), demonstrating the remarkable predictive ability of FIM. We also show that the ligand binding sites (local information) overweight the sequence similarities (global information) in ligand-target binding, and introducing too much global information would be harmful to the predictive ability. Moreover, The derived fragment interaction network can provide the chemical insights on

  5. Nanoparticle ligand presentation for targeting solid tumors.

    PubMed

    Duskey, Jason T; Rice, Kevin G

    2014-10-01

    Among the many scientific advances to come from the study of nanoscience, the development of ligand-targeted nanoparticles to eliminate solid tumors is predicted to have a major impact on human health. There are many reports describing novel designs and testing of targeted nanoparticles to treat cancer. While the principles of the technology are well demonstrated in controlled lab experiments, there are still many hurdles to overcome for the science to mature into truly efficacious targeted nanoparticles that join the arsenal of agents currently used to treat cancer in humans. One of these hurdles is overcoming unwanted biodistribution to the liver while maximizing delivery to the tumor. This almost certainly requires advances in both nanoparticle stealth technology and targeting. Currently, it continues to be a challenge to control the loading of ligands onto polyethylene glycol (PEG) to achieve maximal targeting. Nanoparticle cellular uptake and subcellular targeting of genes and siRNA also remain a challenge. This review examines the types of ligands that have been most often used to target nanoparticles to solid tumors. As the science matures over the coming decade, careful control over ligand presentation on nanoparticles of precise size, shape, and charge will likely play a major role in achieving success. PMID:24927668

  6. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    DOE PAGESBeta

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; Barros, Tiago; Kuriyan, John; Boggon, Titus J.

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain ofmore » FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.« less

  7. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    SciTech Connect

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; Barros, Tiago; Kuriyan, John; Boggon, Titus J.

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

  8. UNC2025, a Potent and Orally Bioavailable MER/FLT3 Dual Inhibitor

    PubMed Central

    2015-01-01

    We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined. PMID:25068800

  9. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells.

    PubMed

    Stanicka, Joanna; Russell, Eileen G; Woolley, John F; Cotter, Thomas G

    2015-04-10

    Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22(phox), a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22(phox) and p22(phox)-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22(phox), and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22(phox) localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22(phox) mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability. PMID:25697362

  10. NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells*

    PubMed Central

    Stanicka, Joanna; Russell, Eileen G.; Woolley, John F.; Cotter, Thomas G.

    2015-01-01

    Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22phox, a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22phox and p22phox-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22phox, and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22phox localize to the nuclear membrane in MV4–11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22phox mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability. PMID:25697362

  11. IMC-EB10, an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.

    PubMed

    Piloto, Obdulio; Nguyen, Bao; Huso, David; Kim, Kyu-Tae; Li, Yiwen; Witte, Larry; Hicklin, Daniel J; Brown, Patrick; Small, Donald

    2006-05-01

    The class III receptor tyrosine kinase FLT3 is expressed on the blasts of >90% of patients with B-lineage acute lymphoblastic leukemias (ALL). In addition, it is expressed at extremely high levels in ALL patients with mixed lineage leukemia rearrangements or hyperdiploidy and is sometimes mutated in these same patients. In this report, we investigate the effects of treating ALL cell lines and primary samples with human anti-FLT3 monoclonal antibodies (mAb) capable of preventing binding of FLT3 ligand. In vitro studies, examining the ability of two anti-FLT3 mAbs (IMC-EB10 and IMC-NC7) to affect FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. FLT3 phosphorylation was consistently inhibited by IMC-NC7 treatment, but in some cell lines, IMC-EB10 actually stimulated FLT3 activation, possibly as a result of antibody-mediated receptor dimerization. Through antibody-dependent, cell-mediated cytotoxicity, such an antibody could still prove efficacious against leukemia cells in vivo. In fact, IMC-EB10 treatment significantly prolonged survival and/or reduced engraftment of several ALL cell lines and primary ALL samples in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This occurred even when IMC-EB10 treatment resulted in FLT3 activation in vitro. Moreover, fluorescence-activated cell sorting and PCR analysis of IMC-EB10-treated NOD/SCID mice surviving 150 days post-leukemic cell injection revealed that FLT3 immunotherapy reduced leukemic engraftment below the level of detection in these assays (<0.001%). Furthermore, in vivo IMC-EB10 treatment did not select for resistant cells, because cells surviving IMC-EB10 treatment remain sensitive to IMC-EB10 cytotoxicity upon retransplantation. In vivo studies involving either partial depletion or activation of natural killer (NK) cells show that most of the cytotoxic effect of IMC-EB10 is mediated through NK cells. Therefore, such an antibody, either

  12. Copy-neutral loss of heterozygosity is prevalent and a late event in the pathogenesis of FLT3/ITD AML.

    PubMed

    Stirewalt, D L; Pogosova-Agadjanyan, E L; Tsuchiya, K; Joaquin, J; Meshinchi, S

    2014-01-01

    Patients with high FLT3 internal tandem duplication allelic ratios (FLT3/ITD-ARs) have a poor prognosis. Single-nucleotide polymorphism/comparative genomic hybridization, single-cell PCR and colony-forming assays were used to evaluate genotypic evolution of high FLT3/ITD-ARs in 85 acute myeloid leukemia (AML) patients. Microarrays were used to examine molecular pathways disrupted in leukemic blasts with high FLT3/ITD-ARs. Copy-neutral loss of heterozygosity (CN-LOH) was identified at the FLT3 locus in diagnostic samples with high FLT3/ITD-ARs (N=11), but not in samples with low FLT3/ITD-ARs (N=24), FLT3-activating loop mutations (N=11) or wild-type FLT3 (N=39). Single-cell assays showed that homozygous FLT3/ITD genotype was present in subsets of leukemic blasts at diagnosis but became the dominant clone at relapse. Less differentiated CD34(+)/CD33(-) progenitor colonies were heterozygous for FLT3/ITD, whereas more differentiated CD34(+)/CD33(+) progenitor colonies were homozygous for FLT3/ITD. Expression profiling revealed that samples harboring high FLT3/ITD-ARs aberrantly expressed genes within the recombination/DNA repair pathway. Thus, the development of CN-LOH at the FLT3 locus, which results in high FLT3/ITD-ARs, likely represents a late genomic event that occurs after the acquisition of the FLT3/ITD. Although the etiology underlying the development of CN-LOH remains to be clarified, the disruption in recombination/DNA repair pathway, which is present before the development of LOH, may have a role. PMID:24786392

  13. Copy-neutral loss of heterozygosity is prevalent and a late event in the pathogenesis of FLT3/ITD AML

    PubMed Central

    Stirewalt, D L; Pogosova-Agadjanyan, E L; Tsuchiya, K; Joaquin, J; Meshinchi, S

    2014-01-01

    Patients with high FLT3 internal tandem duplication allelic ratios (FLT3/ITD-ARs) have a poor prognosis. Single-nucleotide polymorphism/comparative genomic hybridization, single-cell PCR and colony-forming assays were used to evaluate genotypic evolution of high FLT3/ITD-ARs in 85 acute myeloid leukemia (AML) patients. Microarrays were used to examine molecular pathways disrupted in leukemic blasts with high FLT3/ITD-ARs. Copy-neutral loss of heterozygosity (CN-LOH) was identified at the FLT3 locus in diagnostic samples with high FLT3/ITD-ARs (N=11), but not in samples with low FLT3/ITD-ARs (N=24), FLT3-activating loop mutations (N=11) or wild-type FLT3 (N=39). Single-cell assays showed that homozygous FLT3/ITD genotype was present in subsets of leukemic blasts at diagnosis but became the dominant clone at relapse. Less differentiated CD34+/CD33− progenitor colonies were heterozygous for FLT3/ITD, whereas more differentiated CD34+/CD33+ progenitor colonies were homozygous for FLT3/ITD. Expression profiling revealed that samples harboring high FLT3/ITD-ARs aberrantly expressed genes within the recombination/DNA repair pathway. Thus, the development of CN-LOH at the FLT3 locus, which results in high FLT3/ITD-ARs, likely represents a late genomic event that occurs after the acquisition of the FLT3/ITD. Although the etiology underlying the development of CN-LOH remains to be clarified, the disruption in recombination/DNA repair pathway, which is present before the development of LOH, may have a role. PMID:24786392

  14. FLT3 and CDK4/6 inhibitors: signaling mechanisms and tumor burden in subcutaneous and orthotopic mouse models of acute myeloid leukemia.

    PubMed

    Zhang, Yaping; Hsu, Cheng-Pang; Lu, Jian-Feng; Kuchimanchi, Mita; Sun, Yu-Nien; Ma, Ji; Xu, Guifen; Zhang, Yilong; Xu, Yang; Weidner, Margaret; Huard, Justin; D'Argenio, David Z

    2014-12-01

    FLT3(ITD) subtype acute myeloid leukemia (AML) has a poor prognosis with currently available therapies. A number of small molecule inhibitors of FLT3 and/or CDK4/6 are currently under development. A more complete and quantitative understanding of the mechanisms of action of FLT3 and CDK4/6 inhibitors may better inform the development of current and future compounds that act on one or both of the molecular targets, and thus may lead to improved treatments for AML. In this study, we investigated in both subcutaneous and orthotopic AML mouse models, the mechanisms of action of three FLT3 and/or CDK4/6 inhibitors: AMG925 (Amgen), sorafenib (Bayer and Onyx), and quizartinib (Ambit Biosciences). A composite model was developed to integrate the plasma pharmacokinetics of these three compounds on their respective molecular targets, the coupling between the target pathways, as well as the resulting effects on tumor burden reduction in the subcutaneous xenograft model. A sequential modeling approach was used, wherein model structures and estimated parameters from upstream processes (e.g. PK, cellular signaling) were fixed for modeling subsequent downstream processes (cellular signaling, tumor burden). Pooled data analysis was employed for the plasma PK and cellular signaling modeling, while population modeling was applied to the tumor burden modeling. The resulting model allows the decomposition of the relative contributions of FLT3(ITD) and CDK4/6 inhibition on downstream signaling and tumor burden. In addition, the action of AMG925 on cellular signaling and tumor burden was further studied in an orthotopic tumor mouse model more closely representing the physiologically relevant environment for AML. PMID:25326874

  15. FLT3 and NPM1 mutations in Chinese patients with acute myeloid leukemia and normal cytogenetics.

    PubMed

    Wang, Lei; Xu, Wei-lai; Meng, Hai-tao; Qian, Wen-bin; Mai, Wen-yuan; Tong, Hong-yan; Mao, Li-ping; Tong, Yin; Qian, Jie-jing; Lou, Yin-jun; Chen, Zhi-mei; Wang, Yun-gui; Jin, Jie

    2010-10-01

    Mutations of fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) exon 12 genes are the most common abnormalities in adult acute myeloid leukemia (AML) with normal cytogenetics. To assess the prognostic impact of the two gene mutations in Chinese AML patients, we used multiplex polymerase chain reaction (PCR) and capillary electrophoresis to screen 76 AML patients with normal cytogenetics for mutations in FLT3 internal tandem duplication (FLT3/ITD) and exon 12 of the NPM1 gene. FLT3/ITD mutation was detected in 15 (19.7%) of 76 subjects, and NPM1 mutation in 20 (26.3%) subjects. Seven (9.2%) cases were positive for both FLT3/ITD and NPM1 mutations. Significantly more FLT3/ITD aberration was detected in subjects with French-American-British (FAB) M1 (42.8%). NPM1 mutation was frequently detected in subjects with M5 (47.1%) and infrequently in subjects with M2 (11.1%). FLT3 and NPM1 mutations were significantly associated with a higher white blood cell count in peripheral blood and a lower CD34 antigen expression, but not age, sex, or platelet count. Statistical analysis revealed that the FLT3/ITD-positive group had a lower complete remission (CR) rate (53.3% vs. 83.6%). Survival analysis showed that the FLT3/ITD-positive/NPM1 mutation-negative group had worse overall survival (OS) and relapse-free survival (RFS). The FLT3/ITD-positive/NPM1 mutation-positive group showed a trend towards favorable survival compared with the FLT3/ITD-positive/NPM1 mutation-negative group (P=0.069). Our results indicate that the FLT3/ITD mutation might be a prognostic factor for an unfavorable outcome in Chinese AML subjects with normal cytogenetics, while NPM1 mutation may be a favorable prognostic factor for OS and RFS in the presence of FLT3/ITD. PMID:20872983

  16. FLT3 and NPM1 mutations in Chinese patients with acute myeloid leukemia and normal cytogenetics

    PubMed Central

    Wang, Lei; Xu, Wei-lai; Meng, Hai-tao; Qian, Wen-bin; Mai, Wen-yuan; Tong, Hong-yan; Mao, Li-ping; Tong, Yin; Qian, Jie-jing; Lou, Yin-jun; Chen, Zhi-mei; Wang, Yun-gui; Jin, Jie

    2010-01-01

    Mutations of fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) exon 12 genes are the most common abnormalities in adult acute myeloid leukemia (AML) with normal cytogenetics. To assess the prognostic impact of the two gene mutations in Chinese AML patients, we used multiplex polymerase chain reaction (PCR) and capillary electrophoresis to screen 76 AML patients with normal cytogenetics for mutations in FLT3 internal tandem duplication (FLT3/ITD) and exon 12 of the NPM1 gene. FLT3/ITD mutation was detected in 15 (19.7%) of 76 subjects, and NPM1 mutation in 20 (26.3%) subjects. Seven (9.2%) cases were positive for both FLT3/ITD and NPM1 mutations. Significantly more FLT3/ITD aberration was detected in subjects with French-American-British (FAB) M1 (42.8%). NPM1 mutation was frequently detected in subjects with M5 (47.1%) and infrequently in subjects with M2 (11.1%). FLT3 and NPM1 mutations were significantly associated with a higher white blood cell count in peripheral blood and a lower CD34 antigen expression, but not age, sex, or platelet count. Statistical analysis revealed that the FLT3/ITD-positive group had a lower complete remission (CR) rate (53.3% vs. 83.6%). Survival analysis showed that the FLT3/ITD-positive/NPM1 mutation-negative group had worse overall survival (OS) and relapse-free survival (RFS). The FLT3/ITD-positive/NPM1 mutation-positive group showed a trend towards favorable survival compared with the FLT3/ITD-positive/NPM1 mutation-negative group (P=0.069). Our results indicate that the FLT3/ITD mutation might be a prognostic factor for an unfavorable outcome in Chinese AML subjects with normal cytogenetics, while NPM1 mutation may be a favorable prognostic factor for OS and RFS in the presence of FLT3/ITD. PMID:20872983

  17. FLT3 and NPM1 gene mutations in childhood acute myeloblastic leukemia.

    PubMed

    Mukda, Ekchol; Pintaraks, Katsarin; Sawangpanich, Rachchadol; Wiangnon, Surapon; Pakakasama, Samart

    2011-01-01

    Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human hematologic malignancies. Mutations in fms-like tyrosine kinase 3 (FLT3) gene including internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain (TKD) as well as in nucleoplasmin (NPM1) gene are associated with pathogenesis of acute myeloblastic leukemia (AML). Several reports have demonstrated high incidences of the FLT3 and NPM1 mutations in adult AML patients. Since the pathogenesis of pediatric AML is different from that of adult and the FLT3 and NPM1 mutations have not been well characterized in childhood AML. Therefore, the objective of this study was to determine the frequencies of FLT3 and NPM1 mutations in 64 newly diagnosed childhood AML patients. All blood and bone marrow samples were previously diagnosed with AML by using flow cytometry and/or cytochemistry. FLT3-ITD and FLT3-TKD were detected by PCR and PCR-RFLP methods, respectively. The NPM1 mutation was analyzed by PCR and direct DNA sequencing. The FLT3 mutations were detected in 7 of 64 (11.1%), including FLT3-ITD in 4 of 64 (6.3%) and FLT-TKD in 3 of 62 (4.8%). The NPM1 mutation was not detected in this cohort. By multivariate analysis, white blood cell counts, peripheral blood and bone marrow blast cell counts at diagnosis were significantly higher in children with FLT3-ITD (P<0.05). In addition, the median percentage of CD117 was significantly higher in leukemic blast cells with FLT3-ITD than those with wild type (P=0.01). We did not find any FLT3 mutations in children aged less than 5 years. The AML M3 cell type was most frequently associated with FLT3 gene mutations (50%). In conclusion, the FLT3 mutations was found in 11.1% but none of NPM1 mutation was detected in Thai children with AML. These data support the hypothesis of different biology and pathogenesis between adult and childhood AML. PMID:22126574

  18. 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

  19. Nucleophosmin mutation in Southeast Asian acute myeloid leukemia: eight novel variants, FLT3 coexistence and prognostic impact of NPM1/FLT3 mutations.

    PubMed

    Boonthimat, Chetsada; Thongnoppakhun, Wanna; Auewarakul, Chirayu U

    2008-10-01

    NPM1 mutations were investigated in 400 Southeast Asian leukemia patients and were detectable in 105 cases (26.25%) of acute myeloid leukemia but in no cases of acute lymphoid leukemia or chronic myeloid leukemia. Eight novel and 5 known mutations were identified. All predicted novel proteins shared the last five amino acids VSLRK with the similar gain of nuclear exporting signal motif as known variants. Older age, high white blood cell and platelet counts, normal cytogenetics, and CD34-negativity were associated with NPM1 mutation. FLT3 mutation was more frequent in mutant NPM1 than wild-type cases (56.8% vs. 25.6%) whereas RAS and AML1 mutations were rarely found. Overall survival analysis based on the NPM1/FLT3 mutational status revealed a better outcome for the NPM1-positive/FLT3-negative subgroup. We conclude that: i) NPM1 mutation represents a common genetic hallmark in Southeast Asian acute myeloid leukemia with a normal karyotype; ii) NPM1 mutants coexisted mainly with FLT3 mutants, but not RAS or AML1; iii) FLT3 mutation had a negative prognostic impact on patients with mutant NPM1. PMID:18641025

  20. Allogeneic Hematopoietic Stem Cell Transplantation in FLT3-ITD-Positive Acute Myelogenous Leukemia: The Role for FLT3 Tyrosine Kinase Inhibitors Post-Transplantation.

    PubMed

    Schiller, Gary J; Tuttle, Pamela; Desai, Pinkal

    2016-06-01

    In recent years, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become increasingly common in patients with acute myelogenous leukemia (AML) due to improved donor availability and the use of nonmyeloablative regimens. However, despite the potential clinical gains with allo-HSCT, the post-transplantation outcomes for many patients, especially those with high-risk disease, remain dismal. Patients with AML who have internal tandem duplication mutations in the tyrosine kinase receptor FLT3 (FLT3-ITD) face particularly poor outcomes, even after allo-HSCT, which appears to only partially mitigate the poor prognosis associated with this mutation. Experimental treatments to reduce the likelihood of relapse and improve survival following allo-HSCT include maintenance with FLT3-specific tyrosine kinase inhibitors (TKIs), several of which are currently being evaluated in clinical studies. Preliminary data and case reports suggest that FLT3 TKIs can be effective in the post-transplantation setting, particularly for patients with FLT3-ITD mutations. Improvements in donor matching, transplantation procedures, and supportive care have allowed a greater number of patients to undergo allo-HSCT than ever before. For these patients, it is essential to identify effective post-transplantation therapies to reduce the risk of relapse and improve disease-free survival. PMID:26785334

  1. Targeting mitochondrial energy metabolism with TSPO ligands.

    PubMed

    Gut, Philipp

    2015-08-01

    The translocator protein (18 kDa) (TSPO) resides on the outer mitochondrial membrane where it is believed to participate in cholesterol transport and steroid hormone synthesis. Although it is almost ubiquitously expressed, what TSPO does in non-steroidogenic tissues is largely unexplored. Recent studies report changes in glucose homoeostasis and cellular energy production when TSPO function is modulated by selective ligands or by genetic loss-of-function. This review summarizes findings that connect TSPO function with the regulation of mitochondrial energy metabolism. The juxtaposition of TSPO at the cytosolic/mitochondrial interface and the existence of endogenous ligands that are regulated by metabolism suggest that TSPO functions to adapt mitochondrial to cellular metabolism. From a pharmacological perspective the specific up-regulation of TSPO in neuro-inflammatory and injury-induced conditions make TSPO an interesting, druggable target of mitochondrial metabolism. PMID:26551690

  2. Targeting Selectins and Their Ligands in Cancer

    PubMed Central

    Natoni, Alessandro; Macauley, Matthew S.; O’Dwyer, Michael E.

    2016-01-01

    Aberrant glycosylation is a hallmark of cancer cells with increased evidence pointing to a role in tumor progression. In particular, aberrant sialylation of glycoproteins and glycolipids has been linked to increased immune cell evasion, drug evasion, drug resistance, tumor invasiveness, and vascular dissemination, leading to metastases. Hypersialylation of cancer cells is largely the result of overexpression of sialyltransferases (STs). Differentially, humans express twenty different STs in a tissue-specific manner, each of which catalyzes the attachment of sialic acids via different glycosidic linkages (α2-3, α2-6, or α2-8) to the underlying glycan chain. One important mechanism whereby overexpression of STs contributes to an enhanced metastatic phenotype is via the generation of selectin ligands. Selectin ligand function requires the expression of sialyl-Lewis X and its structural isomer sialyl-Lewis A, which are synthesized by the combined action of alpha α1-3-fucosyltransferases, α2-3-sialyltransferases, β1-4-galactosyltranferases, and N-acetyl-β-glucosaminyltransferases. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 are critical to the generation of functional E- and P-selectin ligands and overexpression of these STs have been linked to increased risk of metastatic disease in solid tumors and poor outcome in multiple myeloma. Thus, targeting selectins and their ligands as well as the enzymes involved in their generation, in particular STs, could be beneficial to many cancer patients. Potential strategies include ST inhibition and the use of selectin antagonists, such as glycomimetic drugs and antibodies. Here, we review ongoing efforts to optimize the potency and selectivity of ST inhibitors, including the potential for targeted delivery approaches, as well as evaluate the potential utility of selectin inhibitors, which are now in early clinical development. PMID:27148485

  3. Targeting Selectins and Their Ligands in Cancer.

    PubMed

    Natoni, Alessandro; Macauley, Matthew S; O'Dwyer, Michael E

    2016-01-01

    Aberrant glycosylation is a hallmark of cancer cells with increased evidence pointing to a role in tumor progression. In particular, aberrant sialylation of glycoproteins and glycolipids has been linked to increased immune cell evasion, drug evasion, drug resistance, tumor invasiveness, and vascular dissemination, leading to metastases. Hypersialylation of cancer cells is largely the result of overexpression of sialyltransferases (STs). Differentially, humans express twenty different STs in a tissue-specific manner, each of which catalyzes the attachment of sialic acids via different glycosidic linkages (α2-3, α2-6, or α2-8) to the underlying glycan chain. One important mechanism whereby overexpression of STs contributes to an enhanced metastatic phenotype is via the generation of selectin ligands. Selectin ligand function requires the expression of sialyl-Lewis X and its structural isomer sialyl-Lewis A, which are synthesized by the combined action of alpha α1-3-fucosyltransferases, α2-3-sialyltransferases, β1-4-galactosyltranferases, and N-acetyl-β-glucosaminyltransferases. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 are critical to the generation of functional E- and P-selectin ligands and overexpression of these STs have been linked to increased risk of metastatic disease in solid tumors and poor outcome in multiple myeloma. Thus, targeting selectins and their ligands as well as the enzymes involved in their generation, in particular STs, could be beneficial to many cancer patients. Potential strategies include ST inhibition and the use of selectin antagonists, such as glycomimetic drugs and antibodies. Here, we review ongoing efforts to optimize the potency and selectivity of ST inhibitors, including the potential for targeted delivery approaches, as well as evaluate the potential utility of selectin inhibitors, which are now in early clinical development. PMID:27148485

  4. BET protein antagonist JQ1 is synergistically lethal with FLT3 tyrosine kinase inhibitor (TKI) and overcomes resistance to FLT3-TKI in AML cells expressing FLT-ITD.

    PubMed

    Fiskus, Warren; Sharma, Sunil; Qi, Jun; Shah, Bhavin; Devaraj, Santhana G T; Leveque, Christopher; Portier, Bryce P; Iyer, Swaminathan; Bradner, James E; Bhalla, Kapil N

    2014-10-01

    Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34(+) human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34(+) normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI-resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD-expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI-resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI. PMID:25053825

  5. Differential requirement for wild-type Flt3 in leukemia-initiation among mouse models of human leukemia

    PubMed Central

    Kamezaki, Kenjiro; Luchsinger, Larry; Snoeck, Hans-Willem

    2014-01-01

    FLT3 is one of the most frequently mutated genes in acute leukemias. However, the role in leukemogenesis of wt Flt3, which is highly expressed in many hematological malignancies, is unclear. We show here that in mouse models established by retroviral transduction of leukemic fusion proteins deletion of Flt3 strongly inhibits MLL-ENL and to lesser extent p210BCR-ABL-induced leukemogenesis, but has no effect in MLL-AF9 or AML1-ETO9a models. Flt3 acts at the level of leukemic stem cells (LSCs), as a fraction of LSCs in MLL-ENL, but not in MLL-AF9-induced leukemia, expressed Flt3 in vivo, and Flt3 expression on LSCs was associated with leukemia development in this model. Furthermore, efficiency of MLL-ENL, but not of MLL-AF9-induced leukemia induction was significantly enhanced after transduction of Flt3+ compared to Flt3− wt myeloid progenitors. However, Flt3 is not required for immortalization of bone marrow cells in vitro by MLL-ENL and does not affect colony-formation by MLL-ENL LSCs in vitro, suggesting that in vitro models do not reflect the in vivo biology of MLL-ENL leukemia with respect to Flt3 requirement. We conclude that wt Flt3 plays a role in leukemia initiation in vivo, which is, however, not universal. PMID:24269847

  6. Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines.

    PubMed

    Oveland, Eystein; Wergeland, Line; Hovland, Randi; Lorens, James B; Gjertsen, Bjørn Tore; Fladmark, Kari E

    2012-08-01

    Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines. PMID:22422053

  7. Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells

    PubMed Central

    Abe, Mariko; Pelus, Louis M.; Singh, Pratibha; Hirade, Tomohiro; Onishi, Chie; Purevsuren, Jamiyan; Taketani, Takeshi; Yamaguchi, Seiji; Fukuda, Seiji

    2016-01-01

    Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression. PMID:27387666

  8. Design of ligand-targeted nanoparticles for enhanced cancer targeting

    NASA Astrophysics Data System (ADS)

    Stefanick, Jared F.

    Ligand-targeted nanoparticles are increasingly used as drug delivery vehicles for cancer therapy, yet have not consistently produced successful clinical outcomes. Although these inconsistencies may arise from differences in disease models and target receptors, nanoparticle design parameters can significantly influence therapeutic efficacy. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, this work evaluates the roles of polyethylene glycol (PEG) coating, ethylene glycol (EG) peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size on tumor targeting in a systematic manner. These parameters were analyzed in multiple disease models by targeting human epidermal growth factor receptor 2 (HER2) in breast cancer and very late antigen-4 (VLA-4) in multiple myeloma to demonstrate the widespread applicability of this approach. By increasing the hydrophilicity of the targeting peptide sequence and simultaneously optimizing the EG peptide-linker length, the in vitro cellular uptake of targeted liposomes was significantly enhanced. Specifically, including a short oligolysine chain adjacent to the targeting peptide sequence effectively increased cellular uptake ~80-fold using an EG6 peptide-linker compared to ~10-fold using an EG45 linker. In vivo, targeted liposomes prepared in a traditional manner lacking the oligolysine chain demonstrated similar biodistribution and tumor uptake to non-targeted liposomes. However, by including the oligolysine chain, targeted liposomes using an EG45 linker significantly improved tumor uptake ~8-fold over non-targeted liposomes, while the use of an EG6 linker decreased tumor accumulation and uptake, owing to differences in cellular uptake kinetics, clearance mechanisms, and binding site barrier effects. To further improve tumor targeting and enhance the selectivity of targeted

  9. Tetraspecific ligand for tumor-targeted delivery of nanomaterials.

    PubMed

    Kim, Dongwook; Friedman, Adam D; Liu, Rihe

    2014-07-01

    The polygenetic nature of most cancers emphasizes the necessity of cancer therapies that target multiple essential signaling pathways. However, there is a significant paucity of targeting ligands with multi-specificities for targeted delivery of biomaterials. To address this unmet need, we generated a tetraspecific targeting ligand that recognizes four different cancer biomarkers, including VEGFR2, αvβ3 integrin, EGFR, and HER2 receptors, which have been implicated in numerous malignant tumors. The tetraspecific targeting ligand was constructed by sequentially connecting four targeting ligand subunits via flexible linkers, yielding a fusion protein that can be highly expressed in Escherichia coli and readily purified to near homogeneity. Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI) studies and extensive cellular binding analyses indicated that all the targeting ligand subunits in the tetraspecific fusion protein recognized their target receptors proximately to the corresponding monospecific ligands. The resulting tetraspecific targeting ligand was applied for the delivery of nanomaterials such as gold nanoparticles (AuNPs) for targeted hyperthermic killing of various cancer cell lines with biomarkers of interest expressed. We demonstrate that the tetraspecific ligand can be facilely introduced on the surface of AuNPs and efficient target-dependent killing of cancer cells can be achieved only when the AuNPs are conjugated with the tetraspecific ligand. Significantly, the tetraspecific ligand simultaneously interacts with more than one receptors, such as EGFR and HER2 receptors, when they are expressed on the surface of the same cell, as demonstrated by in vitro binding assays and cell binding analyses. Our results demonstrate that the tetraspecific ligand, through multivalency and synergistic binding, can be readily used to generate various 'smart' biomaterials with greatly broadened tumor targeting range for simultaneous targeting of multiple

  10. Tetraspecific Ligand for Tumor-Targeted Delivery of Nanomaterials

    PubMed Central

    Kim, Dongwook; Friedman, Adam D.; Liu, Rihe

    2014-01-01

    The polygenetic nature of most cancers emphasizes the necessity of cancer therapies that target multiple essential signaling pathways. However, there is a significant paucity of targeting ligands with multi-specificities for targeted delivery of biomaterials. To address this unmet need, we generated a tetraspecific targeting ligand that recognizes four different cancer biomarkers, including VEGFR2, αvβ3 integrin, EGFR, and HER2 receptors, which have been implicated in numerous malignant tumors. The tetraspecific targeting ligand was constructed by sequentially connecting four targeting ligand subunits via flexible linkers, yielding a fusion protein that can be highly expressed in E. coli and readily purified to near homogeneity. Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI) studies and extensive cellular binding analyses indicated that all the targeting ligand subunits in the tetraspecific fusion protein recognized their target receptors proximately to the corresponding monospecific ligands. The resulting tetraspecific targeting ligand was applied for the delivery of nanomaterials such as gold nanoparticles (AuNPs) for targeted hyperthermic killing of various cancer cell lines with biomarkers of interest expressed. We demonstrate that the tetraspecific ligand can be facilely introduced on the surface of AuNPs and efficient target-dependent killing of cancer cells can be achieved only when the AuNPs are conjugated with the tetraspecific ligand. Significantly, the tetraspecific ligand simultaneously interacts with more than one receptors, such as EGFR and HER2 receptors, when they are expressed on the surface of the same cell, as demonstrated by in vitro binding assays and cell binding analyses. Our results demonstrate that the tetraspecific ligand, through multivalency and synergistic binding, can be readily used to generate various ‘smart’ biomaterials with greatly broadened tumor targeting range for simultaneous targeting of multiple

  11. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

    PubMed Central

    Deeb, Kristin K.; Smonskey, Matthew T.; DeFedericis, HanChun; Deeb, George; Sait, Sheila N.J.; Wetzler, Meir; Wang, Eunice S.; Starostik, Petr

    2014-01-01

    In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations. PMID:25379410

  12. FLT3-ITD-associated gene-expression signatures in NPM1-mutated cytogenetically normal acute myeloid leukemia.

    PubMed

    Huang, Liang; Zhou, Kuangguo; Yang, Yunfan; Shang, Zhen; Wang, Jue; Wang, Di; Wang, Na; Xu, Danmei; Zhou, Jianfeng

    2012-08-01

    Concomitance of the FLT3-ITD mutation is associated with poor prognosis in NPM1-mutated cytogenetically normal acute myeloid leukemia (CN-AML) patients, and precise studies on its role in leukemogenesis are needed; these may be elucidated at the molecular level by gene express profiling. In the present study, we built a gene-expression-based classifier using prediction analysis of microarray to characterize the FLT3-ITD signature in NPM1-mutated CN-AML patients, which comprised 10 annotated genes, and demonstrated an overall accuracy of 83.8 % in cross-validation. To characterize the signature in another way, differential expression was revealed for 34 genes by class comparison, and the up-regulation of LAPTM4B and MIR155HG was validated by quantitative RT-PCR in our small cohort of NPM1-mutated CN-AML samples, which appeared to be associated with this specific subtype. The 10-gene classifier and differentially expressed genes identified in this study indicate a potential utility for risk-assessed treatment stratification, and suggest new therapeutic targets for these high-risk AML patients. PMID:22688855

  13. JAK2, complemented by a second signal from c-kit or flt-3, triggers extensive self-renewal of primary multipotential hemopoietic cells

    PubMed Central

    Zhao, Shengming; Zoller, Karen; Masuko, Masayoshi; Rojnuckarin, Ponlapat; Yang, Xuexian O.; Parganas, Evan; Kaushansky, Kenneth; Ihle, James N.; Papayannopoulou, Thalia; Willerford, Dennis M.; Clackson, Tim; Blau, C.Anthony

    2002-01-01

    Defining signals that can support the self-renewal of multipotential hemopoietic progenitor cells (MHPCs) is pertinent to understanding leukemogenesis and may be relevant to developing stem cell-based therapies. Here we define a set of signals, JAK2 plus either c-kit or flt-3, which together can support extensive MHPC self-renewal. Phenotypically and functionally distinct populations of MHPCs were obtained, depending on which receptor tyrosine kinase, c-kit or flt-3, was activated. Self-renewal was abrogated in the absence of STAT5a/b, and in the presence of inhibitors targeting either the mitogen-activated protein kinase or phosphatidylinositol 3′ kinase pathways. These findings suggest that a simple two-component signal can drive MHPC self-renewal. PMID:11980713

  14. [Investigation of FLT3-ITD and NPM1 mutations in patients with myelodysplastic syndrome and mixed myeloid diseases].

    PubMed

    Gritsaev, S V; Martynkevich, I S; Ivanova, M P; Moskalenko, M V; Aksenova, V Iu; Tiranova, S A; Abdulkadyrov, K M

    2010-01-01

    Two FLT3-ITD mutations, one FLT3-TKD) and five NPM1 mutations were detected in 7 patients with de novo myelodysplastic syndrome (MDS) out of 44 cases of MDS and MDS/mixed myeloid diseases. Expression of one of the three investigated mutations was identified: 4 in gene NPM1 (9.1%) and 2--FLT3-ITD (4.5%); simultaneous FLT3-ITD and NPM1 mutation--1 (2.3%); no progression in NPM1 within 9-20 months--3, although with chromosome 7 damage--2. It was suggested that NPM1 mutation without complex karyotype may serve as marker of relatively favorable course. PMID:21395122

  15. Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells.

    PubMed

    Saleh, Ayman M; Taha, Mutasem O; Aziz, Mohammad A; Al-Qudah, Mahmoud A; AbuTayeh, Reem F; Rizvi, Syed A

    2016-06-01

    Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains. PMID:26916980

  16. The Flt3 Internal Tandem Duplication Alters Chemotherapy Response In Vitro and In Vivo in a p53-Dependent Manner

    PubMed Central

    Pardee, Timothy S.; Zuber, Johannes; Lowe, Scott W.

    2011-01-01

    Objective The FLT3 internal tandem duplication (Flt3-ITD) confers a worse prognosis for patients with acute myeloid leukemia (AML); however, the mechanisms involved are unknown. As AML is treated with cytarabine (Ara-C) and an anthracycline we sought to determine the effects of the Flt3-ITD on response to these agents. Methods A genetically defined mouse model of AML was used to examine the effects of the Flt3-ITD on response to cytarabine and doxorubicin in vitro and in vivo. Results In vitro, the Flt3-ITD conferred resistance to doxorubicin and doxorubicin plus Ara-C, but sensitivity to Ara-C alone. This resistance was reversible by the Flt3-ITD inhibitor sorafenib. The Flt3-ITD did not affect DNA damage levels following treatment but was associated with increased levels of p53. The p53 response was critical to the observed changes as the Flt3-ITD had no effect on chemotherapy response in the setting of p53 null AML. In vivo, the Flt3-ITD accelerated engraftment that was partially reversible by Ara-C but not doxorubicin. Additionally, Ara-C provided a significant reduction in disease burden and a survival advantage that was not increased by the addition of doxorubicin. Doxorubicin alone lead to only minimal disease reduction and no survival benefit. Conclusions These data demonstrate that the Flt3-ITD confers sensitivity to cytarabine, but resistance to doxorubicin in a manner that depends on p53. Thus, patients with Flt3-ITD positive AML may not benefit from treatment with an anthracycline. PMID:21288478

  17. Chronic FLT3-ITD Signaling in Acute Myeloid Leukemia Is Connected to a Specific Chromatin Signature

    PubMed Central

    Cauchy, Pierre; James, Sally R.; Zacarias-Cabeza, Joaquin; Ptasinska, Anetta; Imperato, Maria Rosaria; Assi, Salam A.; Piper, Jason; Canestraro, Martina; Hoogenkamp, Maarten; Raghavan, Manoj; Loke, Justin; Akiki, Susanna; Clokie, Samuel J.; Richards, Stephen J.; Westhead, David R.; Griffiths, Michael J.; Ott, Sascha; Bonifer, Constanze; Cockerill, Peter N.

    2015-01-01

    Summary Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML. PMID:26212328

  18. Lack of FLT3-TKD835 gene mutation in toxicity of sulfur mustard in Iranian veterans

    PubMed Central

    Ayatollahi, Hossein; Rafiee, Mohammad; Keramati, Mohammad-Reza; Balali-Mood, Mahdi; Asgharzadeh, Ali; Sadeghian, Mohammad Hadi; Sheikhi, Maryam; Amini, Nafiseh; Zarmehri, Azam Moradi

    2015-01-01

    Objective(s): Sulfur mustard (SM) was used by the Iraqi army against the Iranian troops in the Iran-Iraq war from 1983–1988. This chemical gas affects different organs including the skin, lungs and the hematopoietic system. Any exposure to SM increases the risk of chromosomal breaking, hyperdiploidy and hypodiploidy. Studies have shown that the risk for acute myeloblastic and lymphoblastic leukemia increases in veterans exposed to SM. FLT3 mutations including ITD and TKD mutations had been observed in some cases of leukemia. Therefore, we aimed to investigate the frequency of FLT3-TKD835 mutations in the veterans exposed to SM agent. Materials and Methods: We studied 42 patients who were exposed to SM during the war in Khorasan Razavi province, Mashhad, Iran in 2012. As control group, 30 healthy males were selected from first-degree relatives of the patients. For assessment of TKD835 mutation, DNA was extracted and RFLP-PCR was performed. Results: Analysis of RFLP-PCR data showed no FLT-3 TKD mutation in any of the patients. Conclusion: Although contact with SM can increase the risk of malignancy especially hematologic neoplasms, results of the study show that another mechanism of leukemogenesis, other than FLT3-TKD mutation, may be the reason for increased risk of leukemia in SM toxicity. PMID:26523218

  19. The Successful Complete Remission Induction by Sorafenib Monotherapy in a FLT3-D835Y-Positive Patient with Refractory Acute Monocytic Leukemia.

    PubMed

    Yue, Yanhua; Jin, Song; Xu, Ting; Zhou, Jin; Ma, Liang; Shen, Hongjie; Wu, Depei; Chen, Suning; Miao, Miao

    2016-06-01

    Sorafenib has been shown to be active in AML patients with FLT3-ITD. However, the effect of sorafenib in AML patients with FLT-TKD has never been well determined. Moreover, acquisition of secondary FLT3 TKD mutations, mainly at D835 (D835F/H/V/Y), are recognized as the major mechanisms of resistance of AML patients with FLT3-ITD to sorafenib. It has been reported that sorafenib induced death of cells that expressed the FLT3-ITD or FLT3-D835G but not cells that expressed the FLT3-D835Y point mutant or wild-type FLT3 in vitro. Here, we report the successful complete remission induction by sorafenib monotherapy in a FLT3-D835Y-positive patient with refractory AML-M5 followed by allogeneic stem cell transplantation. PMID:27408351

  20. Internal tandem duplication and tyrosine kinase domain mutations in FLT3 alter the response to daunorubicin in Ba/F3 cells

    PubMed Central

    TAKAHASHI, SHINICHIRO; SHIRAHAMA, KUMI

    2016-01-01

    Internal tandem duplication (ITD) and activating point mutations, mainly at aspartic acid 835 in the tyrosine kinase domain (TKD), are frequently identified in the Fms-related tyrosine kinase 3 (FLT3) receptor gene in acute myeloid leukemia. The ITD in FLT3 (FLT3-ITD) confers resistance to several chemotherapeutic drugs; however, the relative effects of FLT3-ITD and FLT3-TKD mutations on the efficacy of these drugs have not been reported. In the present study, ITD or TKD mutant forms of FLT3 in Ba/F3 cells were expressed, as in the absence of interleukin-3 (IL-3) the growth of these cells is completely dependent on FLT3 oncogenic signals. As a result, the 50% effective dose for daunorubicin was significantly higher in both Ba/F3-FLT3-ITD clones, and also in one of the two Ba/F3-FLT3-TKD clones when cells were cultured without IL-3. This phenomenon was not observed for cytarabine in either Ba/F3-FLT3-ITD or Ba/F3-FLT3-TKD cells. Collectively, these results indicate that ITD and TKD mutations in FLT3 may confer daunorubicin resistance in Ba/F3 cells. PMID:26870340

  1. Allogeneic Transplantation in First Remission Improves Outcomes Irrespective of FLT3-ITD Allelic Ratio in FLT3-ITD-Positive Acute Myelogenous Leukemia.

    PubMed

    Oran, Betül; Cortes, Jorge; Beitinjaneh, Amer; Chen, Hsiang-Chun; de Lima, Marcos; Patel, Keyur; Ravandi, Farhad; Wang, Xuemei; Brandt, Mark; Andersson, Borje S; Ciurea, Stefan; Santos, Fabio P; de Padua Silva, Leandro; Shpall, Elizabeth J; Champlin, Richard E; Kantarjian, Hagop; Borthakur, Gautam

    2016-07-01

    The adverse prognosis of internal tandem duplication in the FMS-like tyrosine kinase 3 gene(s) (FLT3-ITD) in patients with acute myelogenous leukemia (AML) may depend on allelic burden. We compared postremission treatment with chemotherapy and hematopoietic stem cell transplantation (HSCT) in 169 FLT3-ITDmut intermediate cytogenetic risk AML patients with allelic ratio evaluable at diagnosis who achieved first complete remission (CR1) with induction therapy. To minimize selection bias, the analysis was limited to patients who remained in CR1 for at least 4 months (median time to HSCT) after achieving CR1, and propensity score matching was implemented. Sensitivity analysis including patients who remained in CR1 for at least 3 months was applied as well. HSCT in CR1 was associated with longer relapse-free survival (RFS) and overall survival (OS), with 3-year estimated rates of 18% and 24%, respectively (P < .001), for patients receiving chemotherapy and 46% and 54%, respectively (P < .001), for those undergoing HSCT. Multivariate regression models showed that HSCT remained statistically significant with improved RFS and OS independent of FLT3-ITD allelic ratio and NPM1 status. Irrespective of postremission therapy, relapse remains the main reason for treatment failure, with a 3-year incidence of 68% in chemotherapy recipients versus 41% in HSCT recipients. Allogeneic HSCT improved disease outcomes compared with chemotherapy after propensity score matching was applied. The improvement observed for RFS (hazard ratio [HR], 0.55; P = .09) and OS (HR, 0.58; P = .10) with HSCT as postremission therapy in patients who remained in CR1 for at least 4 months did not reach statistical significance; however, the sensitivity analyses including patients who remained in CR1 for at least 3 months showed significant improvement in both RFS (HR, 0.31; P = .002) and OS (HR, 0.27; P = .02) after propensity score matching. Our results indicate that HSCT in CR1 for AML FLT

  2. NPM1, FLT3, and c-KIT mutations in pediatric acute myeloid leukemia in Russian population.

    PubMed

    Yatsenko, Yuliya; Kalennik, Olga; Maschan, Mikhail; Kalinina, Irina; Maschan, Alexey; Nasedkina, Tatyana

    2013-04-01

    We evaluated frequencies of NPM1, FLT3, c-KIT mutations in childhood acute myeloid leukemia (AML) in Russia and assessed prognostic relevance of the mutations. RNA and DNA were extracted from bone marrow samples of 186 (106 male and 80 female) pediatric patients younger than 17 year with de novo AML. Mutations and chromosomal rearrangements were detected by sequencing of a corresponding gene. NPM1 mutations were found in 5.2%, FLT3 mutations in 12.1%, c-KIT mutations in 3.7% of the patients. NPM1 mutations were associated with the absence of chromosomal aberrations (P=0.007) and FLT3/ITD (P=0.018). New data on incidence of c-KIT mutations in various AML subtypes as well as new variations of c-KIT mutations in the exon 8 are presented. The results are compared to previously published studies on NPM1, FLT3, c-KIT mutations in various populations. No statistically significant differences in survival rates between groups with or without of FLT3, NPM1, c-KIT mutations were found (P>0.05). Meanwhile, 4-year overall survival rates were higher in patients having NPM1 mutations comparing with NPM1/WT patients (100% vs. 50%) and in patients having FLT3 mutations comparing with FLT3/WT patients (70% vs. 50%). The data presented contribute to knowledge on incidence and prognostic significance of the mutations in pediatric AML. PMID:23511494

  3. Presence of FLT3-ITD and high BAALC expression are independent prognostic markers in childhood acute myeloid leukemia.

    PubMed

    Staffas, Anna; Kanduri, Meena; Hovland, Randi; Rosenquist, Richard; Ommen, Hans Beier; Abrahamsson, Jonas; Forestier, Erik; Jahnukainen, Kirsi; Jónsson, Ólafur G; Zeller, Bernward; Palle, Josefine; Lönnerholm, Gudmar; Hasle, Henrik; Palmqvist, Lars; Ehrencrona, Hans

    2011-11-24

    Mutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information. PMID:21967978

  4. Bioconjugation Methods for Coupling Targeting Ligands with Fluorescent Dyes.

    PubMed

    Ling, Xiaoxi

    2016-01-01

    Targeted molecular imaging probes are essential tools for visualization of specific molecular processes in cells and living systems. Among these, targeted fluorescent probes are widely used due to the high sensitivity and resolution of fluorescence imaging. The conventional strategy for developing targeted fluorescent probes is to couple targeting ligands with fluorescent dyes by covalent bond via bioconjugation. Here, we describe several commonly used bioconjugation methods, from traditional amide and thiol coupling, to metal-catalyzed coupling reaction and catalyst free cycloaddition. PMID:27283413

  5. Molecular genetic tests for FLT3, NPM1, and CEBPA in acute myeloid leukemia.

    PubMed

    Zhang, Qing; Bai, Shaochun; Vance, Gail H

    2013-01-01

    Patients with acute myeloid leukemia (AML) and a normal karyotype constitute the single largest cytogenetic group of AML. It is important to identify prognostic markers that predict patients' outcome more precisely. The presence of mutations in FLT3 (FMS-like tyrosine kinase 3), NPM1 (Nucleophosmin), and CEBPA (CCAAT/enhancer-binding protein alpha) genes hold prognostic significance in patients with AML and normal cytogenetics. Therefore, mutation identification may help to optimize therapeutic approaches in this group of patients. Polymerase chain reaction (PCR)-based fragment length analysis for mutations in FLT3 and NPM1 has been shown to be a fast and sensitive method, while nucleotide sequencing represents a gold standard for CEBPA heterogeneous mutational screening. We describe both fragment length assay and sequencing methods for mutational analysis of these three genes. PMID:23666693

  6. Acute promyelocytic leukemia harbouring rare FLT3-TKD and WT1 mutations: A case report

    PubMed Central

    LIU, TING-TING; ZENG, KE; WANG, LIN; LIU, TING; NIU, TING

    2015-01-01

    The involvement of the central nervous system (CNS) is rare in acute promyelocytic leukemia (APL). The present study reported the case of a 34-year-old male patient with APL that possessed a rare point mutation (p.Asn841Gly, c.2523C>A) in the tyrosine kinase domain of the FMS-like tyrosine kinase 3 (FLT3) gene and a novel Wilm tumor gene mutation (c.1209_1210insT/p.K404X). The patient suffered central nervous system and systemic relapses twice during systemic and intrathecal chemotherapy. At present, the patient is undergoing alternative induction and consolidation therapies, including the administration of FLT3 inhibitor, tetraarsenic tetrasulfide and novel cytotherapy, and is prepared for salvage allogeneic hematopoietic stem cell transplantion (allo-HSCT). The present study indicated that patients with APL that are at a high risk of relapse and unfavorable gene mutations should receive immediate allo-HSCT, whenever possible. PMID:26622765

  7. Gelatin-coated Gold Nanoparticles as Carriers of FLT3 Inhibitors for Acute Myeloid Leukemia Treatment.

    PubMed

    Suarasan, Sorina; Simon, Timea; Boca, Sanda; Tomuleasa, Ciprian; Astilean, Simion

    2016-06-01

    This study presents the design of a gold nanoparticle (AuNPs)-drug system with improved efficiency for the treatment of acute myeloid leukemia. The system is based on four different FLT3 inhibitors, namely midostaurin, sorafenib, lestaurtinib, and quizartinib, which were independently loaded onto gelatin-coated gold nanoparticles. Detailed investigation of the physicochemical properties of the formed complexes lead to the selection of quizartinib-loaded AuNPs for the in vitro evaluation of the biological effects of the formed complex against OCI-AML3 acute myeloid leukemia cells. Viability tests by MTT demonstrated that the proposed drug complex has improved efficacy when compared with the drug alone. The obtained results constitute a premise for further in vivo investigation of such drug vehicles based on AuNPs. To the best of our knowledge, this is the first study that investigates the delivery of the above-mentioned FLT3 inhibitors via gelatin-coated gold nanoparticles. PMID:26808072

  8. FLT3 and NPM1 mutations in a cohort of AML patients and detection of a novel mutation in tyrosine kinase domain of FLT3 gene from Western India.

    PubMed

    Ghosh, Kanjaksha; Swaminathan, Suchitra; Madkaikar, Manisha; Gupta, Maya; Kerketta, Lily; Vundinti, Baburao

    2012-11-01

    Acute myeloid leukemia (AML) is an aggressive hematological disorder characterized by the loss of ability of the hematopoietic progenitor cells to differentiate and proliferate normally leading to an accumulation of immature myeloid cells in the bone marrow. Several novel molecular genetic aberrations in FLT3 and NPM1 have been shown to have a prognostic impact in AML, particularly in those having normal karyotype. Though there is substantial amount of data on these mutations from western literature, there is surprisingly little data from Indian subcontinent on the frequency of this mutation in AML patients from India. The present study screens a large cohort of non-acute promyelocytic leukemia (APL) AML patients (207 patients) for the presence of FLT3 and NPM1 mutations and further correlates with cytogenetics, immunophenotypic characteristics and with follow-up data wherever available. During the course of study, 56 APL patients were also studied. Briefly, both FLT3 (internal tandem duplication (ITD) in 19.4% and tyrosine kinase domain (TKD) in 9%) and NPM1 mutations were detected in 28.4% of the total non-APL AML patients screened showing distinct correlations with hematologic, immunophenotypic, cytogenetics characteristics and follow-up. With regards to adult APL patients, 22.2 and 32.6% of the patients showed FLT3 and NPM1 mutation, respectively. In the pediatrics age group (<15 years), 23 and 16% of patients with APL showed FLT3 and NPM1 mutation, respectively, while in non-APL patient is this age group, 23% of patients showed both FLT3 and NPM1 mutation. NPM1 mutation was distinctly uncommon in younger age group of patients. In contrast to report elsewhere, most of our FLT3 mutation was in exon 11 rather than in exon 12. FLT3 mutation due to ITD or TKD mutation was detected in 2:1 ratio in our patients and a new TKD mutation was also detected S840G in an M5 patient who did not go into remission and had a short survival of 3 months from diagnosis. Generally

  9. Feature genes predicting the FLT3/ITD mutation in acute myeloid leukemia

    PubMed Central

    LI, CHENGLONG; ZHU, BIAO; CHEN, JIAO; HUANG, XIAOBING

    2016-01-01

    In the present study, gene expression profiles of acute myeloid leukemia (AML) samples were analyzed to identify feature genes with the capacity to predict the mutation status of FLT3/ITD. Two machine learning models, namely the support vector machine (SVM) and random forest (RF) methods, were used for classification. Four datasets were downloaded from the European Bioinformatics Institute, two of which (containing 371 samples, including 281 FLT3/ITD mutation-negative and 90 mutation-positive samples) were randomly defined as the training group, while the other two datasets (containing 488 samples, including 350 FLT3/ITD mutation-negative and 138 mutation-positive samples) were defined as the test group. Differentially expressed genes (DEGs) were identified by significance analysis of the micro-array data by using the training samples. The classification efficiency of the SCM and RF methods was evaluated using the following parameters: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the receiver operating characteristic curve. Functional enrichment analysis was performed for the feature genes with DAVID. A total of 585 DEGs were identified in the training group, of which 580 were upregulated and five were downregulated. The classification accuracy rates of the two methods for the training group, the test group and the combined group using the 585 feature genes were >90%. For the SVM and RF methods, the rates of correct determination, specificity and PPV were >90%, while the sensitivity and NPV were >80%. The SVM method produced a slightly better classification effect than the RF method. A total of 13 biological pathways were overrepresented by the feature genes, mainly involving energy metabolism, chromatin organization and translation. The feature genes identified in the present study may be used to predict the mutation status of FLT3/ITD in patients with AML. PMID:27177049

  10. Feature genes predicting the FLT3/ITD mutation in acute myeloid leukemia.

    PubMed

    Li, Chenglong; Zhu, Biao; Chen, Jiao; Huang, Xiaobing

    2016-07-01

    In the present study, gene expression profiles of acute myeloid leukemia (AML) samples were analyzed to identify feature genes with the capacity to predict the mutation status of FLT3/ITD. Two machine learning models, namely the support vector machine (SVM) and random forest (RF) methods, were used for classification. Four datasets were downloaded from the European Bioinformatics Institute, two of which (containing 371 samples, including 281 FLT3/ITD mutation-negative and 90 mutation‑positive samples) were randomly defined as the training group, while the other two datasets (containing 488 samples, including 350 FLT3/ITD mutation-negative and 138 mutation-positive samples) were defined as the test group. Differentially expressed genes (DEGs) were identified by significance analysis of the microarray data by using the training samples. The classification efficiency of the SCM and RF methods was evaluated using the following parameters: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under the receiver operating characteristic curve. Functional enrichment analysis was performed for the feature genes with DAVID. A total of 585 DEGs were identified in the training group, of which 580 were upregulated and five were downregulated. The classification accuracy rates of the two methods for the training group, the test group and the combined group using the 585 feature genes were >90%. For the SVM and RF methods, the rates of correct determination, specificity and PPV were >90%, while the sensitivity and NPV were >80%. The SVM method produced a slightly better classification effect than the RF method. A total of 13 biological pathways were overrepresented by the feature genes, mainly involving energy metabolism, chromatin organization and translation. The feature genes identified in the present study may be used to predict the mutation status of FLT3/ITD in patients with AML. PMID:27177049

  11. Effect of NPM1 and FLT3 Mutations on the Outcomes of Elderly Patients With Acute Myeloid Leukemia Receiving Standard Chemotherapy

    PubMed Central

    Daver, Naval; Liu Dumlao, Theresa; Ravandi, Farhad; Pierce, Sherry; Borthakur, Gautam; Pemmaraju, Naveen; Nazha, Aziz; Faderl, Stefan; Jabbour, Elias; Garcia-Manero, Guillermo; Cortes, Jorges; Kantarjian, Hagop; Quintás-Cardama, Alfonso

    2014-01-01

    This study analyzes the clinical impact of NPM1 and FLT3 mutations in AML patients 65 years of age or older treated with cytotoxic chemotherapy at our institution. A total of 557 patients were retrospectively reviewed. No difference in survivalwasnoted for patients with isolated FLT3 orNPM1mutations. However, elderlyAMLpatients who were NPM1 mutated and FLT3 wild type had significantly improved survival with cytotoxic chemotherapy. Background The effect of prognostically important gene mutations (MUTs), nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1) and fms-related tyrosine kinase 3 (FLT3), in elderly patients with acute myeloid leukemia (AML) is not well defined. Patients and Methods We analyzed 557 patients, 65 years of age or older with newly diagnosed AML, treated at our institution between 2000 and 2010 with cytotoxic chemotherapy. NPM1 and FLT3 analysis were available in 146 patients (26%) and 388 patients (70%), respectively. Results NPM1 and FLT3 MUTs occurred in 16% and 12% of patients, respectively. No difference in median overall survival was observed between FLT3-MUT and NPM1-MUT patients who received cytotoxic chemotherapy. Outcome was significantly better among patients with NPM1-MUT/FLT3-wild type (WT) genotype (n = 14) compared with patients carrying FLT3/NPM1 genotypes other than NPM1-MUT/FLT3-WT (n = 125). The complete remission rates were 71% and 49%, respectively (P = .11). The median survival was 21.5 months vs. 9.0 months and estimated 2-year survival rates were 51% vs. 38%, respectively (P = .003). NPM1 and FLT3 MUTs appear to occur less frequently in elderly AML patients. The prognostic effect of isolated NPM1- or isolated FLT3-MUT is minimal. Conclusion Elderly AML patients with NPM1-MUT/FLT3-WT genotype have significantly improved outcomes compared with patients with other NPM1/FLT3 genotypes when treated with cytotoxic chemotherapy. PMID:23763915

  12. Prognostic implications of NPM1 mutations and FLT3 internal tandem duplications in Egyptian patients with cytogenetically normal acute myeloid leukemia.

    PubMed

    Shamaa, Sameh; Laimon, Nabil; Aladle, Doaa A; Azmy, Emad; Elghannam, Doaa M; Salem, Dalia A; Taalab, Mona M

    2014-01-01

    Nucleophosmin (NPM1) and fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) gene mutations represent the most frequent molecular aberrations in patients with cytogenetically normal-acute myeloid leukemia (CN-AML). We analyzed the prognostic impact of these mutations and their interactions in adults with CN-AML. NPM1 mutation (NPM1mut) and FLT3-ITD mutation (FLT3-ITD+) were analyzed by polymerase chain reaction and GeneScan assays of bone marrow samples obtained from newly diagnosed 104 CN-AML patients. FLT3-ITD+ and NPM1mut were detected in 36 (34.6%) and 30 (28.8%) out of 104 subjects, respectively, 16 cases (15.4%) had double NPM1mut/FLT3-ITD+. The incidence of FLT3-ITD+ was significantly higher in the NPM1mut group than in the NPM1 wild (NPM1wt) group (P = 0.018). Statistical analysis revealed that isolated NPM1mut group had a better clinical outcomes in terms of higher complete response (CR) rate (P = 0.01) and a trend towards favorable overall survival (OS) and disease-free survival (DFS) (P = 0.28, 0.40, respectively). In contrast, the isolated FLT3-ITD+ group had an unfavorable outcome in terms of lower CR rate (P = 0.12), shorter OS, and DFS (P < 0.0001 for both). The NPM1mut/FLT3-ITD-group had the best OS and DFS, while the NPM1wt/FLT3-ITD+ group had the worst OS and DFS than other groups (NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-) (P < 0.0001 for both). Multivariate Cox regression analysis showed that age and FLT3/ITD+ were independent poor prognostic factors for OS (P = 0.006, <0.0001, respectively), while FLT3/ITD+ was independent predictor for DFS (P = 0.04). However, NPM1mut did not have a significant impact on OS and DFS. In conclusion, adult patients with CN-AML carrying isolated NPM1mut and isolated FLT3-ITD+ exhibit different clinical outcomes than those with NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-. Patients with NPM1mut/FLT3-ITD- had the best prognosis in terms of higher CR, OS, and DFS, while those with NPM1mut/FLT3-ITD+ had the worst

  13. Mutation of NPM1 and FLT3 Genes in Acute Myeloid Leukemia and Their Association with Clinical and Immunophenotypic Features

    PubMed Central

    Chauhan, Pradeep Singh; Ihsan, Rakhshan; Singh, L. C.; Gupta, Dipendra Kumar; Mittal, Vishakha; Kapur, Sujala

    2013-01-01

    Background. Mutations in NPM1 and FLT3 genes represent the most frequent genetic alterations and important diagnostic and prognostic indicators in patients with acute myeloid leukemia (AML). Objective. We investigated the prevalence and clinical characteristics of NPM1 and FLT3 mutations in 161 patients of de novo AML including adults and children. Results. NPM1 mutation was found in 21% and FLT3 mutation in 25% of the AML patients. Thirteen (8%) samples were positive for both NPM1 and FLT3/ITD mutations. Adult patients had significantly higher frequency of NPM1 mutation than children (25.8% versus 8.8%; P = 0.02). Further, NPM1 mutation was found to be more frequent in patients above 45 years of age (P = 0.02). NPM1 mutation was significantly associated with higher platelet count (P = 0.05) and absence of hepatosplenomegaly (P = 0.01), while FLT3/ITD mutation was associated with higher white blood count (P = 0.01). Immunophenotypically, NPM1 mutation was associated with the lack of CD34 (P < 0.001) and HLD-DR expression (P < 0.001), while FLT3/ITD mutation was positively associated with the expression of CD7 (P = 0.04). No correlation was found between NPM1 mutation and fusion gene. Interestingly, FLT3/ITD mutation was found to be inversely associated with AML/ETO fusion gene (P = 0.04). Conclusions. The results suggest that distinct clinical and immunophenotypic characteristics of NPM1 and FLT3/ITD mutations present further insight into the molecular mechanism of leukemogenesis. PMID:24288427

  14. FLT3 and NPM1 mutations in myelodysplastic syndromes: Frequency and potential value for predicting progression to acute myeloid leukemia.

    PubMed

    Bains, Ashish; Luthra, Rajyalakshmi; Medeiros, L Jeffrey; Zuo, Zhuang

    2011-01-01

    We reviewed FLT3 and NPM1 mutation data in a large cohort of patients with myelodysplastic syndrome (MDS). The frequencies of FLT3 and NPM1 mutation were 2.0% and 4.4%, respectively, and mutations were restricted to cases of intermediate- and high-risk MDS. Cytogenetic abnormalities were identified in 46.9% of cases. FLT3 mutations were associated with a complex karyotype (P = .009), whereas NPM1 mutations were associated with a diploid karyotype (P < .001). FLT3 mutation (P < .001) was associated with progression to acute myeloid leukemia (AML), as were a higher bone marrow (BM) blast count (P < .001) and complex cytogenetics (P = .039). No patient with an NPM1 mutation alone had disease that progressed to AML. Cox proportional regression multivariate analysis indicated that FLT3 mutation, NPM1 mutation, complex cytogenetics, BM blast count, pancytopenia, and age were independent factors that correlated with progression-free survival. We conclude that FLT3 and NPM1 mutations are rare in MDS, but assessment of mutation status is potentially useful for predicting progression to AML. PMID:21173125

  15. The juxtamembrane domain in ETV6/FLT3 is critical for PIM-1 up-regulation and cell proliferation

    SciTech Connect

    Vu, Hoang Anh; Xinh, Phan Thi; Kano, Yasuhiko; Tokunaga, Katsushi; Sato, Yuko

    2009-06-05

    We recently reported that the ETV6/FLT3 fusion protein conferred interleukin-3-independent growth on Ba/F3 cells. The present study has been conducted to assess role of the juxtamembrane domain of FLT3 for signal transduction and cell transformation. The wild-type ETV6/FLT3 fusion protein in transfected cells was a constitutively activated tyrosine kinase that led to up-regulation of PIM-1 and activations of STAT5, AKT, and MAPK. Deletion of the juxtamembrane domain abrogated interleukin-3-independent growth of the transfected cells and PIM-1 up-regulation, whereas it retained compatible levels of phosphorylations of STAT5, AKT, and MAPK. Further deletion of N-terminal region of the tyrosine kinase I domain of FLT3 completely abolished these phosphorylations. Our data indicate that the juxtamembrane domain of FLT3 in ETV6/FLT3 fusion protein is critical for cell proliferation and PIM-1 up-regulation that might be independent of a requirement for signaling through STAT5, MAPK, and AKT pathways.

  16. FLT3-ITD Knock-in Impairs Hematopoietic Stem Cell Quiescence/Homeostasis, Leading to Myeloproliferative Neoplasm

    PubMed Central

    Chu, S. Haihua; Heiser, Diane; Li, Li; Kaplan, Ian; Collector, Michael; Huso, David; Sharkis, Saul J; Civin, Curt; Small, Don

    2013-01-01

    SUMMARY Internal tandem duplication (ITD) mutations within the FMS-Like Tyrosine Kinase gene (FLT3) render the receptor constitutively active, driving proliferation and survival in leukemic blasts. Expression of Flt3-ITD from the endogenous promoter in a murine knock-in model results in progenitor expansion and a myeloproliferative neoplasm. In this study, we show that this expansion begins with over-proliferation within a compartment of normally quiescent long-term hematopoietic stem cells (LT-HSCs), which become rapidly depleted. This depletion is reversible upon treatment with the small molecule inhibitor Sorafenib, which also ablates the disease. Although the normal LT-HSC has been defined as Flt3-negative by flow cytometric detection, we demonstrate that Flt3 is capable of playing a role within this compartment by examining the effects of constitutively activated Flt3-ITD. This indicates an important link between stem cell quiescence/homeostasis and myeloproliferative disease while also giving novel insight into the emergence of FLT3-ITD mutations in the evolution of leukemic transformation. PMID:22958930

  17. Prognostic significance of FLT3-ITD in pediatric acute myeloid leukemia: a meta-analysis of cohort studies.

    PubMed

    Wu, Xiaoli; Feng, Xuefeng; Zhao, Xiaoqing; Ma, Futian; Liu, Na; Guo, Hongming; Li, Chaonan; Du, Huan; Zhang, Baoxi

    2016-09-01

    The purpose of the study was to assess the effect of the internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD) on the outcome in pediatric acute myeloid leukemia (AML) patients. We identified eligible studies from several databases including PubMed, Embase, and Cochrane Central Register of Controlled Trials (CENTRAL) (from January 1995 to July 2015). Ten studies of 1661 pediatric patients with AML were included in exploring the relationship between the FLT3-ITD and overall survival (OS)/event free survival (EFS). Pediatric patients with AML with FLT3-ITD had worse OS [HR = 2.19 (1.60-3.01)]/EFS [HR = 1.70 (1.37-2.11)] than those patients without FLT3-ITD. Furthermore, FLT3-ITD had unfavorable effect on OS/EFS in the subgroups of NOS, uni/multivariate model, number of patients, the length of following-up, and patient source. The findings of this meta-analysis indicated that FLT3-ITD had negative impact on pediatric patients with AML. PMID:27435859

  18. Targeted delivery to bone and mineral deposits using bisphosphonate ligands.

    PubMed

    Cole, Lisa E; Vargo-Gogola, Tracy; Roeder, Ryan K

    2016-04-01

    The high concentration of mineral present in bone and pathological calcifications is unique compared with all other tissues and thus provides opportunity for targeted delivery of pharmaceutical drugs, including radiosensitizers and imaging probes. Targeted delivery enables accumulation of a high local dose of a therapeutic or imaging contrast agent to diseased bone or pathological calcifications. Bisphosphonates (BPs) are the most widely utilized bone-targeting ligand due to exhibiting high binding affinity to hydroxyapatite mineral. BPs can be conjugated to an agent that would otherwise have little or no affinity for the sites of interest. This article summarizes the current state of knowledge and practice for the use of BPs as ligands for targeted delivery to bone and mineral deposits. The clinical history of BPs is briefly summarized to emphasize the success of these molecules as therapeutics for metabolic bone diseases. Mechanisms of binding and the relative binding affinity of various BPs to bone mineral are introduced, including common methods for measuring binding affinity in vitro and in vivo. Current research is highlighted for the use of BP ligands for targeted delivery of BP conjugates in various applications, including (1) therapeutic drug delivery for metabolic bone diseases, bone cancer, other bone diseases, and engineered drug delivery platforms; (2) imaging probes for scintigraphy, fluorescence, positron emission tomography, magnetic resonance imaging, and computed tomography; and (3) radiotherapy. Last, and perhaps most importantly, key structure-function relationships are considered for the design of drugs with BP ligands, including the tether length between the BP and drug, the size of the drug, the number of BP ligands per drug, cleavable tethers between the BP and drug, and conjugation schemes. PMID:26482186

  19. Association of cup-like nuclei in blasts with FLT3 and NPM1 mutations in acute myeloid leukemia.

    PubMed

    Park, Borae G; Chi, Hyun-Sook; Jang, Seongsoo; Park, Chan-Jeoung; Kim, Dae-Young; Lee, Jung-Hee; Lee, Je-Hwan; Lee, Kyoo-Hyung

    2013-04-01

    The purpose of this study was to investigate the correlation of mutations of the fms-like tyrosine kinase (FLT3) and nucleophosmin (NPM1) genes with the cup-like nuclear morphology of blasts in patients with acute myeloid leukemia (AML). We retrospectively reviewed peripheral blood (PB) and bone marrow (BM) slides of 208 patients prepared at the time of diagnosis of AML based on the results of testing for mutations of both NPM1 exon 12 and FLT3. We investigated the association between this phenotype and hematologic findings, disease markers, and mutations in NPM1 exon 12, FLT3-internal tandem duplication (ITD), and tyrosine kinase domain (TKD) genes. Cup-like nuclei were found in 44 patients (21.2 %) diagnosed with AML. This morphology was associated with high blast counts in the PB and BM; AML type, especially AML M1 (FAB classification); low CD34 expression; and mutation of FLT3-ITD, -TKD, NPM1 regardless of other mutations (p < 0.05 for all). However, FLT3-ITD or TKD mutation alone (nine cases, p = 0.228) was not associated, and NPM1 mutation alone (14 cases, p = 0.036) was weakly associated with cup-like nuclei. Mutation of both NPM1 and FLT3-ITD or TKD (17 cases, p < 0.001) was strongly correlated with the cup-like nuclear morphology. AML with cup-like nuclei is strongly associated with co-occurring mutations of both NPM1 and FLT3-ITD or TKD. Therefore, testing for both mutations is recommended for patients with the cup-like nuclear morphology. PMID:23238897

  20. Prognostic impact of white blood cell count in intermediate risk acute myeloid leukemia: relevance of mutated NPM1 and FLT3-ITD

    PubMed Central

    de Jonge, Hendrik J.M.; Valk, Peter J.M.; de Bont, Eveline S.J.M.; Schuringa, Jan Jacob; Ossenkoppele, Gert; Vellenga, Edo; Huls, Gerwin

    2011-01-01

    Background High white blood cell count at presentation is an unfavorable prognostic factor for treatment outcome in intermediate cytogenetic risk acute myeloid leukemia. Since the impact of white blood cell count on outcome of subgroups defined by the molecular markers NPMc+ and FLT3-internal tandem duplication (ITD) is unknown, we addressed this issue. Design and Methods We studied the effect of white blood cell count on outcome in a clinically and molecularly well-defined cohort of 525 patients with acute myeloid leukemia using these molecular markers. In addition, since an increased white blood cell count has been associated with an increased FLT3-ITD/FLT3 (wild-type) ratio, we investigated whether the effect of white blood cell count on outcome could be explained by the FLT3-ITD/FLT3 ratio. Results This analysis revealed that white blood cell count had no impact on outcome in patients with the genotypic combinations ‘NPMc+ without FLT3-ITD’ and ‘NPM1 wild-type with or without FLT3-ITD’. In contrast, white blood cell count had a significant impact on complete remission rate (P=0.034), event-free survival (P=0.009) and overall survival (P<0.001) in patients with the genotypic combination ‘NPMc+ with FLT3-ITD’. A FLT3-ITD/FLT3 ratio greater than 1 was also associated with a reduced complete remission rate (P=0.066) and significantly reduced event-free survival (P= 0.001) and overall survival (P=0.001) in patients with the genotypic combination ‘NPMc+ with FLT3-ITD’. Multivariable analysis revealed that white blood cell count and FLT3-ITD/FLT3 ratio were independent prognostic indicators for outcome in the subgroup with the genotypic combination ‘NPMc+ with FLT3-ITD’. Conclusions Our results demonstrate that both high white blood cell count and FLT3-ITD/FLT3 ratio are prognostic factors in patients with acute myeloid leukemia with the genotypic combination ‘NPMc+ with FLT3-ITD'. PMID:21606167

  1. [Correlation of NPM1, FLT3-ITD mutations with leukocyte count and myeloblasts percentage in AML patients with normal karyotype].

    PubMed

    Su, Long; Li, Wei; Cui, Jiu-Wei; Tan, Ye-Hui; Yang, Yan; Liu, Xiao-Liang; Yu, Ping; Hu, Rui-Ping; Wang, Li-Li; Gao, Su-Jun

    2013-06-01

    This study was aimed to investigate the correlation of NPM1 and FLT3-ITD mutations with leukocyte count in peripheral blood and bone marrow blasts in patients with acute myeloid leukemia (AML). Fifty-one acute myeloid leukemia patients with normal karyotype from January 2009 to December 2011 were enrolled in this study. The clinical data of 51 cases were analyzed retrospectively. Out of 52 cases 22 were male, and 29 were female. The median age was 47 years old (ranged from 14 to 83 years old). The de novo patients were examined by bone marrow cytomorphology and blood routine analysis. Polymerase chain reaction was used to analyze the NPM1 and FLT3-ITD mutations. The results showed that the patients with NPM1 mutations had higher leukocyte count compared with those without mutations (30.7×10(9)/L vs 8.6×10(9)/L, P = 0.002). FLT3-ITD mutation was related to higher leukocyte count (42.38×10(9)/L vs 11.45×10(9)/L without mutation, P = 0.033) and blasts (74.0% vs 60.25% without mutation, P = 0.036). The leukocyte count and percentage of bone marrow blasts were lowest in the patients with neither mutations, and gradually increasing in the NPM1(-) mutation, FLT3-ITD(-) mutation, and NPM1(+) mutation, FLT3-ITDI(+) mutation, and NPM1(+)/FLT3-ITD(+) mutation groups (P < 0.05). The patients tended to have NPM1 (P = 0.002) and FLT3-ITD (P = 0.033) mutations when their leukocyte counts were more than 12.55×10(9)/L and 37.85×10(9)/L, respectively. Those with bone marrow blast more than 72.25% showed higher rate of FLT3-ITD mutation (P = 0.008). Patients with NPM1 mutations had higher complete remission rate than those without NPM1 mutation (78.13% vs 40.0%, χ(2) = 4.651, P = 0.031) after remission induction therapy. It is concluded that both NPM1 and FLT3-ITD mutations are linked to higher leukocyte count and blast percentage, suggesting that both mutations may be associated with increased proliferation of leukemia cells, and may have a synergistic function in stimulating

  2. Simultaneous detection of NPM1 and FLT3-ITD mutations by capillary electrophoresis in acute myeloid leukemia.

    PubMed

    Noguera, N I; Ammatuna, E; Zangrilli, D; Lavorgna, S; Divona, M; Buccisano, F; Amadori, S; Mecucci, C; Falini, B; Lo-Coco, F

    2005-08-01

    Mutations in the Nucleophosmin (NPM1) gene have been recently described to occur in about one-third of acute myeloid leukemias (AML) and represent the most frequent genetic alteration currently known in this subset. These mutations generate an elongated NPM1 protein that localizes aberrantly in the cytoplasm. In analogy with Flt3 alterations, NPM1 mutations are mostly detectable in AML with normal karyotype and their recognition may be relevant to identify distinct response to treatment. Hence, in addition to conventional karyotyping and RT-PCR of fusion genes, combined analysis of both Flt3 and NPM1 mutations will be increasingly relevant in the genetic diagnosis work-up of AML. We developed a multiplex RT-PCR assay followed by capillary electrophoresis to simultaneously analyze NPM1 and Flt3 gene alterations (NFmPCR assay). The assay was validated in leukemic cell RNAs extracted from 38 AML patients, which had been previously characterized for Flt3 status by conventional RT-PCR. Direct sequencing of NPM1 RT-PCR products was carried out in 15 cases to verify results obtained by capillary electrophoresis. Both NPM1 sequencing and conventional RT-PCR Flt3 results showed 100% concordance with the results of the NFmPCR assay. We suggest that this assay may be introduced in routine analysis of genetic alterations in AML. PMID:15973451

  3. Reversal of FLT3 mutational status and sustained expression of NPM1 mutation in paired presentation, and relapse samples in a patient with acute myeloid leukemia.

    PubMed

    Radojkovic, Milica; Tosic, Natasa; Colovic, Natasa; Ristic, Slobodan; Pavlovic, Sonja; Colovic, Milica

    2012-01-01

    We report a case of de novo acute myeloid leukemia (AML) with unstable FLT3 gene mutations and stable NPM1 mutation. FLT3/D835 and NPM1 (Type A) mutations were detected upon diagnosis. During the relapse, the FLT3/D835 mutation changed to an FLT3/ITD mutation while the NPM1 (Type A) mutation was retained. Cytogenetic analyses showed the normal karyotype at diagnosis and relapse. Our findings raise interesting questions about the significance of these mutations in the leukemogenic process, about their stability during the evolution of the disease, and regarding the selection of appropriate molecular markers for the monitoring of minimal residual disease. PMID:22585616

  4. Arsenic trioxide in front-line therapy of acute promyelocytic leukemia (C9710): prognostic significance of FLT3 mutations and complex karyotype

    PubMed Central

    Poiré, Xavier; Moser, Barry K.; Gallagher, Robert E.; Laumann, Kristina; Bloomfield, Clara D.; Powell, Bayard L.; Koval, Gregory; Gulati, Kabir; Holowka, Nicholas; Larson, Richard A.; Tallman, Martin S.; Appelbaum, Frederick R.; Sher, Dorie; Willman, Cheryl; Paietta, Elisabeth; Stock, Wendy

    2014-01-01

    The addition of arsenic trioxide (ATO) to frontline therapy of acute promyelocytic leukemia (APL) has been shown to result in significant improvements in disease-free survival (DFS). FLT3 mutations are frequently observed in APL, but its prognostic significance remains unclear. We analyzed 245 newly diagnosed adult patients with APL treated on intergroup trial C9710 and evaluated previously defined biological and prognostic factors and their relationship to FLT3 mutations and to additional karyotypic abnormalities. FLT3 mutations were found in 48% of patients, including 31% with an internal tandem duplication (FLT3-ITD), 14% with a point mutation (FLT3-D835) and 2% with both mutations. The FLT3-ITD mutant level was uniformly low, <0.5. Neither FLT3 mutation had an impact on remission rate, induction death rate, DFS or overall survival (OS). The addition of ATO consolidation improved outcomes regardless of FLT3 mutation type or level, initial white blood cell count, PML–RARA isoform type or transcript level. The presence of a complex karyotype was strongly associated with an inferior OS independently of post-remission treatment. In conclusion, the addition of ATO to frontline therapy overcomes the impact of previously described adverse prognostic factors including FLT3 mutations. However, complex karyotype is strongly associated with an inferior OS despite ATO therapy. PMID:24160850

  5. Arsenic trioxide in front-line therapy of acute promyelocytic leukemia (C9710): prognostic significance of FLT3 mutations and complex karyotype.

    PubMed

    Poiré, Xavier; Moser, Barry K; Gallagher, Robert E; Laumann, Kristina; Bloomfield, Clara D; Powell, Bayard L; Koval, Gregory; Gulati, Kabir; Holowka, Nicholas; Larson, Richard A; Tallman, Martin S; Appelbaum, Frederick R; Sher, Dorie; Willman, Cheryl; Paietta, Elisabeth; Stock, Wendy

    2014-07-01

    The addition of arsenic trioxide (ATO) to frontline therapy of acute promyelocytic leukemia (APL) has been shown to result in significant improvements in disease-free survival (DFS). FLT3 mutations are frequently observed in APL, but its prognostic significance remains unclear. We analyzed 245 newly diagnosed adult patients with APL treated on intergroup trial C9710 and evaluated previously defined biological and prognostic factors and their relationship to FLT3 mutations and to additional karyotypic abnormalities. FLT3 mutations were found in 48% of patients, including 31% with an internal tandem duplication (FLT3-ITD), 14% with a point mutation (FLT3-D835) and 2% with both mutations. The FLT3-ITD mutant level was uniformly low, < 0.5. Neither FLT3 mutation had an impact on remission rate, induction death rate, DFS or overall survival (OS). The addition of ATO consolidation improved outcomes regardless of FLT3 mutation type or level, initial white blood cell count, PML-RARA isoform type or transcript level. The presence of a complex karyotype was strongly associated with an inferior OS independently of post-remission treatment. In conclusion, the addition of ATO to frontline therapy overcomes the impact of previously described adverse prognostic factors including FLT3 mutations. However, complex karyotype is strongly associated with an inferior OS despite ATO therapy. PMID:24160850

  6. The FLT3ITD mRNA level has a high prognostic impact in NPM1 mutated, but not in NPM1 unmutated, AML with a normal karyotype.

    PubMed

    Schneider, Friederike; Hoster, Eva; Unterhalt, Michael; Schneider, Stephanie; Dufour, Annika; Benthaus, Tobias; Mellert, Gudrun; Zellmeier, Evelyn; Kakadia, Purvi M; Bohlander, Stefan K; Feuring-Buske, Michaela; Buske, Christian; Braess, Jan; Heinecke, Achim; Sauerland, Maria C; Berdel, Wolfgang E; Büchner, Thomas; Wörmann, Bernhard J; Hiddemann, Wolfgang; Spiekermann, Karsten

    2012-05-10

    The impact of a FLT3-internal tandem duplication (FLT3ITD) on prognosis of patients with acute myeloid leukemia (AML) is dependent on the ratio of mutated to wild-type allele. In 648 normal karyotype (NK) AML patients, we found a significant independent effect of the quantitative FLT3ITD mRNA level--measured as (FLT3ITD/wtFLT3)/(FLT3ITD/wtFLT3+1)--on outcome. Moreover, this effect was clearly seen in 329 patients with a mutated NPM1 gene (NPM1+), but not in 319 patients without a NPM1 mutation (wtNPM1). In a multivariate Cox regression model, the quantitative FLT3ITD mRNA level showed an independent prognostic impact on overall survival (OS) and relapse-free survival (RFS) only in the NPM1+ subgroup (OS: hazard ratio, 5.9; [95% confidence interval [CI]: 3.1-11.2]; RFS: hazard ratio, 7.5 [95% CI: 3.4-16.5]). The FLT3ITD mRNA level contributes to relapse risk stratification and might help to guide postremission therapy in NPM1-mutated AML. PMID:22374696

  7. Immunotoxins, ligand-toxin conjugates and molecular targeting.

    PubMed

    Soria, M

    1989-01-01

    Biotechnology provides tools for therapeutic exploitation following advances in the elucidation of protein-to-cell and cell-to-cell interactions. Molecular targeting of bacterial and plant toxins to the desired district of action can be achieved through effector molecules like monoclonal antibodies or protein ligands. Biochemical conjugation of these effectors to SO-6, a single-chain Ribosome Inactivating Protein from Saponaria officinalis, yielded powerful cytotoxic agents that are attractive candidates for therapeutic evaluation. Cloning of the gene for this plant toxin has been achieved. Technologies for expression of protein ligands, such as apolipoproteins or several growth factors, are available in recombinant microorganisms, providing adequate partners for the assembly of targeted chimaeras. Domain engineering of structural and functional regions in effector proteins is now possible and will be carried out with the available technologies to improve existing therapy. PMID:2698471

  8. Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells.

    PubMed

    Han, Lina; Qiu, Peng; Zeng, Zhihong; Jorgensen, Jeffrey L; Mak, Duncan H; Burks, Jared K; Schober, Wendy; McQueen, Teresa J; Cortes, Jorge; Tanner, Scott D; Roboz, Gail J; Kantarjian, Hagop M; Kornblau, Steven M; Guzman, Monica L; Andreeff, Michael; Konopleva, Marina

    2015-04-01

    Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may

  9. Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells

    PubMed Central

    Han, Lina; Qiu, Peng; Zeng, Zhihong; Jorgensen, Jeffrey L; Mak, Duncan H; Burks, Jared K; Schober, Wendy; McQueen, Teresa J; Cortes, Jorge; Tanner, Scott D; Roboz, Gail J; Kantarjian, Hagop M; Kantarjian, Hagop M; Kornblau, Steven M; Guzman, Monica L; Andreeff, Michael; Konopleva, Marina

    2015-01-01

    Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor (SCF) and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34+ subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demontrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which

  10. Acute myeloid leukemia in patients older than 75: prognostic impact of FLT3-ITD and NPM1 mutations.

    PubMed

    Hirsch, Pierre; Qassa, Ghazi; Marzac, Christophe; Tang, Ruoping; Perrot, Jean-Yves; Isnard, Françoise; Mohty, Mohamad; Marie, Jean Pierre; Legrand, Ollivier

    2015-01-01

    The benefit associated with chemotherapy in older patients with acute myeloid leukemia (AML) is debated. The prognostic impact of molecular mutations in these patients is unknown. We identified 79 patients with AML aged 75 years or over. Forty-two received chemotherapy and 37 supportive care only. In intensively treated patients, overall survival was longer (p < 0.001). Achieving complete remission was associated with longer survival (p < 0.001). NPM1 mutations tended to be associated with a higher complete remission rate (p = 0.12). In multivariate analysis, FLT3-ITD was associated with poorer survival (p = 0.049). Patients harboring FLT3-ITD and no NPM1 mutation had a poorer prognosis than others (p = 0.02). Intensive treatments can benefit a portion of elderly patients. FLT3-ITD and NPM1 mutational status might be useful for prognosis stratification. PMID:24724782

  11. How reliable are ligand-centric methods for Target Fishing?

    NASA Astrophysics Data System (ADS)

    Peon, Antonio; Dang, Cuong; Ballester, Pedro

    2016-04-01

    Computational methods for Target Fishing (TF), also known as Target Prediction or Polypharmacology Prediction, can be used to discover new targets for small-molecule drugs. This may result in repositioning the drug in a new indication or improving our current understanding of its efficacy and side effects. While there is a substantial body of research on TF methods, there is still a need to improve their validation, which is often limited to a small part of the available targets and not easily interpretable by the user. Here we discuss how target-centric TF methods are inherently limited by the number of targets that can possibly predict (this number is by construction much larger in ligand-centric techniques). We also propose a new benchmark to validate TF methods, which is particularly suited to analyse how predictive performance varies with the query molecule. On average over approved drugs, we estimate that only five predicted targets will have to be tested to find two true targets with submicromolar potency (a strong variability in performance is however observed). In addition, we find that an approved drug has currently an average of eight known targets, which reinforces the notion that polypharmacology is a common and strong event. Furthermore, with the assistance of a control group of randomly-selected molecules, we show that the targets of approved drugs are generally harder to predict.

  12. From Toxins Targeting Ligand Gated Ion Channels to Therapeutic Molecules

    PubMed Central

    Nasiripourdori, Adak; Taly, Valérie; Grutter, Thomas; Taly, Antoine

    2011-01-01

    Ligand-gated ion channels (LGIC) play a central role in inter-cellular communication. This key function has two consequences: (i) these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii) they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted. PMID:22069709

  13. Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

    PubMed

    Fuchs, Adrian V; Tse, Brian W C; Pearce, Amanda K; Yeh, Mei-Chun; Fletcher, Nicholas L; Huang, Steve S; Heston, Warren D; Whittaker, Andrew K; Russell, Pamela J; Thurecht, Kristofer J

    2015-10-12

    Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle. PMID:26335533

  14. CD123 immunohistochemical expression in acute myeloid leukemia is associated with underlying FLT3-ITD and NPM1 mutations.

    PubMed

    Rollins-Raval, Marian; Pillai, Raju; Warita, Katsuhiko; Mitsuhashi-Warita, Tomoko; Mehta, Rohtesh; Boyiadzis, Michael; Djokic, Miroslav; Kant, Jeffrey A; Roth, Christine G

    2013-05-01

    FLT3-ITD and NPM1 mutation testing in acute myeloid leukemia (AML) plays an important role in prognostic risk stratification, especially within the intermediate cytogenetic risk group. Molecular studies require adequate fresh material and are typically performed on a dedicated aspirate specimen, which may not be available in all cases. Prior flow cytometric studies have suggested an association between CD123 overexpression in AML and FLT3-ITD and/or NPM1 mutations; however, the immunohistochemical (IHC) correlate is unknown. We assessed CD123 IHC expression in 157 AML bone marrow biopsies and/or marrow particle preparations, and correlated with the morphologic, immunophenotypic, and cytogenetic features and with the presence of FLT3-ITD and NPM1 mutations. We found that CD123 IHC expression, seen in 40% of AML, occurred across a wide spectrum of 2008 World Health Organization subtypes and was most frequent within the intermediate risk group. As compared with CD123 IHC-AML, CD123 IHC+AML demonstrated higher marrow blast percentages (median 69%), monocytic differentiation (33/63 cases), and CD34 negativity (29/63 cases). Eighty-three percent (25/30) FLT3-ITD-mutated AML were CD123+ (P<0.0001) and 62% (18/29) NPM1-mutated cases were CD123 IHC+ (P=0.0052) with negative predictive values of 95% for FLT3-ITD and 88% for NPM1. CD123 IHC+AML presents with characteristic pathologic features, some of which may be related to underlying FLT3-ITD and/or NPM1 mutations. PMID:22914610

  15. gp130 receptor ligands as potential therapeutic targets for obesity

    PubMed Central

    Febbraio, Mark A.

    2007-01-01

    Obesity and its related cluster of pathophysiologic conditions including insulin resistance, glucose intolerance, dyslipidemia, and hypertension are recognized as growing threats to world health. It is now estimated that 10% of the world’s population is overweight or obese. As a result, new therapeutic options for the treatment of obesity are clearly warranted. Recent research has focused on the role that gp130 receptor ligands may play as potential therapeutic targets in obesity. One cytokine in particular, ciliary neurotrophic factor (CNTF), acts both centrally and peripherally and mimics the biologic actions of the appetite control hormone leptin, but unlike leptin, CNTF appears to be effective in obesity and as such may have therapeutic potential. In addition, CNTF suppresses inflammatory signaling cascades associated with lipid accumulation in liver and skeletal muscle. This review examines the potential role of gp130 receptor ligands as part of a therapeutic strategy to treat obesity. PMID:17404609

  16. Programmed death-1 & its ligands: promising targets for cancer immunotherapy.

    PubMed

    Shrimali, Rajeev K; Janik, John E; Abu-Eid, Rasha; Mkrtichyan, Mikayel; Khleif, Samir N

    2015-01-01

    Novel strategies for cancer treatment involving blockade of immune inhibitors have shown significant progress toward understanding the molecular mechanism of tumor immune evasion. The preclinical findings and clinical responses associated with programmed death-1 (PD-1) and PD-ligand pathway blockade seem promising, making these targets highly sought for cancer immunotherapy. In fact, the anti-PD-1 antibodies, pembrolizumab and nivolumab, were recently approved by the US FDA for the treatment of unresectable and metastatic melanoma resistant to anticytotoxic T-lymphocyte antigen-4 antibody (ipilimumab) and BRAF inhibitor. Here, we discuss strategies of combining PD-1/PD-ligand interaction inhibitors with other immune checkpoint modulators and standard-of-care therapy to break immune tolerance and induce a potent antitumor activity, which is currently a research area of key scientific pursuit. PMID:26250412

  17. Ligand-targeted liposome design: challenges and fundamental considerations.

    PubMed

    Noble, Gavin T; Stefanick, Jared F; Ashley, Jonathan D; Kiziltepe, Tanyel; Bilgicer, Basar

    2014-01-01

    Nanomedicine, particularly liposomal drug delivery, has expanded considerably over the past few decades, and several liposomal drugs are already providing improved clinical outcomes. Liposomes have now progressed beyond simple, inert drug carriers and can be designed to be highly responsive in vivo, with active targeting, increased stealth, and controlled drug-release properties. Ligand-targeted liposomes (LTLs) have the potential to revolutionize the treatment of cancer. However, these highly engineered liposomes generate new problems, such as accelerated clearance from circulation, compromised targeting owing to non-specific serum protein binding, and hindered tumor penetration. This article highlights recent challenges facing LTL strategies and describes the advanced design elements used to circumvent them. PMID:24210498

  18. The prognostic impact of FLT3-ITD and NPM1 mutations in patients with relapsed acute myeloid leukemia and intermediate-risk cytogenetics

    PubMed Central

    How, J; Sykes, J; Minden, M D; Gupta, V; Yee, K W L; Schimmer, A D; Schuh, A C; Kamel-Reid, S; Brandwein, J M

    2013-01-01

    Internal tandem duplication of the fms-like tyrosine kinase-3 gene (FLT3-ITD) and nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis, but less is known about their utility to predict outcomes at relapse. We retrospectively analysed outcomes of 70 patients with relapsed, intermediate-risk karyotype AML who received a uniform reinduction regimen, with respect to FLT3-ITD and NPM1 mutation status and first complete remission (CR1) duration. CR1 duration, but not molecular status, was significantly correlated with CR2 rate. On univariate analysis, patients with mutated FLT3-ITD (FLT3+) had significantly worse overall survival (OS) compared with those with neither an NPM1 nor FLT3-ITD mutation (NPM1-/FLT3-). On multivariate analysis, shorter CR1 duration was significantly correlated with inferior OS at relapse (P<0.0001), while FLT3 and NPM1 mutation status and age were not significantly correlated with OS. Patients who subsequently underwent allogeneic stem cell transplant (alloSCT) had a superior OS regardless of CR1 duration, but outcomes were better in patients with CR1 duration>12 months. In intermediate-risk karyotype AML patients receiving reinduction, CR1 duration remains the most important predictor of OS at relapse; FLT3-ITD and NPM1 status are not independent predictors of survival. PMID:23708641

  19. Clinical implications of non-A-type NPM1 and FLT3 mutations in patients with normal karyotype acute myeloid leukemia.

    PubMed

    Park, Borae G; Chi, Hyun-Sook; Park, Seo-Jin; Min, Sook Kyoung; Jang, Seongsoo; Park, Chan-Jeoung; Kim, Dae-Young; Lee, Jung-Hee; Lee, Je-Hwan; Lee, Kyoo-Hyung

    2012-01-01

    Mutations in the nucleophosmin (NPM1) and fms-like tyrosine kinase-3 (FLT3) genes are the most commonly observed mutations in patients with normal-karyotype acute myeloid leukemia (AML-NK). We analyzed the prognostic effects and interactions of these mutations in 201 AML-NK patients. NPM1 and FLT3 mutations were found in 38.3 and 24.9% of AML-NK patients, respectively. NPM1 mutations (NPM1mut), especially in patients without FLT3 mutations (FLT3mut), were associated with a favorable outcome. However, NPM1mut did not affect survival. FLT3mut tended to be associated with a poor survival outcome. FLT3mut showed no prognostic effects in patients with A-type NPM1mut. However, FLT3mut were associated with a significantly worse prognosis in patients with non-A-type NPM1mut. The prognostic interaction between the NPM1 and FLT3 mutations was significant in patients with non-A-type NPM1mut. PMID:22104247

  20. Fms like tyrosine kinase (FLT3) and nucleophosmin 1 (NPM1) mutations in de novo normal karyotype acute myeloid leukemia (AML).

    PubMed

    Dunna, Nageswara Rao; Rajappa, Senthil; Digumarti, Raghunadharao; Vure, Sugunakar; Kagita, Sailaja; Damineni, Surekha; Rao, V R; Yadav, Satish Kumar; Ravuri, Rajasekhara Reddy; Satti, Vishnupriya

    2010-01-01

    Mutations in FLT3 and NPM1 are important prognostic factors in AML, influencing outcome in normal karyotype cases. We here analysed incidences of FLT3/ITD, D 835 and NPM1 mutations in patients with de novo normal karyotype AML using PCR and gene sequencing, along with laboratory parameters and treatment outcomes. There were 128 patients with a median age of 45 years (range, 19-65). FLT3/ITD mutations were detected in 26 (20.3%), FLT3/D835 in 8 (6.2%) and NPM1 in 22 (17.1%). The incidence of FLT3/ITD was higher in those with elevated lactate dehydrogenase (LDH) and peripheral blasts (p=< 0.002, < 0.001) while NPM1 mutations or both NPM1 and FLT3/ITD was more common in elevated total leukocyte counts (TLC), LDH and peripheral blasts (p=<0.0001). Complete response and disease free survival were lower in those with FLT3/ITD mutations (p=0.04, 0.03). The incidence of FLT3 and NPM1 mutations was found to be low in Indian patients with normal karyotype AML. PMID:21338238

  1. The prognostic impact of FLT3-ITD and NPM1 mutations in patients with relapsed acute myeloid leukemia and intermediate-risk cytogenetics.

    PubMed

    How, J; Sykes, J; Minden, M D; Gupta, V; Yee, K W L; Schimmer, A D; Schuh, A C; Kamel-Reid, S; Brandwein, J M

    2013-01-01

    Internal tandem duplication of the fms-like tyrosine kinase-3 gene (FLT3-ITD) and nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis, but less is known about their utility to predict outcomes at relapse. We retrospectively analysed outcomes of 70 patients with relapsed, intermediate-risk karyotype AML who received a uniform reinduction regimen, with respect to FLT3-ITD and NPM1 mutation status and first complete remission (CR1) duration. CR1 duration, but not molecular status, was significantly correlated with CR2 rate. On univariate analysis, patients with mutated FLT3-ITD (FLT3+) had significantly worse overall survival (OS) compared with those with neither an NPM1 nor FLT3-ITD mutation (NPM1-/FLT3-). On multivariate analysis, shorter CR1 duration was significantly correlated with inferior OS at relapse (P<0.0001), while FLT3 and NPM1 mutation status and age were not significantly correlated with OS. Patients who subsequently underwent allogeneic stem cell transplant (alloSCT) had a superior OS regardless of CR1 duration, but outcomes were better in patients with CR1 duration>12 months. In intermediate-risk karyotype AML patients receiving reinduction, CR1 duration remains the most important predictor of OS at relapse; FLT3-ITD and NPM1 status are not independent predictors of survival. PMID:23708641

  2. How Reliable Are Ligand-Centric Methods for Target Fishing?

    PubMed Central

    Peón, Antonio; Dang, Cuong C.; Ballester, Pedro J.

    2016-01-01

    Computational methods for Target Fishing (TF), also known as Target Prediction or Polypharmacology Prediction, can be used to discover new targets for small-molecule drugs. This may result in repositioning the drug in a new indication or improving our current understanding of its efficacy and side effects. While there is a substantial body of research on TF methods, there is still a need to improve their validation, which is often limited to a small part of the available targets and not easily interpretable by the user. Here we discuss how target-centric TF methods are inherently limited by the number of targets that can possibly predict (this number is by construction much larger in ligand-centric techniques). We also propose a new benchmark to validate TF methods, which is particularly suited to analyse how predictive performance varies with the query molecule. On average over approved drugs, we estimate that only five predicted targets will have to be tested to find two true targets with submicromolar potency (a strong variability in performance is however observed). In addition, we find that an approved drug has currently an average of eight known targets, which reinforces the notion that polypharmacology is a common and strong event. Furthermore, with the assistance of a control group of randomly-selected molecules, we show that the targets of approved drugs are generally harder to predict. The benchmark and a simple target prediction method to use as a performance baseline are available at http://ballester.marseille.inserm.fr/TF-benchmark.tar.gz. PMID:27148522

  3. DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias.

    PubMed

    Yang, Liubin; Rodriguez, Benjamin; Mayle, Allison; Park, Hyun Jung; Lin, Xueqiu; Luo, Min; Jeong, Mira; Curry, Choladda V; Kim, Sang-Bae; Ruau, David; Zhang, Xiaotian; Zhou, Ting; Zhou, Michael; Rebel, Vivienne I; Challen, Grant A; Göttgens, Berthold; Lee, Ju-Seog; Rau, Rachel; Li, Wei; Goodell, Margaret A

    2016-06-13

    DNMT3A, the gene encoding the de novo DNA methyltransferase 3A, is among the most frequently mutated genes in hematologic malignancies. However, the mechanisms through which DNMT3A normally suppresses malignancy development are unknown. Here, we show that DNMT3A loss synergizes with the FLT3 internal tandem duplication in a dose-influenced fashion to generate rapid lethal lymphoid or myeloid leukemias similar to their human counterparts. Loss of DNMT3A leads to reduced DNA methylation, predominantly at hematopoietic enhancer regions in both mouse and human samples. Myeloid and lymphoid diseases arise from transformed murine hematopoietic stem cells. Broadly, our findings support a role for DNMT3A as a guardian of the epigenetic state at enhancer regions, critical for inhibition of leukemic transformation. PMID:27300438

  4. 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

  5. The STAT5 Inhibitor Pimozide Displays Efficacy in Models of Acute Myelogenous Leukemia Driven by FLT3 Mutations

    PubMed Central

    Nelson, Erik A.; Walker, Sarah R.; Xiang, Michael; Weisberg, Ellen; Bar-Natan, Michal; Barrett, Rosemary; Liu, Suiyang; Kharbanda, Surender; Christie, Amanda L.; Nicolais, Maria; Griffin, James D.; Stone, Richard M.; Kung, Andrew L.

    2012-01-01

    Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myelogenous leukemia (AML) containing the FLT3 internal tandem duplication (ITD) mutation. FLT3 ITD is a constitutively active tyrosine kinase that drives the activation of STAT5, leading to the growth and survival of AML cells. Although there has been some success in identifying tyrosine kinase inhibitors that block the function of FLT3 ITD, there remains a continued need for effective treatment of this disease. We have identified the psychotropic drug pimozide as an effective inhibitor of STAT5 function. Pimozide inhibits the tyrosine phosphorylation of STAT5, leading to the death of AML cells through the induction of apoptosis. Pimozide shows a combinatorial effect with the tyrosine kinase inhibitors midostaurin (PKC412) and sunitinib in the inhibition of STAT5 tyrosine phosphorylation and the induction of apoptosis. Significantly, pimozide reduces the tumor burden in a mouse model of FLT3-driven AML. Therefore, identifying STAT5 inhibitors may provide a new avenue for the treatment of AML, and these may be effective alone or in combination with tyrosine kinase inhibitors. PMID:23264850

  6. Next-generation sequencing of FLT3 internal tandem duplications for minimal residual disease monitoring in acute myeloid leukemia

    PubMed Central

    Hélevaut, Nathalie; Rodriguez, Céline; Quief, Sabine; Sebda, Shéhérazade; Renneville, Aline; Nibourel, Olivier; Rousselot, Philippe; Gruson, Bérengère; Dombret, Hervé; Castaigne, Sylvie; Preudhomme, Claude

    2015-01-01

    Minimal Residual Disease (MRD) detection can be used for early intervention in relapse, risk stratification, and treatment guidance. FLT3 ITD is the most common mutation found in AML patients with normal karyotype. We evaluated the feasibility of NGS with high coverage (up to 2.4.106 PE fragments) for MRD monitoring on FLT3 ITD. We sequenced 37 adult patients at diagnosis and various times of their disease (64 samples) and compared the results with FLT3 ITD ratios measured by fragment analysis. We found that NGS could detect variable insertion sites and lengths in a single test for several patients. We also showed mutational shifts between diagnosis and relapse, with the outgrowth of a clone at relapse different from that dominant at diagnosis. Since NGS is scalable, we were able to adapt sensitivity by increasing the number of reads obtained for follow-up samples, compared to diagnosis samples. This technique could be applied to detect biological relapse before its clinical consequences and to better tailor treatments through the use of FLT3 inhibitors. Larger cohorts should be assessed in order to validate this approach. PMID:26078355

  7. Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

    PubMed Central

    Barragán, Eva; Montesinos, Pau; Camos, Mireia; González, Marcos; Calasanz, Maria J.; Román-Gómez, José; Gómez-Casares, Maria T.; Ayala, Rosa; López, Javier; Fuster, Óscar; Colomer, Dolors; Chillón, Carmen; Larrayoz, María J.; Sánchez-Godoy, Pedro; González-Campos, José; Manso, Félix; Amador, Maria L.; Vellenga, Edo; Lowenberg, Bob; Sanz, Miguel A.

    2011-01-01

    Background Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. Design and Methods We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. Results FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. Conclusions FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens. PMID:21685470

  8. [Detection of NPM1, FLT3 and C-KIT mutations in acute myeloid leukemia and their prognostic analysis].

    PubMed

    Li, Ling; Lyu, Xiao-Dong; Mi, Rui-Hua; Ding, Jing; Chen, Lin; Wang, Qian; Yin, Qing-Song; Hu, Jie-Ying; Fan, Rui-Hua; Wei, Xu-Dong

    2013-06-01

    This study was aimed to evaluate the frequencies and prognostic significance of the nucleophosmin 1 (NPM1) mutation, the fms-like tyrosine kinase 3 (FLT3) mutation and c-KIT mutation in acute myeloid leukemia (AML) and to explore their relevance to clinical characteristics, cytogenetics and survival. Genomic DNA from 78 newly diagnosed AML from August 2010 to October 2012 was screened by PCR and sequencing or capillary electrophoresis (CE) for NPM1, FLT3 and c-KIT mutations. The results showed that the incidence of NPM1 mutation was 14.1% in AML patients and 26.7% in normal karyotype AML patients. NPM1 mutant cases were significantly associated with old age (P < 0.05), high peripheral white cell count and platelet counts (P < 0.05) and low expression of CD34 (P < 0.05), but no statistic difference was found in sex, percentage of bone marrow blasts, Hb, expression of CD117 and HLA-DR, complete remission rate, overall survival and relapse rate (P > 0.05). The prevalences of FLT3-ITD and FLT3-TKD mutations were 11.5% (9/78) and 3.8% (3/78) respectively, and no one patient has both of the two mutations. Patients with FLT3-ITD mutation had higher white blood cell counts and percentage of in bone marrow blasts (P < 0.05), and lower overall survival (P < 0.05), more relative to normal karyotype (P < 0.05), while no statistic difference was found in sex, age, platelet count, Hb level, complete remission rate and relapse rate (P > 0.05). No statistic analysis was performed due to the cases of less FLT3-TKD mutation. C-KIT mutation accounts for 7.7% (6/78). Patients with C-KIT mutation had a higher percentage in abnormal karyotype (P < 0.05), and higher relapse rate (P < 0.05), and lower overall survival, whereas no statistic difference was found in sex, age, percentage of bone marrow blasts, peripheral blood cell count, complete remission rate (P > 0.05). It is concluded that the detection of NPM1, FLT3 and C-KIT mutations may contribute to guiding treatment and evaluating

  9. [Simultaneous detection of FLT3-ITD and NPM1 gene mutations in acute myeloid leukemia by double PCR].

    PubMed

    Zhang, Ze-Chuan; Lu, Quan-Yi; Zhao, Jiang-Ning; Chen, Ya-Mei; Li, Zhi-Peng

    2011-06-01

    Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML. A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3, with the aim of detecting almost all reported mutations. After optimization, a touchdown PCR was chosen for the multiplex PCR procedure, with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively. The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products. The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio. All the positive mutated samples were confirmed by sequencing. The results showed that 17 patients with NPM1 mutation, 15 patients with FLT3-ITD mutation, 6 patients with both NPM1 and FLT3-ITD mutations were found among 93 patents. 7 patients with M₂, 4 patients with M₄, 5 patients with M₅ and 1 patients with M₆ were found out of 17 patients with NPM1 mutation, in which 10 patients were male and 7 patients were female, 15 patients were with type A, 1 patients was with type B and 1 patients was with type Nm, strikingly 1 CML patient in blast crisis was found to carry a type A mutation. Among 15 patients with FLT3-ITD mutation 1 patient with M₁, 8 patients with M₂, 2 patients with M₂, 2 patients with M₃, 1 patient with M₄, 3 patients with M₅ were found, in which 5 patients were male and 10 patients were female. Sequencing results further confirmed the accuracy and reliability of this method. It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated. This method is fast, easy, accurate and capable to calculate the mutant/wild-type ratio. PMID:21729557

  10. Comprehensive genomic analysis reveals FLT3 activation and a therapeutic strategy for a patient with relapsed adult B-lymphoblastic leukemia.

    PubMed

    Griffith, Malachi; Griffith, Obi L; Krysiak, Kilannin; Skidmore, Zachary L; Christopher, Matthew J; Klco, Jeffery M; Ramu, Avinash; Lamprecht, Tamara L; Wagner, Alex H; Campbell, Katie M; Lesurf, Robert; Hundal, Jasreet; Zhang, Jin; Spies, Nicholas C; Ainscough, Benjamin J; Larson, David E; Heath, Sharon E; Fronick, Catrina; O'Laughlin, Shelly; Fulton, Robert S; Magrini, Vincent; McGrath, Sean; Smith, Scott M; Miller, Christopher A; Maher, Christopher A; Payton, Jacqueline E; Walker, Jason R; Eldred, James M; Walter, Matthew J; Link, Daniel C; Graubert, Timothy A; Westervelt, Peter; Kulkarni, Shashikant; DiPersio, John F; Mardis, Elaine R; Wilson, Richard K; Ley, Timothy J

    2016-07-01

    The genomic events responsible for the pathogenesis of relapsed adult B-lymphoblastic leukemia (B-ALL) are not yet clear. We performed integrative analysis of whole-genome, whole-exome, custom capture, whole-transcriptome (RNA-seq), and locus-specific genomic assays across nine time points from a patient with primary de novo B-ALL. Comprehensive genome and transcriptome characterization revealed a dramatic tumor evolution during progression, yielding a tumor with complex clonal architecture at second relapse. We observed and validated point mutations in EP300 and NF1, a highly expressed EP300-ZNF384 gene fusion, a microdeletion in IKZF1, a focal deletion affecting SETD2, and large deletions affecting RB1, PAX5, NF1, and ETV6. Although the genome analysis revealed events of potential biological relevance, no clinically actionable treatment options were evident at the time of the second relapse. However, transcriptome analysis identified aberrant overexpression of the targetable protein kinase encoded by the FLT3 gene. Although the patient had refractory disease after salvage therapy for the second relapse, treatment with the FLT3 inhibitor sunitinib rapidly induced a near complete molecular response, permitting the patient to proceed to a matched-unrelated donor stem cell transplantation. The patient remains in complete remission more than 4 years later. Analysis of this patient's relapse genome revealed an unexpected, actionable therapeutic target that led to a specific therapy associated with a rapid clinical response. For some patients with relapsed or refractory cancers, this approach may indicate a novel therapeutic intervention that could alter outcome. PMID:27181063

  11. Phase I Trial of Maintenance Sorafenib after Allogeneic Hematopoietic Stem Cell Transplantation for FLT3-ITD AML

    PubMed Central

    Chen, Yi-Bin; Li, Shuli; Lane, Andrew A.; Connolly, Christine; Del Rio, Candice; Valles, Betsy; Curtis, Morgan; Ballen, Karen; Cutler, Corey; Dey, Bimalangshu R.; El-Jawahri, Areej; Fathi, Amir T.; Ho, Vincent T.; Joyce, Amy; McAfee, Steven; Rudek, Michelle; Rajkhowa, Trivikram; Verselis, Sigitas; Antin, Joseph H.; Spitzer, Thomas R.; Levis, Mark; Soiffer, Robert

    2014-01-01

    The FLT3-ITD mutation is associated with a high relapse rate for patients with AML even after allogeneic hematopoietic stem cell transplantation (HSCT). Sorafenib is a tyrosine kinase inhibitor which inhibits the FLT3 tyrosine kinase and has shown encouraging activity in FLT3-ITD AML. We conducted a phase I trial of maintenance sorafenib after HSCT in patients with FLT3-ITD AML (ClinicalTrials.gov NCT01398501). Patients received a variety of conditioning regimens and graft sources. A dose escalation 3+3 cohort design was used to define the maximum tolerated dose (MTD) with an additional 10 patients treated at the MTD. Sorafenib was initiated between days 45 and 120 after HSCT continued for twelve 28-day cycles. Twenty-two patients were enrolled (status at HSCT: CR1=16, CR2=3, refractory=3). The MTD was established at 400 mg BID with one DLT observed (pericardial effusion). Two patients died of transplant-related causes, both unrelated to sorafenib. Two patients stopped sorafenib after relapse and 5 stopped due to attributable toxicities after the DLT period. Median follow-up for surviving patients is 16.7 months after HSCT (range, 8.1–35.0). There was one case of grade II acute GVHD after starting sorafenib and the 12-month cumulative incidence of chronic GVHD was 38% (90% CI, 21%–56%). For all patients, one-year progression-free survival (PFS) is 85% (90% CI, 66%–94%) and one-year overall survival (OS) is 95% (90% CI, 79%–99%) after HSCT. For patients in CR1 / CR2 prior to HSCT (n=19), one-year PFS is 95% (90% CI, 76%–99%) and one-year OS is 100% with only one patient who has relapsed. Sorafenib is safe after HSCT for FLT3-ITD AML and merits further investigation for the prevention of relapse. PMID:25239228

  12. Intra-Articular Fms-Like Tyrosine Kinase 3 Ligand Expression Is a Driving Force in Induction and Progression of Arthritis

    PubMed Central

    Dehlin, Mats; Bokarewa, Maria; Rottapel, Robert; Foster, Simon J.; Magnusson, Mattias; Dahlberg, Leif E.; Tarkowski, Andrej

    2008-01-01

    Background One of the hallmarks of rheumatoid arthritis (RA) is hyperplasia and inflammation of the synovial tissue being characterized by in situ occurrence of highly differentiated leukocytes. Fms-like tyrosine kinase 3 (Flt3) has a crucial role in hematopoiesis, regulation of cell proliferation, differentiation and apoptosis. Typically, Flt3 is expressed on early myeloid and lymphoid progenitors and is activated by its soluble ligand (Flt3-L). The highly differentiated cellular pattern in the synovium of the RA patients made us hypothesize that Flt3-L, with its ability to induce proliferation and differentiation, could be of importance in induction and/or progression of arthritis. Methodology/Principal Findings To investigate occurrence of Flt3-L in RA we have measured its levels in matched serum and synovial fluid samples from 130 patients and 107 controls. To analyse the pro-inflammatory role of Flt3-L, we continuously overexpressed this protein locally in healthy mouse joints using homologous B-cell line transfected with Flt3-L gene. Additionally, recombinant Flt3-L was instillated intra-articularly in combination with peptidoglycans, a Toll Like Receptor 2-ligand with stong arthritogenic properties. Our results show significantly higher levels of Flt3-L in the synovial fluid as compared to serum levels in RA subjects (p = 0.0001). In addition, RA synovial fluid levels of Flt-3-L were significantly higher than these obtained from synovial fluids originating from non-inflammatory joint diseases (p = 0.022). Intra-articular administration of B-cell line transfected with Flt3-L gene resulted in highly erosive arthritis while inoculation of the same B-cell line without hyperexpression of Flt3-L did not induce erosivity and only in a minority of cases caused synovial proliferation! Flt3-ligand potentiated peptidoglycan induced arthritis as compared to mice injected with peptidoglycan alone (p<0.05). Conclusions/Significance Our findings indicate that Flt3

  13. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia.

    PubMed

    Gale, Rosemary E; Green, Claire; Allen, Christopher; Mead, Adam J; Burnett, Alan K; Hills, Robert K; Linch, David C

    2008-03-01

    An internal tandem duplication in the fms-like tyrosine kinase 3 gene (FLT3/ITD) is associated with poor prognosis in acute myeloid leukemia (AML), but the impact of mutant level, size, and interaction with nucleophosmin 1 (NPM1) mutations remains controversial. We evaluated these characteristics in a large cohort of young adult AML patients. There was a highly significant trend for worsening in relapse risk (RR) and overall survival (OS) with increasing FLT3/ITD mutant level (P < .001 for both), and even in the low level mutant group (1%-24% of total FLT3 alleles), RR was significantly worse than in the FLT3 wild-type (WT) group (P < .001). In multivariate analysis, mutant level was the most powerful prognostic factor for RR. Mutant size and number had no significant impact on outcome. The beneficial impact of an NPM1 mutation on RR and OS was seen in FLT3/ITD(+) as well as FLT3/WT patients; both markers were highly significant independent predictors of outcome (P < .001). Stratification using both markers identified 3 prognostic groups: good (FLT3/ITD(-)NPM1(+)), intermediate (FLT3/ITD(-)NPM1(-) or FLT3/ITD(+)NPM1(+)), and poor (FLT3/ITD(+)NPM1(-)). Patients with high FLT3/ITD mutant level (greater than 50%) or FLT3/ITD(+) in the absence of an NPM1 mutation may be good candidates for more experimental therapeutic approaches. PMID:17957027

  14. Screening and Optimization of Ligand Conjugates for Lysosomal Targeting

    PubMed Central

    Meerovich, Igor; Koshkaryev, Alexander; Thekkedath, Ritesh; Torchilin, Vladimir P.

    2011-01-01

    The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide good rate of co-localization well the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared with plain liposomes. These results were additionally confirmed by the flow cytometry of the intact cells treated with liposomes loaded with with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside, a specific substrate for the intralysosomal β-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands – rhodamine B DSPE-PEG2k-amide and 6-(3-(DSPE-PEG2k)-thioureido) rhodamine B – demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the

  15. Amount of bone marrow blasts is strongly correlated to NPM1 and FLT3-ITD mutation rate in AML with normal karyotype.

    PubMed

    Haferlach, Torsten; Bacher, Ulrike; Alpermann, Tamara; Haferlach, Claudia; Kern, Wolfgang; Schnittger, Susanne

    2012-01-01

    FLT3-ITDs are linked to higher leukocytes/blasts in acute myeloid leukemia. To evaluate the effect of NPM1 mutations, we correlated NPM1mut status with morphology in 805 adult normal karyotype AML. NPM1mut were found in 391/805 (48.6%), FLT3-ITD in 219/805 (27.2%). Frequencies of FLT3-ITD and NPM1mut cases were continuously increasing by blast decades: NPM1mut from 38/123 (30.9%) in 20-29% blast decade to 70/103 (68.0%) in 90-100% decade (p<0.001), FLT3-ITDs from 15/123 (12.2%) to 58/103 (56.3%) (p<0.001). Mean WBC count was highest in NPM1-mut/FLT3-ITD-positive and lowest in NPM1-wildtype/FLT3-ITD-negative patients (p<0.0001); similar for BM blasts. Therefore, FLT3-ITD and NPM1mut might synergistically stimulate blast proliferation. PMID:21621842

  16. A review of the ligands and related targeting strategies for active targeting of paclitaxel to tumours.

    PubMed

    Li, Juan; Wang, Fengshan; Sun, Deqing; Wang, Rongmei

    2016-08-01

    It has been 30 years since the discovery of the anti-tumour property of paclitaxel (PTX), which has been successfully applied in clinic for the treatment of carcinomas of the lungs, breast and ovarian. However, PTX is poorly soluble in water and has no targeting and selectivity to tumour tissue. Recent advances in active tumour targeting of PTX delivery vehicles have addressed some of the issues related to lack of solubility in water and non-specific toxicities associated with PTX. These PTX delivery vehicles are designed for active targeting to specific cancer cells by the addition of ligands for recognition by specific receptors/antigens on cancer cells. This article will focus on various ligands and related targeting strategies serving as potential tools for active targeting of PTX to tumour tissues, illustrating their use in different tumour models. This review also highlights the need of further studies on the discovery of receptors in different cells of specific organ and ligands with binding efficiency to these specific receptors. PMID:26878228

  17. Rule of five in 2015 and beyond: Target and ligand structural limitations, ligand chemistry structure and drug discovery project decisions.

    PubMed

    Lipinski, Christopher A

    2016-06-01

    The rule of five (Ro5), based on physicochemical profiles of phase II drugs, is consistent with structural limitations in protein targets and the drug target ligands. Three of four parameters in Ro5 are fundamental to the structure of both target and drug binding sites. The chemical structure of the drug ligand depends on the ligand chemistry and design philosophy. Two extremes of chemical structure and design philosophy exist; ligands constructed in the medicinal chemistry synthesis laboratory without input from natural selection and natural product (NP) metabolites biosynthesized based on evolutionary selection. Exceptions to Ro5 are found mostly among NPs. Chemistry chameleon-like behavior of some NPs due to intra-molecular hydrogen bonding as exemplified by cyclosporine A is a strong contributor to NP Ro5 outliers. The fragment derived, drug Navitoclax is an example of the extensive expertise, resources, time and key decisions required for the rare discovery of a non-NP Ro5 outlier. PMID:27154268

  18. Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors.

    PubMed

    Aráoz, Rómulo; Vilariño, Natalia; Botana, Luis M; Molgó, Jordi

    2010-07-01

    Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents. PMID:20238109

  19. Target gene delivery from targeting ligand conjugated chitosan-PEI copolymer for cancer therapy.

    PubMed

    Nam, Joung-Pyo; Nah, Jae-Woon

    2016-01-01

    In this study, we designed a novel carrier which was having low cytotoxicity, site-specific target function, and high transfection efficiency using low molecular weight water soluble O-carboxymethyl chitosan (OCMCh), branched low molecular weight poly(ethyleneimine) (bPEI), and targeting ligand (epitope type, HER-2/neu). OCMCh/bPEI/targeting ligand, HPOCP copolymer, and targeting ligand-modified polyamphoteric polymer, and were prepared by chemical reaction and characterized by (1)H NMR and FT-IR. The binding affinity, protecting efficiency, and releasing ability of gene/HPOCP polyplex were confirmed by gel retardation assay. The pDNA(pEGFP)/HPOCP polyplexes showed high gene transfection efficiency in HCT 119 cell. In addition, siRNA/HPOCP polyplexes formed spherical shape and have particle sizes from 100 to 300nm. The siRNA/HPOCP polyplexes have lower cytotoxicity than PEI in the all of siRNA concentrations ranging from 0 to 2μg/μL in HEK 293 cells. The cell viability of siRNA/HPOCP polyplexes was performed in SK-Br3 cells with VEGF siRNA or BCL2 siRNA. In addition, confocal laser-scanning microscopy and flow cytometry assay were performed for cellular localization and cellular uptake efficiency of siRNA/HPOCP polyplexes. The results of the present study demonstrate that HPOCP copolymer is a good candidate as gene delivery carriers for gene delivery system or gene therapy. PMID:26453863

  20. The prevalence and clinical profiles of FLT3-ITD, FLT3-TKD, NPM1, C-KIT, DNMT3A, and CEBPA mutations in a cohort of patients with de novo acute myeloid leukemia from southwest China.

    PubMed

    Gou, Haimei; Zhou, Juan; Ye, Yuanxin; Hu, Xuejiao; Shang, Mengqiao; Zhang, Jingya; Zhao, Zhenzhen; Peng, Wu; Zhou, Yanhong; Zhou, Yi; Song, Xingbo; Lu, Xiaojun; Ying, Binwu

    2016-06-01

    While a substantial amount of data on gene mutations related to acute myeloid leukemia (AML) prognosis from western and other populations have been reported, these studies largely describe one or two genes. Additionally, in southwest China, only insufficient data exist regarding FLT3-ITD, FLT3-TKD, NPM1, C-KIT, DNMT3A, and CEBPA mutations have been widely used in clinical settings. Therefore, a comprehensive study about these mutations of clinical importance in the prognosis of AML in western China is necessary. In a cohort of 255 patients with de novo AML, we retrospectively analyzed the prevalence of the six gene mutations, and then we assessed the results in conjunction with clinical characteristics and treatment responses. As for the frequencies of these mutations, the NPM1 mutation occurred most frequently (17.7 %; 42/237), followed by the CEBPA mutation (15.0 %; 19/127) and the FLT3-ITD mutation (10.2 %; 25/244). The frequencies of the FLT3-TKD, DNMT3A, and C-KIT mutations were 3.7 % (9/234), 4.0 % (9/225) and 4.2 % (10/238), respectively. These mutations were closely related to clinical characteristics including FAB classification, gender and age, hemogram, blasts (%), fusion genes, and immunophenotypes. Additionally, a higher complete remission (CR) rate was found in NPM1-mutated patients. The occurrence of these mutations is variable among different countries and regions worldwide, which may provide clues to the etiology of AML. Besides, we identified new clinical characteristics that advance our understanding of these mutations and further clarify the involvement of these mutations in the development of leukemia. PMID:26676635

  1. Examination of the FLT3 and NPM1 mutational status in patients with acute myeloid leukemia from southeastern Poland

    PubMed Central

    Zmorzyński, Szymon; Michalak-Wojnowska, Małgorzata; Wąsik-Szczepanek, Ewa; Filip, Agata A.

    2016-01-01

    Introduction Acute myeloid leukemia (AML) is a genetically heterogeneous disease at both the cytogenetic and molecular levels. In AML cells many chromosomal aberrations are observed, some of them being characteristic of a particular subtype of patients, and others being less significant. Besides chromosomal abnormalities, the leukemic cells can have a variety of mutations involving individual genes. The aim of this work was to investigate the frequencies of molecular alterations with the focus on FLT3-ITD and NPM1 mutations in AML patients of different age groups living in a southeastern region of Poland. Material and methods The study group comprised 50 consecutive AML patients. We analyzed bone marrow samples by conventional cytogenetics. Cytogenetic evaluation in selected cases was complemented by the FISH technique. The internal tandem mutation in the FLT3 gene was identified using polymerase chain reaction (PCR), and the NPM1 mutation was assessed by direct nucleotide sequencing. Results The studies using classical cytogenetics showed chromosomal aberrations in 32 (64%) patients. In 18 cases no changes in the karyotype were found by conventional karyotyping. FLT3-ITD mutation was detected in 4 (8%) patients and mutation of NPM1 in 3 patients with AML (6%). Conclusions The incidence of both mutations in our study group was lower than described elsewhere. We have confirmed that FLT3-ITD occurred more commonly in older patients and it was associated with shorter overall survival. By contrast, mutation of exon 12 of the NPM1 gene seems to be a good prognostic factor in AML patients with normal karyotype. PMID:26925127

  2. Metabolic alterations and drug sensitivity of tyrosine kinase inhibitor resistant leukemia cells with a FLT3/ITD mutation.

    PubMed

    Huang, Amin; Ju, Huai-Qiang; Liu, Kaiyan; Zhan, Guilian; Liu, Daolu; Wen, Shijun; Garcia-Manero, Guillermo; Huang, Peng; Hu, Yumin

    2016-07-28

    Internal tandem duplication (ITD) of the juxtamembrane region of FMS-like tyrosine kinase-3 (FLT3) receptor is a common type of mutation in adult acute myeloid leukemia (AML), and patient response to FLT3 inhibitors appears to be transient due to the emergence of drug resistance. We established two sorafenib-resistant cell lines carrying FLT3/ITD mutations, including the murine BaF3/ITD-R and human MV4-11-R cell lines. Gene expression profile analysis of the resistant and parental cells suggests that the highest ranked molecular and cellular functions of the differentially expressed genes are related to mitochondrial dysfunction. Both murine and human resistant cell lines display a longer doubling time, along with a significant inhibition of mitochondrial respiratory chain activity and substantial upregulation of glycolysis. The sorafenib-resistant cells exhibit increased expression of a majority of glycolytic enzymes, including hexokinase 2, which is also highly expressed in the mitochondrial fraction and is associated with resistance to apoptotic cell death. The sorafenib-resistant cells are collaterally sensitive to a number of glycolytic inhibitors including 2-deoxyglucose and 3-bromopyruvate propylester. Our study reveals a metabolic signature of sorafenib-resistant cells and suggests that glycolytic inhibition may override such resistance and warrant further clinical investigation. PMID:27132990

  3. A Flt3 and Ras-dependent Pathway Primes B Cell Development by Inducing A State of IL7-responsiveness

    PubMed Central

    Li, Lin-Xi; Goetz, Christine A.; Katerndahl, Casey D.S.; Sakaguchi, Nobuo; Farrar, Michael A.

    2009-01-01

    Ras plays an important role in B cell development. However, the stage at which Ras governs B cell development remains unclear. Moreover, the upstream receptors and downstream effectors of Ras that govern B cell differentiation remain undefined. Using mice that express a dominant negative form of Ras, we demonstrate that Ras-mediated signaling plays a critical role in the development of common lymphoid progenitors (CLP). This developmental block parallels that found in flt3−/− mice, suggesting that Flt3 is an important upstream activator of Ras in early B cell progenitors. Ras inhibition impaired proliferation of CLP and pre-pro-B cells but not pro-B cells. Rather, Ras promotes STAT5-dependent pro-B cell differentiation by enhancing IL7Rα levels and suppressing socs2 and socs3 expression. Our results suggest a model in which Flt3/Ras-dependent signals play a critical role in B cell development by priming early B cell progenitors for subsequent STAT5-dependent B cell differentiation. PMID:20065110

  4. The surface molecular signature of leukemic cells is associated with NPM1 mutations and FLT3 -ITD in patients with de novo acute myeloid leukemia.

    PubMed

    Su, Long; Gao, Su-Jun; Li, Wei; Tan, Ye-Hui; Cui, Jiu-Wei; Han, Wei

    2014-01-01

    Certain molecular mutations are associated with signs of cell morphology and differentiation in acute myeloid leukemia (AML). However, only limited data are available for the detailed analysis of such correlations. In this study, AML patients were classified into 4 subsets according to CD34, HLA-DR and CD11c expression levels. Significantly low CD34 antigen expression was observed in nucleophosmin (NPM1)-mutated patients and in those with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD). No correlations were observed among NPM1 mutations, FLT3-ITD and monocytic morphology in patients without CD34 expression. Both NPM1 mutations and FLT3-ITD were absent in cluster IIb patients (CD34(+)CD11c(-)). The associations among NPM1 mutations, FLT3-ITD and the surface molecular signature of leukemic cells may offer beneficial information about the pathogenesis of AML. PMID:24192815

  5. Detection of NPM1 exon 12 mutations and FLT3 – internal tandem duplications by high resolution melting analysis in normal karyotype acute myeloid leukemia

    PubMed Central

    Tan, Angela YC; Westerman, David A; Carney, Dennis A; Seymour, John F; Juneja, Surender; Dobrovic, Alexander

    2008-01-01

    Background Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45–60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples. Results We developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29) were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results. Conclusion HRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases. PMID:18664261

  6. Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients

    PubMed Central

    Zuffa, Elisa; Franchini, Eugenia; Papayannidis, Cristina; Baldazzi, Carmen; Simonetti, Giorgia; Testoni, Nicoletta; Abbenante, Maria Chiara; Paolini, Stefania; Sartor, Chiara; Parisi, Sarah; Marconi, Giovanni; Cattina, Federica; Bochicchio, Maria Teresa; Venturi, Claudia; Ottaviani, Emanuela; Cavo, Michele; Martinelli, Giovanni

    2015-01-01

    FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2–2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients. PMID:26384303

  7. Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to post-remission therapy.

    PubMed

    Pratcorona, Marta; Brunet, Salut; Nomdedéu, Josep; Ribera, Josep Maria; Tormo, Mar; Duarte, Rafael; Escoda, Lourdes; Guàrdia, Ramon; Queipo de Llano, M Paz; Salamero, Olga; Bargay, Joan; Pedro, Carmen; Martí, Josep Maria; Torrebadell, Montserrat; Díaz-Beyá, Marina; Camós, Mireia; Colomer, Dolors; Hoyos, Montserrat; Sierra, Jorge; Esteve, Jordi

    2013-04-01

    Risk associated to FLT3 internal tandem duplication (FLT3-ITD) in patients with acute myeloid leukemia (AML) may depend on mutational burden and its interaction with other mutations. We analyzed the effect of FLT3-ITD/FLT3 wild-type (FLT3wt) ratio depending on NPM1 mutation (NPM1mut) in 303 patients with intermediate-risk cytogenetics AML treated with intensive chemotherapy. Among NPM1mut patients, FLT3wt and low ratio (<0.5) subgroups showed similar overall survival, relapse risk, and leukemia-free survival, whereas high ratio (≥0.5) patients had a worse outcome. In NPM1wt AML, FLT3-ITD subgroups showed a comparable outcome, with higher risk of relapse and shortened overall survival than FLT3wt patients. Allogeneic stem cell transplantation in CR1 was associated with a reduced relapse risk in all molecular subgroups with the exception of NPM1mut AML with absent or low ratio FLT3-ITD. In conclusion, effect of FLT3 burden is modulated by NPM1 mutation, especially in patients with a low ratio. PMID:23377436

  8. Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis

    PubMed Central

    Doolittle, Elizabeth; Peiris, Pubudu M.; Doron, Gilad; Goldberg, Amy; Tucci, Samantha; Rao, Swetha; Shah, Shruti; Sylvestre, Meilyn; Govender, Priya; Turan, Oguz; Lee, Zhenghong; Schiemann, William P.; Karathanasis, Efstathios

    2015-01-01

    Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvβ3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection. PMID:26203676

  9. Heptameric targeting ligands against EGFR and HER2 with high stability and avidity.

    PubMed

    Kim, Dongwook; Yan, Yitang; Valencia, C Alexander; Liu, Rihe

    2012-01-01

    Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, Z(EGFR) and Z(HER2), were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric Z(EGFR) and Z(HER2) ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents.. PMID:22912791

  10. Label-free integrative pharmacology on-target of opioid ligands at the opioid receptor family

    PubMed Central

    2013-01-01

    Background In vitro pharmacology of ligands is typically assessed using a variety of molecular assays based on predetermined molecular events in living cells. Many ligands including opioid ligands pose the ability to bind more than one receptor, and can also provide distinct operational bias to activate a specific receptor. Generating an integrative overview of the binding and functional selectivity of ligands for a receptor family is a critical but difficult step in drug discovery and development. Here we applied a newly developed label-free integrative pharmacology on-target (iPOT) approach to systematically survey the selectivity of a library of fifty-five opioid ligands against the opioid receptor family. All ligands were interrogated using dynamic mass redistribution (DMR) assays in both recombinant and native cell lines that express specific opioid receptor(s). The cells were modified with a set of probe molecules to manifest the binding and functional selectivity of ligands. DMR profiles were collected and translated to numerical coordinates that was subject to similarity analysis. A specific set of opioid ligands were then selected for quantitative pharmacology determination. Results Results showed that among fifty-five opioid ligands examined most ligands displayed agonist activity in at least one opioid receptor expressing cell line under different conditions. Further, many ligands exhibited pathway biased agonism. Conclusion We demonstrate that the iPOT effectively sorts the ligands into distinct clusters based on their binding and functional selectivity at the opioid receptor family. PMID:23497702

  11. Age-dependent frequencies of NPM1 mutations and FLT3-ITD in patients with normal karyotype AML (NK-AML).

    PubMed

    Schneider, Friederike; Hoster, Eva; Schneider, Stephanie; Dufour, Annika; Benthaus, Tobias; Kakadia, Purvi M; Bohlander, Stefan K; Braess, Jan; Heinecke, Achim; Sauerland, Maria C; Berdel, Wolfgang E; Buechner, Thomas; Woermann, Bernhard J; Feuring-Buske, Michaela; Buske, Christian; Creutzig, Ursula; Thiede, Christian; Zwaan, Michel C; van den Heuvel-Eibrink, Marry M; Reinhardt, Dirk; Hiddemann, Wolfgang; Spiekermann, Karsten

    2012-01-01

    Prognosis of AML in elderly patients is poor due to adverse patient characteristics and comorbidities. In addition, disease-associated parameters reveal differences between older and younger patients with AML. Survival in normal karyotype AML (NK-AML) is influenced by different clinical and molecular markers. The aim of this work was to investigate the frequencies of molecular markers in patients with NK-AML with a focus on NPM1 mutations and FLT3-ITD in different age groups. In the present study, we analyzed the frequencies of mutations of NPM1 and FLT3-ITD in a cohort of 1,321 adult patients and 148 children with AML treated within the AMLCG99, the AML98, and AML04 trials and their distribution in different age groups. Additionally, the frequencies of mutations in CEBPA genes, FLT3-TKD, and MLL-PTD were analyzed in the cohort with NK-AML (n = 729). Our data show that the presence of mutations of NPM1 (from 60% to 40%) and FLT3-ITD (from 50% to 20%) significantly decreased with age in adult AML. Consequently, the proportion of NPM1-/FLT3-ITD- patients increased with age. The decreasing frequency of NPM1 mutations in elderly patients was paralleled by a reduced complete remission (CR) rate in the elderly of 55% compared to 80% in the younger patients. By contrast, the frequencies of other gene mutations, like FLT3-TKD and MLL-PTD, and mutations in CEBPA were not age-dependent. The decreasing frequency of the favorable NPM1 mutations with increasing age may partially explain the worse outcome in the elderly patients. Furthermore, the increasing amount of elderly patients without NPM1 mutations or FLT3-ITD suggests that other molecular and clinical risk factors may influence prognosis in this age group. PMID:21744003

  12. CUPLIKE NUCLEI (PROMINENT NUCLEAR INVAGINATIONS) IN ACUTE MYELOID LEUKEMIA ARE HIGHLY ASSOCIATED WITH FLT3-ITD AND NPM1 MUTATIONS

    PubMed Central

    Chen, Weina; Konoplev, Sergej; Medeiros, L. Jeffrey; Koeppen, Hartmut; Leventaki, Vasiliki; Vadhan-Raj, Saroj; Jones, Dan; Kantarjian, Hagop M.; Falini, Brunangelo; Bueso-Ramos, Carlos E.

    2012-01-01

    BACKGROUND A small subset of acute myeloid leukemia (AML) cases has cuplike nuclei. Others have shown that these neoplasms have distinctive clinicopathologic and molecular features. METHODS We searched for AML cases with cuplike nuclei at our institution over a 10-year interval. We used a strict definition for cuplike nuclei, ≥10% blasts with nuclear invaginations ≥25% of the nuclear area. We reviewed relevant data and compared the results with a control group of AML without cuplike nuclei. RESULTS We identified 22 cases of AML with cuplike nuclei, classified as AML without maturation (FAB AML M1). Compared with a control group AML M1cases, AMLs with cuplike nuclei were significantly associated with FLT3-ITD mutations (86 vs. 38%, p=0.002), NPM1 mutations (86 vs. 19%, p<0.0001), both mutations (77% vs. 14%, p<0.0001), normal karyotype (86 vs. 40%, p=0.003), bone marrow blast count (90% vs. 84%, p=0.016), myeloperoxidase positivity (95% vs. 30% blasts, p=0.001), higher D-dimer levels (>5000 vs. 569 ng/mL, p=0.001), and absence of CD7 (91% vs. 52%, p=0.007), CD34 (82% vs. 5%, p<0.0001), and HLA-DR (59% vs. 10%, p=0.001). There were no differences in age, sex, or peripheral blood counts. The positive predictive value of recognizing AML with cuplike nuclei for FLT3-ITD, NPM1, and both mutations was 81%, 86%, and 77%, respectively. CONCLUSIONS Cuplike nuclei in AML are highly associated with the presence of NPM1 and FLT3-ITD mutations and a number of clinicopathologic and immunophenotypic features. The recognition of AML with cuplike nuclei may be helpful in streamlining the workup of these neoplasms. PMID:19672946

  13. [Correlation of adult AML Npm1 mutations with prognosis and its relationship with gene mutation of FLT3 and CEBPA].

    PubMed

    Bao, Li-Yan; Wang, Ji-Shi

    2010-02-01

    This study was aimed to investigate the correlation of 12th exon mutations in the npm1 gene with prognosis of adult AML patients and to explore the relationship of 12th exon mutation with other gene mutations. The specimen of bone marrow and peripheral blood from AML patients, the informations of medical history, symptoms, related image examinations, blood routine examination, NAP, oxygen saturation level in artery blood and EPO level in serum were collected; the bcr/abl fusion gene was detected by routine examination of bone marrow + biopsy + chromosome mapping + FISH. The patients were typed according to WHO classification. The DNA in cells was extracted, the npm1 gene mutation was detected by allele specific PCR combined were the sequencing. The results indicated that the npm1 heterozygote gene mutation was found in 72 out of 150 AML patients with normal cytogenetics (48%, 72/150). 48% patients showed a frameshift mutation in the C-terminal region of the NPM1 protein. The AML patients with npm1 gene mutation had specific clinical, phenotypic and genetic characteristics. The statistical analysis demonstrated the relationship between npm1 and flt3 ITDs. The patients with npm1 mutation showed a better response to induction therapy, furthermore, the overall survival (OS) rate of patients without flt3 ITD mutation was enhanced. The multivariate analysis demonstrated that the npm1 gene mutation and cebpa mutation positively correlated to the OS rate, and the correlation of flt3 mutation to OS rate showed negative. It is concluded that npm1 mutation is a favorable independent prognostic factor for adult AML patients with normal cytogenetics under conditions without FIT3 gene mutation. PMID:20137111

  14. Prognostic significance of FLT3 internal tandem duplication and NPM1 mutations in acute myeloid leukemia in an unselected patient population.

    PubMed

    Suzuki, Rikio; Onizuka, Makoto; Kojima, Minoru; Shimada, Masako; Okamura, Kaori; Fukagawa, Satomi; Tsuboi, Kosuke; Kikuchi, Ako; Kobayashi, Hiroyuki; Shintani, Ayumi; Ogawa, Yoshiaki; Kawada, Hiroshi; Hotta, Tomomitsu; Ando, Kiyoshi

    2007-12-01

    Mutations in the fms-like tyrosine kinase 3 (FLT3) gene containing an internal tandem duplication (FLT3/ITD) or mutations in the nucleophosmin 1 gene (NPM1) are thought to be prognostic indicators in acute myeloid leukemia (AML). Previous studies suggested that FLT3/ITD mutation indicates a poor prognosis and that NPM1 mutation indicates a more favorable one, but these studies were often performed with selected patient populations. We investigated the clinical significance of these mutations at our institution with an unselected group of patients with newly diagnosed AML. This group included patients > or =60 years old and those with a poor performance status. Using polymerase chain reaction and sequencing analyses, we detected FLT3/ITD mutations in 12 patients (20.0%) and NPM1 mutations in 7 patients (11.7%) among a group of 60 patients. There was a nonsignificant trend for FLT3/ITD mutation to be associated with a poorer predicted overall survival (OS) probability in this population. In contrast, OS was significantly higher in patients with wild-type NPM1 than in patients with NPM1 mutation, both for all AML patients and for AML patients with a normal karyotype. In this general and unselected AML patient population, NPM1 mutation was not a prognostic indicator of a favorable outcome. PMID:18192111

  15. MiR-424 and miR-155 deregulated expression in cytogenetically normal acute myeloid leukaemia: correlation with NPM1 and FLT3 mutation status

    PubMed Central

    2012-01-01

    Background MicroRNA have a central role in normal haematopoiesis and are deregulated in acute myeloid leukaemia (AML). The purpose of the study was to investigate by qRT-PCR the expression of miRNAs involved in myeloid differentiation (miR-424, miR-155, miR-223, miR-17-5p) in 48 patients with cytogenetically normal AML well characterized for NPM1 and/or FLT3 mutations. Three types of normalization were used for the data validation. Findings We found that miR-424 was down-modulated in AMLs with NPM1mutA regardless of FLT3 status. On the contrary, miR-155 showed up-regulation in patients with FLT3 internal tandem duplications (ITD) with or without NPM1 mutations. No significant associations were found by analyzing miR-223 and miR-17-5p in relation to FLT3 and NPM1 status. Conclusions This study supports the view that major genetic subsets of CN-AML are associated with distinct miRNA signatures and suggests that miR-424 and miR-155 deregulation is involved in the pathogenesis of CN-AML with NPM1 and FLT3-ITD mutations, respectively. PMID:22681934

  16. Metformin synergistically sensitizes FLT3-ITD-positive acute myeloid leukemia to sorafenib by promoting mTOR-mediated apoptosis and autophagy.

    PubMed

    Wang, Fangfang; Liu, Zuofeng; Zeng, Jisha; Zhu, Hongyan; Li, Jingjing; Cheng, Xiaomin; Jiang, Tao; Zhang, Li; Zhang, Chuanfen; Chen, Tie; Liu, Ting; Jia, Yongqian

    2015-12-01

    Mutations of Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), accounting for approximately 30% of patients with acute myeloid leukemia (AML), results in poor therapeutic efficacy and short survival. Sorafenib, an oral multikinase inhibitor, can inhibit FLT3 and improve clinical outcome of FLT3 mutated leukemia. Our current studies have shown that, the antidiabetic drug metformin also exerts anti-leukemic effect by activating p-AMPK and synergistically sensitizes FLT3 mutated AML to sorafenib. Both agents suppress cell proliferation in a dose-dependent manner and induce apoptosis via cell cycle arrest, but does not obviously modulate autophagy marker, light chain 3 (LC3). Mechanistically, in the presence of metformin, the anticancer potential of sorafenib, accompanying with increased LC3 levels, is found to be synergistically enhanced with the remarkably reduced protein expression of the mTOR/p70S6K/4EBP1 pathway, while not appreciably altering cell cycle. Overall, these results show metformin in aid of sorafenib may represent a promising and attractive strategy for the treatment of FLT3-ITD mutated AML. PMID:26505133

  17. Evaluation of Docking Target Functions by the Comprehensive Investigation of Protein-Ligand Energy Minima

    PubMed Central

    Oferkin, Igor V.; Katkova, Ekaterina V.; Sulimov, Alexey V.; Kutov, Danil C.; Sobolev, Sergey I.; Voevodin, Vladimir V.; Sulimov, Vladimir B.

    2015-01-01

    The adequate choice of the docking target function impacts the accuracy of the ligand positioning as well as the accuracy of the protein-ligand binding energy calculation. To evaluate a docking target function we compared positions of its minima with the experimentally known pose of the ligand in the protein active site. We evaluated five docking target functions based on either the MMFF94 force field or the PM7 quantum-chemical method with or without implicit solvent models: PCM, COSMO, and SGB. Each function was tested on the same set of 16 protein-ligand complexes. For exhaustive low-energy minima search the novel MPI parallelized docking program FLM and large supercomputer resources were used. Protein-ligand binding energies calculated using low-energy minima were compared with experimental values. It was demonstrated that the docking target function on the base of the MMFF94 force field in vacuo can be used for discovery of native or near native ligand positions by finding the low-energy local minima spectrum of the target function. The importance of solute-solvent interaction for the correct ligand positioning is demonstrated. It is shown that docking accuracy can be improved by replacement of the MMFF94 force field by the new semiempirical quantum-chemical PM7 method. PMID:26693223

  18. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  19. Development of a successive targeting liposome with multi-ligand for efficient targeting gene delivery

    PubMed Central

    Ma, Kun; Shen, Haijun; Shen, Song; Xie, Men; Mao, Chuanbin; Qiu, Liyan; Jin, Yi

    2012-01-01

    Background A successful gene delivery system needs to breakthrough several barriers to allow efficient transgenic expression. In the present study, successive targeting liposomes (STL) were constructed by integrating various targeting groups into a nanoparticle to address this issue. Methods Polyethylenimine (PEI) 1800-triamcinolone acetonide (TA) with nuclear targeting capability was synthesized by a two-step reaction. Lactobionic acid was connected with cholesterol to obtain a compound of [(2-lactoylamido) ethylamino]formic acid cholesterol ester (CHEDLA) with hepatocyte-targeting capability. The liposome was modified with PEI 1800-TA and CHEDLA to prepare successive targeting liposome (STL). Its physicochemical properties and transfection efficiency were investigated both in vitro and in vivo. Results The diameter of STL was approximately 100 nm with 20 mV of potential. The confocal microscopy observation and potential assay verified that lipid bilayer of STL was decorated with PEI 1800-TA. Cytotoxicity of STL was significantly lower than that of PEI 1800-TA and PEI 25K. The transfection efficiency of 10% CHEDLA STL in HepG2 cells was the higher than of the latter two with serum. Its transfection efficiency was greatly reduced with excessive free galactose, indicating that STL was absorbed via galactose receptor-mediated endocytosis. The in vivo study in mice showed that 10% CHEDLA STL had better transgenic expression in liver than the other carriers. Conclusions STL with multi-ligand was able to overcome the various barriers to target nucleus and special cells and present distinctive transgenic expression. Therefore, it has a great potential for gene therapy as a nonviral carrier. PMID:21574214

  20. Cellular Protection using Flt3 and PI3Kα inhibitors demonstrates multiple mechanisms of oxidative glutamate toxicity

    PubMed Central

    Kang, Yunyi; Tiziani, Stefano; Park, Goonho; Kaul, Marcus; Paternostro, Giovanni

    2014-01-01

    Glutamate-induced oxidative stress is a major contributor to neurodegenerative diseases. Here we identify small molecule inhibitors of this process. We screen a kinase inhibitor library on neuronal cells and identify Flt3 and PI3Kα inhibitors as potent protectors against glutamate toxicity. Both inhibitors prevented reactive oxygen species (ROS) generation, mitochondrial hyperpolarization, and lipid peroxidation in neuronal cells, but they do so by distinct molecular mechanisms. The PI3Kα inhibitor protects cells by inducing partial restoration of depleted glutathione levels and accumulation of intracellular amino acids, whereas the Flt3 inhibitor prevents lipid peroxidation, a key mechanism of glutamate-mediated toxicity. We also demonstrate that glutamate toxicity involves a combination of ferroptosis, necrosis, and AIF-dependent apoptosis. We confirm the protective effect by using multiple inhibitors of these kinases and multiple cell types. Our results not only identify compounds that protect against glutamate-stimulated oxidative stress, but also provide new insights into the mechanisms of glutamate toxicity in neurons. PMID:24739485

  1. Theranostic Nanoparticles Carrying Doxorubicin Attenuate Targeting Ligand Specific Antibody Responses Following Systemic Delivery

    PubMed Central

    Yang, Emmy; Qian, Weiping; Cao, Zehong; Wang, Liya; Bozeman, Erica N.; Ward, Christina; Yang, Bin; Selvaraj, Periasamy; Lipowska, Malgorzata; Wang, Y. Andrew; Mao, Hui; Yang, Lily

    2015-01-01

    Understanding the effects of immune responses on targeted delivery of nanoparticles is important for clinical translations of new cancer imaging and therapeutic nanoparticles. In this study, we found that repeated administrations of magnetic iron oxide nanoparticles (IONPs) conjugated with mouse or human derived targeting ligands induced high levels of ligand specific antibody responses in normal and tumor bearing mice while injections of unconjugated mouse ligands were weakly immunogenic and induced a very low level of antibody response in mice. Mice that received intravenous injections of targeted and polyethylene glycol (PEG)-coated IONPs further increased the ligand specific antibody production due to differential uptake of PEG-coated nanoparticles by macrophages and dendritic cells. However, the production of ligand specific antibodies was markedly inhibited following systemic delivery of theranostic nanoparticles carrying a chemotherapy drug, doxorubicin. Targeted imaging and histological analysis revealed that lack of the ligand specific antibodies led to an increase in intratumoral delivery of targeted nanoparticles. Results of this study support the potential of further development of targeted theranostic nanoparticles for the treatment of human cancers. PMID:25553097

  2. Mutation analysis of JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in Chinese patients with myeloproliferative neoplasms.

    PubMed

    Wang, Min; He, Na; Tian, Tian; Liu, Lu; Yu, Shuang; Ma, Daoxin

    2014-01-01

    Since the discovery of JAK2V617F tyrosine kinase-activating mutation, several genes have been found mutated in myeloproliferative neoplasms (MPNs). FLT3-ITD, NPM1, and DNMT3A mutations frequently occurred in AML patients and have been found conferred with myeloproliferative neoplasms in mouse model. Therefore, we sought to search for mutations in JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in 129 cases including 120 classic MPN cases and 9 MDS/MPN cases. JAK2V617F mutation was found in 60% of the 120 classic MPNs. However, none of the patients displayed FLT3-ITD and NPM1 mutations; only 2 patients harbored DNMT3A R882 mutation. Further studies including whole-genome sequence will be conducted to investigate the possible involvement of these genes in MPN. PMID:24895580

  3. Mutation Analysis of JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in Chinese Patients with Myeloproliferative Neoplasms

    PubMed Central

    Wang, Min; He, Na; Tian, Tian; Liu, Lu; Yu, Shuang; Ma, Daoxin

    2014-01-01

    Since the discovery of JAK2V617F tyrosine kinase-activating mutation, several genes have been found mutated in myeloproliferative neoplasms (MPNs). FLT3-ITD, NPM1, and DNMT3A mutations frequently occurred in AML patients and have been found conferred with myeloproliferative neoplasms in mouse model. Therefore, we sought to search for mutations in JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in 129 cases including 120 classic MPN cases and 9 MDS/MPN cases. JAK2V617F mutation was found in 60% of the 120 classic MPNs. However, none of the patients displayed FLT3-ITD and NPM1 mutations; only 2 patients harbored DNMT3A R882 mutation. Further studies including whole-genome sequence will be conducted to investigate the possible involvement of these genes in MPN. PMID:24895580

  4. [The efficacy of sorafenib to prevent relapse in patients with FLT3-ITD mutation positive acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation].

    PubMed

    Zu, Y L; Zhang, Y L; Zhou, J; Han, L J; Zhao, H F; Gui, R R; Hou, Y J; Song, Y P

    2016-08-01

    To study the efficacy of sorafenib to prevent relapse in patients with FLT3-ITD mutation positive acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 7 cases with FLT3-ITD positive AML have received allo-HSCT in our department from May 2013 to January 2015. Six cases were administrated with sorafenib after hematopoietic reconstruction. Another patient relapsed on day 192 past allo-HSCT, then she started to use sorafenib after remission of re-induction regimens. Five patients survived. The median progression free survival was 280(126-366)day. This study suggests that sorafenib might prevent relapse past allo-HSCT and improve survival in patients with FLT3-ITD positive AML. PMID:27480560

  5. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid

    PubMed Central

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php PMID:26896846

  6. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid.

    PubMed

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php. PMID:26896846

  7. FMS-Like Tyrosine Kinase 3 Ligand Treatment of Mice Aggravates Acute Lung Injury in Response to Streptococcus pneumoniae: Role of Pneumolysin

    PubMed Central

    Brumshagen, Christina; Maus, Regina; Bischof, Andrea; Ueberberg, Bianca; Bohling, Jennifer; Osterholzer, John J.; Ogunniyi, Abiodun D.; Paton, James C.; Welte, Tobias

    2012-01-01

    FMS-like tyrosine kinase-3 ligand (Flt3L) is a dendritic cell (DC) growth and differentiation factor with potential in antitumor therapies and antibacterial immunization strategies. However, the effect of systemic Flt3L treatment on lung-protective immunity against bacterial infection is incompletely defined. Here, we examined the impact of deficient (in Flt3L knockout [KO] mice), normal (in wild-type [WT] mice), or increased Flt3L availability (in WT mice pretreated with Flt3L for 3, 5, or 7 days) on lung DC subset profiles and lung-protective immunity against the major lung-tropic pathogen, Streptococcus pneumoniae. Although in Flt3L-deficient mice the numbers of DCs positive for CD11b (CD11bpos DCs) and for CD103 (CD103pos DCs) were diminished, lung permeability, a marker of injury, was unaltered in response to S. pneumoniae. In contrast, WT mice pretreated with Flt3L particularly responded with increased numbers of CD11bpos DCs and with less pronounced numbers of CD103pos DCs and impaired bacterial clearance and with increased lung permeability following S. pneumoniae challenge. Notably, infection of Flt3L-pretreated mice with S. pneumoniae lacking the pore-forming toxin, pneumolysin (PLY), resulted in substantially less lung CD11bpos DCs activation and reduced lung permeability. Collectively, this study establishes that Flt3L treatment enhances the accumulation of proinflammatory activated lung CD11bpos DCs which contribute to acute lung injury in response to PLY released by S. pneumoniae. PMID:23006850

  8. PPAR Ligands Function as Suppressors That Target Biological Actions of HMGB1

    PubMed Central

    Chen, Tianhui

    2016-01-01

    High mobility group box 1 (HMGB1), which has become one of the most intriguing molecules in inflammatory disorders and cancers and with which ligand-activated peroxisome proliferator-activated receptors (PPARs) are highly associated, is considered as a therapeutic target. Of particular interest is the fact that certain PPAR ligands have demonstrated their potent anti-inflammatory activities and potential anticancer effects. In this review article we summarize recent experimental evidence that PPAR ligands function as suppressors that target biological actions of HMGB1, including intracellular expression, receptor signaling cascades, and extracellular secretion of HMGB1 in cell lines and/or animal models. We also propose the possible mechanisms underlying PPAR involvement in inflammatory disorders and discuss the future therapeutic value of PPAR ligands targeting HMGB1 molecule for cancer prevention and treatment. PMID:27563308

  9. Immunophenotyping with CD135 and CD117 predicts the FLT3, IL-7R and TLX3 gene mutations in childhood T-cell acute leukemia.

    PubMed

    Noronha, Elda Pereira; Andrade, Francianne Gomes; Zampier, Carolina; de Andrade, Camilla F C G; Terra-Granado, Eugênia; Pombo-de-Oliveira, Maria S

    2016-03-01

    With the combination of immunophenotyping and molecular tests, it is still a challenge to identify the characteristics of T cell acute lymphoblastic leukemia (T-ALL) associated with distinct outcomes. This study tests the possible correlation of cellular expression of CD135 and CD117 with somatic gene mutations in T-ALL. One hundred sixty-two samples were tested, including 143 at diagnosis, 15 from T-lymphoblastic lymphoma at relapse, and four relapse samples from sequential follow-up of T-ALL. CD135 and CD117 monoclonal antibodies were included in the T-ALL panel of flow cytometry. The percentage of cells positivity and the median fluorescence intensity were correlated with gene mutational status. STIL-TAL1, TLX3, FLT3 and IL7R mutations were tested using standard techniques. STIL-TAL1 was found in 24.8%, TLX3 in 12%, IL7R in 10% and FLT3-ITD in 5% of cases. FLT3 and IL7R mutations were mutually exclusive, as were FLT3-ITD and STIL-TAL1. Associations of CD135(high) (p<0.01), CD117(intermediate/high) (p=0.02) and FLT3-ITD, CD117(low) with IL7R(mutated) (p<0.01) and CD135(high) with TLX3(pos) were observed. We conclude that the addition of CD135 and CD117 to the diagnosis can predict molecular aberrations in T-ALL settings, mainly segregating patients with FLT3-ITD, who would benefit from treatment with inhibitors of tyrosine. PMID:26852660

  10. Role of autologous hematopoietic stem cell transplantation according to the NPM1/FLT3-ITD molecular status for cytogenetically normal AML patients: a GOELAMS study.

    PubMed

    Guièze, Romain; Cornillet-Lefebvre, Pascale; Lioure, Bruno; Blanchet, Odile; Pigneux, Arnaud; Recher, Christian; Bonmati, Caroline; Fegueux, Nathalie; Bulabois, Claude-Eric; Bouscary, Didier; Vey, Norbert; Delain, Martine; Turlure, Pascal; Himberlin, Chantal; Harousseau, Jean-Luc; Dreyfus, Francois; Béné, Marie C; Ifrah, Norbert; Chevallier, Patrice

    2012-12-01

    The choice of postremission therapy for acute myeloid leukemia (AML) patients is now based on the blasts' cytogenetic and molecular profile. However, the potential benefit of autologous hematopoietic stem cell transplantation (auto-HSCT) according to the NPM1/FLT3-ITD status has been poorly studied in AML patients with a normal karyotype (NK). Therefore, we evaluated the NPM1/FLT3-ITD molecular status in 135 NK-AML patients treated by allogeneic HSCT (allo-HSCT), auto-HSCT, or chemotherapy as consolidation therapy within the GOELAMS LAM-2001 trial. In univariate analyzes, 4-year leukemia-free survival (LFS) and overall survival (OS) were significantly higher for NPM1+/FLT3-ITD- patients compared with patients presenting another molecular profile (61 vs. 43% and 72 vs. 48%, P = 0.02 and P = 0.01, respectively). In the NPM1+/FLT3-ITD- subgroup, there was no benefit for allo-HSCT or auto-HSCT vs. chemotherapy (4-year LFS: 71, 56, and 60%; 4-year OS: 73, 71, and 60%, respectively; P = NS). For patients with other NPM1/FLT3-ITD molecular profiles, allo-HSCT was found to be the best consolidation therapy, whereas auto-HSCT was associated with a better outcome when compared with chemotherapy (allo-HSCT-, auto-HSCT-, and chemotherapy-related 4-year LFS: 68, 44, and 36%, P = 0.004; 4-year OS: 68, 52, and 29%, respectively, P = 0.02). Our study indicates that allo-HSCT and auto-HSCT provide similar outcomes compared with chemotherapy as consolidation for NPM1+/FLT3-ITD- NK-AML patients. For NK-AML patients with an adverse molecular profile, auto-HSCT could represent an alternative therapeutic approach when no human leukocyte antigen-matched allogeneic donor is available. PMID:22911473

  11. Establishment of xenotransplantation model of human CN-AML with FLT3-ITD (mut) /NPM1 (-) in NOD/SCID mice.

    PubMed

    Shang, Zhen; Wang, Jue; Wang, Di; Xiao, Min; Li, Tong-juan; Wang, Na; Huang, Liang; Zhou, Jian-feng

    2013-06-01

    Patients with FLT3-ITD (mut) /NPM1 (-) cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD (mut) /NPM1 (-) CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells. The FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD (mut) /NPM1 (-) CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies. PMID:23771655

  12. Multifunctional Transmembrane Protein Ligands for Cell-Specific Targeting of Plasma Membrane-Derived Vesicles.

    PubMed

    Zhao, Chi; Busch, David J; Vershel, Connor P; Stachowiak, Jeanne C

    2016-07-01

    Liposomes and nanoparticles that bind selectively to cell-surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single-domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering. PMID:27294846

  13. Key Targets for Multi-Target Ligands Designed to Combat Neurodegeneration

    PubMed Central

    Ramsay, Rona R.; Majekova, Magdalena; Medina, Milagros; Valoti, Massimo

    2016-01-01

    HIGHLIGHTS Compounds that interact with multiple targets but minimally with the cytochrome P450 system (CYP) address the many factors leading to neurodegeneration.Acetyl- and Butyryl-cholineEsterases (AChE, BChE) and Monoamine Oxidases A/B (MAO A, MAO B) are targets for Multi-Target Designed Ligands (MTDL).ASS234 is an irreversible inhibitor of MAO A >MAO B and has micromolar potency against the cholinesterases.ASS234 is a poor CYP substrate in human liver, yielding the depropargylated metabolite.SMe1EC2, a stobadine derivative, showed high radical scavenging property, in vitro and in vivo giving protection in head trauma and diabetic damage of endothelium.Control of mitochondrial function and morphology by manipulating fission and fusion is emerging as a target area for therapeutic strategies to decrease the pathological outcome of neurodegenerative diseases. Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to “dirty drugs” for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson's Disease's (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are

  14. Key Targets for Multi-Target Ligands Designed to Combat Neurodegeneration.

    PubMed

    Ramsay, Rona R; Majekova, Magdalena; Medina, Milagros; Valoti, Massimo

    2016-01-01

    HIGHLIGHTS Compounds that interact with multiple targets but minimally with the cytochrome P450 system (CYP) address the many factors leading to neurodegeneration.Acetyl- and Butyryl-cholineEsterases (AChE, BChE) and Monoamine Oxidases A/B (MAO A, MAO B) are targets for Multi-Target Designed Ligands (MTDL).ASS234 is an irreversible inhibitor of MAO A >MAO B and has micromolar potency against the cholinesterases.ASS234 is a poor CYP substrate in human liver, yielding the depropargylated metabolite.SMe1EC2, a stobadine derivative, showed high radical scavenging property, in vitro and in vivo giving protection in head trauma and diabetic damage of endothelium.Control of mitochondrial function and morphology by manipulating fission and fusion is emerging as a target area for therapeutic strategies to decrease the pathological outcome of neurodegenerative diseases. Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to "dirty drugs" for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson's Disease's (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are simply

  15. Rational and Modular Design of Potent Ligands Targeting the RNA that Causes Myotonic Dystrophy 2

    PubMed Central

    Lee, Melissa M.; Pushechnikov, Alexei; Disney, Matthew D.

    2009-01-01

    Most ligands targeting RNA are identified through screening a therapeutic target for binding members of a ligand library. A potential alternative way to construct RNA binders is through rational design using information about the RNA motifs ligands prefer to bind. Herein, we describe such an approach to design modularly assembled ligands targeting the RNA that causes myotonic dystrophy type 2 (DM2), a currently untreatable disease. A previous study identified that 6′-N-5-hexynoate kanamycin A (1) prefers to bind 2×2 nucleotide, pyrimidine-rich RNA internal loops. Multiple copies of such loops were found in the RNA hairpin that causes DM2. The 1 ligand was then modularly displayed on a peptoid scaffold with varied number and spacing to target several internal loops simultaneously. Modularly assembled ligands were tested for binding to a series of RNAs and for inhibiting the formation of the toxic DM2 RNA-muscleblind protein (MBNL-1) interaction. The most potent ligand displays three 1 modules, each separated by four spacing submonomers, and inhibits the formation of the RNA-protein complex with an IC50 of 25 nM. This ligand is higher affinity and more specific for binding DM2 RNA than MBNL-1. It binds the DM2 RNA at least 20-times more tightly than related RNAs and 15-fold more tightly than MBNL-1. A related control peptoid displaying 6′-N-5-hexynoate neamine (2) is >100-fold less potent at inhibiting the RNA-protein interaction and binds to DM2 RNA >125-fold more weakly. Uptake studies into a mouse myoblast cell line also show that the most potent ligand is cell permeable. PMID:19348464

  16. Potent graft-versus-leukemia effect after reduced-intensity allogeneic SCT for intermediate-risk AML with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD.

    PubMed

    Labouré, Gaëlle; Dulucq, Stéphanie; Labopin, Myriam; Tabrizi, Reza; Guérin, Estelle; Pigneux, Arnaud; Lafarge, Xavier; Leguay, Thibaut; Bouabdallah, Krimo; Dilhuydy, Marie-Sarah; Duclos, Cédric; Lascaux, Axelle; Marit, Gérald; Mahon, François-Xavier; Boiron, Jean-Michel; Milpied, Noël; Vigouroux, Stéphane

    2012-12-01

    To investigate the role of reduced-intensity allogeneic (RIC-allo) stem cell transplant (SCT) as postremission therapy in adult intermediate-risk patients with acute myelogenous leukemia (AML) with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD, we conducted a single-center retrospective study between January 2001 and December 2010. Sixty-six patients were included: 37 treated with RIC-alloSCT and 29 with nonallogeneic SCT therapies. Both groups were comparable concerning age, WBC count at diagnosis, gender, karyotype, genotype, and number of courses of chemotherapy to reach complete remission (CR1). Median follow-up after CR1 was 37 months (range, 11-112 months) and 48 months (range, 9-83 months) in the allo and no-allo groups, respectively. In the allo versus no-allo groups, the 3-year cumulative incidence of relapse (CIR) rates were 25% ± 8% versus 61% ± 9%; P = .005. The 3-year nonrelapse mortality (NRM), overall survival (OS), and relapse-free survival (RFS) were 22% ± 7% versus 4% ± 4% (P = .005), 52% ± 9% versus 44% ± 10% (P = .75), and 53% ± 9% versus 35% ± 9% (P = .28), respectively. Multivariate analysis indicated that CIR was reduced by allo (hazard ratio [HR], 0.32; P = .01). A landmark analysis performed at day 185 after CR1 confirmed a lower CIR after allo. RIC-allo reduces the risk of relapse, suggesting a potent graft-versus-leukemia (GVL) effect in these patients at a high risk of relapse. PMID:22766221

  17. 3,3'-Diindolylmethane Inhibits Flt3L/GM-CSF-induced-bone Marrow-derived CD103+ Dendritic Cell Differentiation Regulating Phosphorylation of STAT3 and STAT5

    PubMed Central

    Choi, Ah-Jeong; Kim, Soo-Ji; Jeong, So-Yeon

    2015-01-01

    The intestinal immune system maintains oral tolerance to harmless antigens or nutrients. One mechanism of oral tolerance is mediated by regulatory T cell (Treg)s, of which differentiation is regulated by a subset of dendritic cell (DC)s, primarily CD103+ DCs. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, plays an important role in regulating immunity. The intestines are exposed to various AhR ligands, including endogenous metabolites and phytochemicals. It was previously reported that AhR activation induced tolerogenic DCs in mice or in cultures of bone marrow-derived DCs. However, given the variety of tolerogenic DCs, which type of tolerogenic DCs is regulated by AhR remains unknown. In this study, we found that AhR ligand 3,3'-diindolylmethane (DIM) inhibited the development of CD103+ DCs from mouse bone marrow cells stimulated with Flt3L and GM-CSF. DIM interfered with phosphorylation of STAT3 and STAT5 inhibiting the expression of genes, including Id2, E2-2, IDO-1, and Aldh1a2, which are associated with DC differentiation and functions. Finally, DIM suppressed the ability of CD103+ DCs to induce Foxp3+ Tregs. PMID:26770182

  18. Multi-ligand functionalized particle design for cell targeting and drug delivery.

    PubMed

    Yoon, Jung Hyun; Kim, Dae Kyung; Na, Miso; Lee, Sei Young

    2016-06-01

    Particle-based delivery systems encompass some of the most promising techniques for therapeutic drug delivery. In particular, multi-functional nanovector systems permit diverse functions such as efficient drug/imaging agent loading and unloading and increased target specificity. To enhance the efficiency of delivery systems, particle size and shape can be altered and specific ligands can be conjugated to the particles to promote interactions with receptors expressed on target cells. Moreover, to maximize efficiency and specificity, multiple types of ligands can be conjugated to the particle surface. To analyze the multi-ligand-receptor mediated adhesion process, we developed a stochastic model considering diverse biophysical parameters, including non-specific interactions, ligand-receptor specific interactions, kinetic affinity between ligand and receptor, hydrodynamic force and particle size. The results demonstrate that limited contact area restricts the probability of adhesion such that multiple ligand-receptor pairs do not always show enhanced adhesion characteristics. To optimize the effect of multiple ligand-receptor pairs, biophysical parameters must be considered. PMID:27100957

  19. Novel ligands that target the mitochondrial membrane protein mitoNEET

    PubMed Central

    Bieganski, Robert M.; Yarmush, Martin L.

    2012-01-01

    Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET. PMID:21531159

  20. Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia.

    PubMed

    Ånensen, N; Hjelle, S M; Van Belle, W; Haaland, I; Silden, E; Bourdon, J-C; Hovland, R; Taskén, K; Knappskog, S; Lønning, P E; Bruserud, Ø; Gjertsen, B T

    2012-03-22

    The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms β and γ expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication. PMID:21860418

  1. Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes

    PubMed Central

    Machado-Neto, João Agostinho; Traina, Fabiola; Lazarini, Mariana; de Melo Campos, Paula; Pagnano, Katia Borgia Barbosa; Lorand-Metze, Irene; Costa, Fernando Ferreira; Olalla Saad, Sara T

    2011-01-01

    INTRODUCTION: Myelodysplastic syndromes encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress toward acute myeloid leukemia. The accumulation of genetic alterations is closely associated with the progression of myelodysplastic syndromes toward acute myeloid leukemia. OBJECTIVE: To investigate the presence of mutations in the points most frequent for mutations (hotspot mutations) in phosphatidylinositol-3-kinase (PI3K), Janus kinase 2 (JAK2), FMS-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1), which are involved in leukemia and other cancers, in a population of Brazilian MDS patients. METHODS: Fifty-one myelodysplastic syndromes patients were included in the study. According to French-American-British classification, the patients were distributed as follows: 31 with refractory anemia, 8 with refractory anemia with ringed sideroblasts, 7 with refractory anemia with excess blasts, 3 with refractory anemia with excess blasts in transformation and 2 with chronic myelomonocytic leukemia. Bone marrow samples were obtained and screened for the presence of hotspot mutations using analysis based on amplification with the polymerase chain reaction, sequencing, fragment size polymorphisms or restriction enzyme digestion. All patients were screened for mutations at the time of diagnosis, and 5 patients were also screened at the time of disease progression. RESULTS: In the genes studied, no mutations were detected in the patients at the time of diagnosis. One patient with chronic myelomonocytic leukemia was heterozygous for a Janus kinase 2 mutation after disease progression. CONCLUSIONS: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression. PMID:21789382

  2. Case report of FLT3-ITD-positive AML patient 11 years after living donor liver transplantation.

    PubMed

    Taniai, N; Yoshida, H; Kawano, Y; Uchida, E

    2014-04-01

    With the increasing number of long-term survivors of living donor liver transplantation, the occurrence of secondary cancer is sometimes reported. Solid tumors such as lymphomas are mainly observed. However, only 8 cases of leukemia have been reported so far. For patients younger than 15 years old, leukemia developed in 4 within 3 years after the liver transplantation, whereas acute lymphoblastic leukemia developed in only 1 patient. This is the first case report of a patient in whom FLT3-ITD-positive acute myeloid leukemia (AML) developed more than 10 years after living donor liver transplantation for congenital biliary atresia. AML developed in a 14-year-old boy 11 years after living donor liver transplantation from his father. The patient received the transplant at the age of 3 years and was treated with tacrolimus and methylprednisolone for transplant rejection. Eleven years posttransplantation, he visited the hospital with general malaise and anemia. Blood tests revealed an elevated white blood cell count of 60,100/μL, and the patient was diagnosed with AML. Chromosome analysis revealed a t(6; 9) (p23 q34) translocation; moreover, genetic testing revealed a FLT3-ITD-positive mutation. We started treatment in accordance with the Tokyo Children's Cancer Study Group AML99 protocol. With chemotherapy treatment, the patient achieved complete remission. After chemotherapy, we performed stem cell transplantation from his father. Other patients were reported in relatively early stages after liver transplantation, but our case was more than 10 years posttransplantation. The association with the onset of congenital bile duct atresia and leukemia is still not clear, but we consider the possibility that long-term immunosuppressive drugs contribute to developing leukemia. PMID:24767404

  3. NPM1 mutations are more stable than FLT3 mutations during the course of disease in patients with acute myeloid leukemia.

    PubMed

    Palmisano, Michela; Grafone, Tiziana; Ottaviani, Emanuela; Testoni, Nicoletta; Baccarani, Michele; Martinelli, Giovanni

    2007-09-01

    NPM1 mutations have been reported to be the most frequent mutations in acute myeloid leukemia (AML). They are associated with a wide spectrum of morphologic subtypes of AML, normal karyotype and FLT3 mutations. The high frequency of NPM1 mutations might provide a suitable marker for monitoring residual disease of AML. PMID:17768124

  4. The value of molecular stratification for CEBPA(DM) and NPM1(MUT) FLT3(WT) genotypes in older patients with acute myeloid leukaemia.

    PubMed

    Dickson, Glenda J; Bustraan, Sophia; Hills, Robert K; Ali, Akbar; Goldstone, Anthony H; Burnett, Alan K; Linch, David C; Gale, Rosemary E

    2016-02-01

    Older adult patients (≥60 years) with acute myeloid leukaemia (AML) are generally considered to be poor-risk and there is limited information available regarding risk stratification based on molecular characterization in this age group, particularly for the double-mutant CEBPA (CEBPA(DM) ) genotype. To investigate whether a molecular favourable-risk genotype can be identified, we investigated CEBPA, NPM1 and FLT3 status and prognostic impact in a cohort of 301 patients aged 60 years or more with intermediate-risk cytogenetics, all treated intensively. Overall survival (OS) at 1 year was highest in the 12 patients (4%) that were CEBPA(DM) compared to the 76 (28%) with a mutant NPM1 and wild-type FLT3 (NPM1(MUT) FLT3(WT) ) genotype or all other patients (75%, 54%, 33% respectively), with median survival 15·2, 13·6 and 6·6 months, although the benefit was short-term (OS at 3 years 17%, 29%, 12% respectively). Combination of the CEBPA(DM) and NPM1(MUT) FLT3(WT) genotype patients defined a molecular group with favourable prognosis (P < 0·0001 in multivariate analysis), with 57% of patients alive at 1 year compared to 33% for all other patients. Knowledge of genotype in older cytogenetically intermediate-risk patients might influence therapy decisions. PMID:26847745

  5. AB119. Induction of suppressor of cytokine signaling-3 in FLT3-ITD positive MV4-11 acute myeloid leukemia cells in response to 5-Azacytidine and Trichostatin A

    PubMed Central

    Johan, Muhammad Farid; Jusoh, Siti Asmaa Mat

    2015-01-01

    Background and objective Suppressor of cytokine signaling-3 (SOCS-3) has been shown to be an important candidate in molecular therapeutic strategies in management of acute myeloid leukemia (AML), particularly in patients carrying FLT3-ITD mutation. SOCS-3 suppresses cytokine signalling by inhibiting the activity of Janus Kinase-2 (JAK-2), and by competing with signal transducer and activator of transcription (STAT) molecules that leads to underexpression. The study aims to determine the epigenetically silence genes in AML cells carrying a FLT3-ITD mutation and epigenetically expressed genes afer treatment with demethylating agent and histone deacetylase inhibitor. Methods MV4-11, a FLT3-ITD positive AML cell line was treated with epigenetic modulating agents; 5-azacytidine (5-Aza, a DNA demethylating agent) and Trichostatin A (TSA, a histone deacetylase inhibitor) at IC50 concentrations. One-Color Microarray-based expression analysis (Agilent SurePrint Technology) was utilized and the data was collected and analyzed by Genespring 12.6 software. The gene expression datasets were subjected to pathway analysis by online DAVID tool (http://david.abcc.ncifcrf.gov/) using KEGG pathway database. The microarray results were validated by quantitative real-time PCR to determine the relative quantification (RQ) values. Results Microarray analysis detected 1,291 expressed genes related to drug interactions. Pathway analysis by KEGG database revealed that the 1,291 genes were: 21 genes from MAPK pathway, 19 genes from pathways in cancers, 17 genes from cytokine-cytokine receptor interaction, 12 from focal adhesion, 12 from regulation of action cytoskeleton, 10 genes from JAK/STAT pathway, 10 genes from Calcium signalling and several other pathways with less than 10 genes involved. Among the 10 genes in JAK/STAT pathway, SOCS-3 was highly expressed in 5-Aza and TSA with 66.24 and 147.43 folds (Genespring analysis, Benjamini Hochberg, P<0.05), respectively compared to untreated

  6. Phage Peptide Libraries As a Source of Targeted Ligands

    PubMed Central

    Nemudraya, A. A.; Richter, V. A.; Kuligina, E. V.

    2016-01-01

    One of the dominant trends in modern pharmacology is the creation of drugs that act directly on the lesion focus and have minimal toxicity on healthy tissues and organs. This problem is particularly acute in relation to oncologic diseases. Short tissue- and organ-specific peptides capable of delivering drugs to the affected organ or tissue are considered promising targeted agents that can be used in the diagnosis and therapy of diseases, including cancer. The review discusses in detail the technology of phage display as a method for obtaining specific targeted peptide agents and offers examples of their use in diagnostic and clinical practice. PMID:27099784

  7. Improvement in clinical outcome of FLT3 ITD mutated acute myeloid leukemia patients over the last one and a half decade.

    PubMed

    Badar, Talha; Kantarjian, Hagop M; Nogueras-Gonzalez, Graciela M; Borthakur, Gautam; Garcia Manero, Guillermo; Andreeff, Michael; Konopleva, Marina; Kadia, Tapan M; Daver, Naval; Wierda, William G; Luthra, Raja; Patel, Keyur; Oran, Betul; Champlin, Richard; Ravandi, Farhad; Cortes, Jorge E

    2015-11-01

    AML with FLT3 ITD mutations are associated with poor outcome. We reviewed outcomes of patients with FLT3 ITD mutated AML to investigate trends over time. We analyzed 224 AML patients (excluding patients with core binding factor and acute promyelocytic leukemia) referred to our institution between 2000 and 2014. Patients were divided into five cohorts by era: 2000-2002 (Era 1, n = 19), 2003-2005 (Era 2, n = 41), 2006-2008 (Era 3, n = 53), 2009-2011 (Era 4, n = 55), and 2012-2014 (Era 5, n = 56) to analyze differences in outcome. The baseline characteristics were not statistically different across Eras. The response rate (CR/CRp) from Era 1-5 was 68%, 49%, 72%, 73%, and 75%, respectively. The overall response rate (all Eras) with chemotherapy alone versus chemotherapy plus FLT3 inhibitor was 67% and 72.5%, respectively (P = 0.4). The median time to relapse was 6, 3.6, 7.9, 8.1 months and not reached from Eras 1 through 5, respectively (P = 0.001). The median OS has improved: 9.6, 7.6, 14.4, 15.7, and 17.8 month from Eras 1-5, respectively (P = <0.001). Stem cell transplant as a time-dependent variable, showed better OS in the univariate analysis (HR: 0.57, 95% CI: 0.39-0.84, P = 0.004) but did not retained its significance in multivariate analysis (HR: 0.75, 95% CI: 0.50-1.13, P = 0.16). Our data suggest improvement in outcome of FLT3 ITD mutated AML patients over the last 15 years. This is probably due to improvement in treatment strategies, including but not limited to integration of FLT3 inhibitors and increased use of SCT. PMID:26299958

  8. The mammalian tachykinin ligand-receptor system: an emerging target for central neurological disorders

    PubMed Central

    Pantaleo, Nick; Chadwick, Wayne; Park, Sung-Soo; Wang, Liyun; Zhou, Yu; Martin, Bronwen; Maudsley, Stuart

    2010-01-01

    Our understanding of the complex signaling neurophysiology of the central nervous system has facilitated the exploration of potential novel receptor-ligand system targets for disorders of this most complex organ. In recent years, many relatively neglected receptor-ligand systems have been re-evaluated with respect to their ability to potently modulate discrete tracts in the central nervous system. One such system is the tachykinin (previously neurokinin) system. The multiple heptahelical G protein-coupled receptors and neuropeptide ligands that comprise this system may be significantly involved in more central nervous systems actions than previously thought, including sleep disorders, amyotrophic lateral sclerosis, Alzheimer’s and Machado-Joseph disease. The development of our understanding of the role of the tachykinin receptor-ligand system in higher order central functions is likely to allow the creation of more specific and selective tachykinin-related neurotherapeutics. PMID:20632965

  9. Aptamers: Active Targeting Ligands for Cancer Diagnosis and Therapy

    PubMed Central

    Wu, Xu; Chen, Jiao; Wu, Min; Zhao, Julia Xiaojun

    2015-01-01

    Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment. PMID:25699094

  10. A new target ligand Ser-Glu for PEPT1-overexpressing cancer imaging.

    PubMed

    Dai, Tongcheng; Li, Na; Zhang, Lingzhi; Zhang, Yuanxing; Liu, Qin

    2016-01-01

    Nanoparticles functionalized with active target ligands have been widely used for tumor-specific diagnosis and therapy. The target ligands include antibodies, peptides, proteins, small molecules, and nucleic acid aptamers. Here, we utilize dipeptide Ser-Glu (DIP) as a new ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for pancreatic cancer target imaging. We demonstrate that in the first step, Ser-Glu-conjugated NPs (NPs-DIP) efficiently bind to AsPC-1 and in the following NPs-DIP are internalized into AsPC-1 in vitro. The peptide transporter 1 inhibition experiment reveals that the targeting effects mainly depend on the specific binding of DIP to peptide transporter 1, which is remarkably upregulated in pancreatic cancer cells compared with varied normal cells. Furthermore, NPs-DIP specifically accumulate in the site of pancreatic tumor xenograft and are further internalized into the tumor cells in vivo after intravenous administration, indicating that DIP successfully enhanced nanoparticles internalization efficacy into tumor cells in vivo. This work establishes Ser-Glu to be a new tumor-targeting ligand and provides a promising tool for future tumor diagnostic or therapeutic applications. PMID:26811678