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Sample records for mll mixed lineage

  1. WDR5 Intearcts with Mixed Lineage Leukemia (MLL) Protein via the Histone H3-binding Pocket

    SciTech Connect

    Song, J.; Kingston, R

    2008-01-01

    WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.

  2. Photoperiod Influences Growth and mll (Mixed-Lineage Leukaemia) Expression in Atlantic Cod

    PubMed Central

    Nagasawa, Kazue; Giannetto, Alessia; Fernandes, Jorge M. O.

    2012-01-01

    Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. However, the molecular mechanisms underlying this phenomenon are currently unknown but it is likely that epigenetic regulation by methyltransferases is involved. The MLL (mixed-lineage leukaemia) family comprises histone methyltransferases that play a critical role in regulating gene expression during early development in mammals. So far, these genes have received scant attention in teleost fish. In the present study, the mean weight of Atlantic cod juveniles reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod conditions for 120 days. We newly determined cDNA sequences of five mll (mll1, mll2, mll3a, mll4b and mll5) and two setd1 (setd1a and setd1ba) paralogues from Atlantic cod. Phylogenetic analysis revealed that the cod genes clustered within the appropriate mll clade and comparative mapping of mll paralogues showed that these genes lie within a region of conserved synteny among teleosts. All mll and setd1 genes were highly expressed in gonads and fast muscle of adult cod, albeit at different levels, and they were differentially regulated with photoperiod in muscle of juvenile fish. Following only one day of exposure to constant light, mll1, mll4b and setd1a were up to 57% lower in these fish compared to the natural photoperiod group. In addition, mRNA expression of myogenic regulatory factors (myog and myf-5) and pax7 in fast muscle was also affected by different photoperiod conditions. Notably, myog was significantly elevated in the continuous illumination group throughout the time course of the experiment. The absence of a day/night cycle is associated with a generalised decrease in mll expression concomitant with an increase in myog transcript levels in fast muscle of Atlantic cod, which may be involved in the observed epigenetic regulation of growth by photoperiod in this species. PMID:22590633

  3. Crystal Structure of Menin Reveals Binding Site for Mixed Lineage Leukemia (MLL) Protein

    SciTech Connect

    Murai, Marcelo J.; Chruszcz, Maksymilian; Reddy, Gireesh; Grembecka, Jolanta; Cierpicki, Tomasz

    2014-10-02

    Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostella menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an {alpha}-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two {alpha}-helical bundles and covered by a {beta}-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.

  4. On the mechanism of multiple lysine methylation by the human mixed lineage leukemia protein-1 (MLL1) core complex.

    PubMed

    Patel, Anamika; Dharmarajan, Venkatasubramanian; Vought, Valarie E; Cosgrove, Michael S

    2009-09-01

    Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked approximately 600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex. PMID:19556245

  5. Mixed Lineage Leukemia 5 (MLL5) Protein Stability Is Cooperatively Regulated by O-GlcNac Transferase (OGT) and Ubiquitin Specific Protease 7 (USP7)

    PubMed Central

    Ding, Xiaodan; Jiang, Wei; Zhou, Peipei; Liu, Lulu; Wan, Xiaoling; Yuan, Xiujie; Wang, Xizi; Chen, Miao; Chen, Jun; Yang, Jing; Kong, Chao; Li, Bin; Peng, Chao; Wong, Catherine C. L.; Hou, Fajian; Zhang, Yan

    2015-01-01

    Mixed lineage leukemia 5 (MLL5) protein is a trithorax family histone 3 lysine 4 (H3K4) methyltransferase that regulates diverse biological processes, including cell cycle progression, hematopoiesis and cancer. The mechanisms by which MLL5 protein stability is regulated have remained unclear to date. Here, we showed that MLL5 protein stability is cooperatively regulated by O-GlcNAc transferase (OGT) and ubiquitin-specific protease 7 (USP7). Depletion of OGT in cells led to a decrease in the MLL5 protein level through ubiquitin/proteasome-dependent proteolytic degradation, whereas ectopic expression of OGT protein suppressed MLL5 ubiquitylation. We further identified deubiquitinase USP7 as a novel MLL5-associated protein using mass spectrometry. USP7 stabilized the MLL5 protein through direct binding and deubiquitylation. Loss of USP7 induced degradation of MLL5 protein. Conversely, overexpression of USP7, but not a catalytically inactive USP7 mutant, led to decreased ubiquitylation and increased MLL5 stability. Co-immunoprecipitation and co-immunostaining assays revealed that MLL5, OGT and USP7 interact with each other to form a stable ternary complex that is predominantly located in the nucleus. In addition, upregulation of MLL5 expression was correlated with increased expression of OGT and USP7 in human primary cervical adenocarcinomas. Our results collectively reveal a novel molecular mechanism underlying regulation of MLL5 protein stability and provide new insights into the functional interplay among O-GlcNAc transferase, deubiquitinase and histone methyltransferase. PMID:26678539

  6. Leukemogenic rearrangements at the mixed lineage leukemia gene (MLL)—multiple rather than a single mechanism

    PubMed Central

    Gole, Boris; Wiesmüller, Lisa

    2015-01-01

    Despite manifold efforts to achieve reduced-intensity and -toxicity regimens, secondary leukemia has remained the most severe side effect of chemotherapeutic cancer treatment. Rearrangements involving a short telomeric <1 kb region of the mixed lineage leukemia (MLL) gene are the most frequently observed molecular changes in secondary as well as infant acute leukemia. Due to the mode-of-action of epipodophyllotoxins and anthracyclines, which have widely been used in cancer therapy, and support from in vitro experiments, cleavage of this MLL breakpoint cluster hotspot by poisoned topoisomerase II was proposed to trigger the molecular events leading to malignant transformation. Later on, clinical patient data and cell-based studies addressing a wider spectrum of stimuli identified cellular stress signaling pathways, which create secondary DNA structures, provide chromatin accessibility, and activate nucleases other than topoisomerase II at the MLL. The MLL destabilizing signaling pathways under discussion, namely early apoptotic DNA fragmentation, transcription stalling, and replication stalling, may all act in concert upon infection-, transplantation-, or therapy-induced cell cycle entry of hematopoietic stem and progenitor cells (HSPCs), to permit misguided cleavage and error-prone DNA repair in the cell-of-leukemia-origin. PMID:26161385

  7. On the Mechanism of Multiple Lysine Methylation by the Human Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex*♦

    PubMed Central

    Patel, Anamika; Dharmarajan, Venkatasubramanian; Vought, Valarie E.; Cosgrove, Michael S.

    2009-01-01

    Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked ∼600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex. PMID:19556245

  8. High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein–Protein Interaction

    PubMed Central

    2015-01-01

    The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ∼288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules. PMID:24472025

  9. High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein-Protein Interaction

    SciTech Connect

    He, Shihan; Senter, Timothy J.; Pollock, Jonathan; Han, Changho; Upadhyay, Sunil Kumar; Purohit, Trupta; Gogliotti, Rocco D.; Lindsley, Craig W.; Cierpicki, Tomasz; Stauffer, Shaun R.; Grembecka, Jolanta

    2014-10-02

    The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ~288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

  10. Prognostic Significance of Mixed-Lineage Leukemia (MLL) Gene Detected by Real-Time Fluorescence Quantitative PCR Assay in Acute Myeloid Leukemia.

    PubMed

    Huang, Sai; Yang, Hua; Li, Yan; Feng, Cong; Gao, Li; Chen, Guo-Feng; Gao, Hong-Hao; Huang, Zhi; Li, Yong-Hui; Yu, Li

    2016-01-01

    BACKGROUND The overall prognosis of acute myeloid leukemia (AML) patients with mixed-lineage leukemia (MLL) gene-positivity is unfavorable. In this study, we evaluated the expression levels of the MLL gene in AML patients. MATERIAL AND METHODS We enrolled 68 MLL gene-positive patients out of 433 newly diagnosed AML patients, and 216 bone marrow samples were collected. Real-time fluorescence quantitative PCR (RQ-PCR) was used to precisely detect the expression levels of the MLL gene. RESULTS We divided 41 patients into 2 groups according to the variation of MRD (minimal residual disease) level of the MLL gene. Group 1 (n=22) had a rapid reduction of MRD level to ≤10^-4 in all samples collected in the first 3 chemotherapy cycles, while group 2 (n=19) had MRD levels constantly >10^-4 in all samples collected in the first 3 chemotherapy cycles. Group 1 had a significantly better overall survival (p=0.001) and event-free survival (p=0.001) compared to group 2. Moreover, the patients with >10^-4 MRD level before the start of HSCT (hematopoietic stem cell transplantation) had worse prognosis and higher risk of relapse compared to patients with ≤10^-4 before the start of HSCT. CONCLUSIONS We found that a rapid reduction of MRD level to ≤10^-4 appears to be a prerequisite for better overall survival and event-free survival during the treatment of AML. The MRD levels detected by RQ-PCR were basically in line with the clinical outcome and may be of great importance in guiding early allogeneic HSCT (allo-HSCT) treatment. PMID:27561414

  11. The Superiority of Allogeneic Hematopoietic Stem Cell Transplantation Over Chemotherapy Alone in the Treatment of Acute Myeloid Leukemia Patients with Mixed Lineage Leukemia (MLL) Rearrangements

    PubMed Central

    Yang, Hua; Huang, Sai; Zhu, Cheng-Ying; Gao, Li; Zhu, Hai-Yan; Lv, Na; Jing, Yu; Yu, Li

    2016-01-01

    Background Acute myeloid leukemia (AML) patients with mixed lineage leukemia (MLL) gene rearrangements always had a very poor prognosis. In this study, we report the incidence of MLL rearrangements in AML patients using gene analysis, as well as the clinical significance and prognostic features of these rearrangements. Material/Methods This retrospective study took place from April 2008 to November 2011 in the People’s Liberation Army General Hospital. A total 433 AML patients were screened by multiple nested reverse transcription polymerase chain reaction (RT-PCR) to determine the incidence of the 11 MLL gene rearrangements. There were 68 cases of MLL gene rearrangements, for a positive rate of 15.7%. A total of 24 patients underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and 34 patients received at least 4 cycles of chemotherapy. Ten patients were lost to follow-up. Results The median follow-up was 29 months. The complete remission (CR) rate was 85.4%. The overall survival (OS) was 57.4±5.9 months for the Allo-HSCT group and 21.0±2.1 months for the chemotherapy group. The Allo-HSCT group had superior survival compared with the chemotherapy group (5-year OS: 59±17% vs. 13±8%, P<0.01; 5-year disease-free survival [DFS]: 65±10% vs. 40±16%, P>0.05). Multivariate analysis showed that transplantation, platelets >50×109/L at onset, and CR are associated with a better OS in MLL rearranged AML patients. Patients with thrombocytopenia and extramedullary involvement were prone to relapse. Conclusions Our results suggest that Allo-HSCT is superior to chemotherapy alone for treating MLL rearranged AML patients. Patients treated with Allo-HSCT have a better prognosis and a longer survival. CR is an independent prognostic factor for OS, and extramedullary involvement is an independent prognostic factor for DFS. MLL rearranged AML patients with thrombocytopenia at onset <50×109 had very bad OS and DFS. PMID:27373985

  12. The Superiority of Allogeneic Hematopoietic Stem Cell Transplantation Over Chemotherapy Alone in the Treatment of Acute Myeloid Leukemia Patients with Mixed Lineage Leukemia (MLL) Rearrangements.

    PubMed

    Yang, Hua; Huang, Sai; Zhu, Cheng-Ying; Gao, Li; Zhu, Hai-Yan; Lv, Na; Jing, Yu; Yu, Li

    2016-01-01

    BACKGROUND Acute myeloid leukemia (AML) patients with mixed lineage leukemia (MLL) gene rearrangements always had a very poor prognosis. In this study, we report the incidence of MLL rearrangements in AML patients using gene analysis, as well as the clinical significance and prognostic features of these rearrangements. MATERIAL AND METHODS This retrospective study took place from April 2008 to November 2011 in the People's Liberation Army General Hospital. A total 433 AML patients were screened by multiple nested reverse transcription polymerase chain reaction (RT-PCR) to determine the incidence of the 11 MLL gene rearrangements. There were 68 cases of MLL gene rearrangements, for a positive rate of 15.7%. A total of 24 patients underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and 34 patients received at least 4 cycles of chemotherapy. Ten patients were lost to follow-up. RESULTS The median follow-up was 29 months. The complete remission (CR) rate was 85.4%. The overall survival (OS) was 57.4±5.9 months for the Allo-HSCT group and 21.0±2.1 months for the chemotherapy group. The Allo-HSCT group had superior survival compared with the chemotherapy group (5-year OS: 59±17% vs. 13±8%, P<0.01; 5-year disease-free survival [DFS]: 65±10% vs. 40±16%, P>0.05). Multivariate analysis showed that transplantation, platelets >50×10^9/L at onset, and CR are associated with a better OS in MLL rearranged AML patients. Patients with thrombocytopenia and extramedullary involvement were prone to relapse. CONCLUSIONS Our results suggest that Allo-HSCT is superior to chemotherapy alone for treating MLL rearranged AML patients. Patients treated with Allo-HSCT have a better prognosis and a longer survival. CR is an independent prognostic factor for OS, and extramedullary involvement is an independent prognostic factor for DFS. MLL rearranged AML patients with thrombocytopenia at onset <50×10^9 had very bad OS and DFS. PMID:27373985

  13. Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1).

    PubMed

    Getlik, Matthäus; Smil, David; Zepeda-Velázquez, Carlos; Bolshan, Yuri; Poda, Gennady; Wu, Hong; Dong, Aiping; Kuznetsova, Ekaterina; Marcellus, Richard; Senisterra, Guillermo; Dombrovski, Ludmila; Hajian, Taraneh; Kiyota, Taira; Schapira, Matthieu; Arrowsmith, Cheryl H; Brown, Peter J; Vedadi, Masoud; Al-Awar, Rima

    2016-03-24

    WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5. PMID:26958703

  14. Automethylation activities within the mixed lineage leukemia-1 (MLL1) core complex reveal evidence supporting a "two-active site" model for multiple histone H3 lysine 4 methylation.

    PubMed

    Patel, Anamika; Vought, Valarie E; Swatkoski, Stephen; Viggiano, Susan; Howard, Benny; Dharmarajan, Venkatasubramanian; Monteith, Kelsey E; Kupakuwana, Gillian; Namitz, Kevin E; Shinsky, Stephen A; Cotter, Robert J; Cosgrove, Michael S

    2014-01-10

    The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a "two-active site" model for multiple H3K4 methylation by the MLL1 core complex. PMID:24235145

  15. Automethylation Activities within the Mixed Lineage Leukemia-1 (MLL1) Core Complex Reveal Evidence Supporting a “Two-active Site” Model for Multiple Histone H3 Lysine 4 Methylation*

    PubMed Central

    Patel, Anamika; Vought, Valarie E.; Swatkoski, Stephen; Viggiano, Susan; Howard, Benny; Dharmarajan, Venkatasubramanian; Monteith, Kelsey E.; Kupakuwana, Gillian; Namitz, Kevin E.; Shinsky, Stephen A.; Cotter, Robert J.; Cosgrove, Michael S.

    2014-01-01

    The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a “two-active site” model for multiple H3K4 methylation by the MLL1 core complex. PMID:24235145

  16. A Novel Non-SET Domain Multi-subunit Methyltransferase Required for Sequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex*

    PubMed Central

    Patel, Anamika; Vought, Valarie E.; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.

    2011-01-01

    Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex. PMID:21106533

  17. A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.

    PubMed

    Patel, Anamika; Vought, Valarie E; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S

    2011-02-01

    Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex. PMID:21106533

  18. Mixed Lineage Leukemia 5 (MLL5) Protein Regulates Cell Cycle Progression and E2F1-responsive Gene Expression via Association with Host Cell Factor-1 (HCF-1)*

    PubMed Central

    Zhou, Peipei; Wang, Zhilong; Yuan, Xiujie; Zhou, Cuihong; Liu, Lulu; Wan, Xiaoling; Zhang, Feng; Ding, Xiaodan; Wang, Chuangui; Xiong, Sidong; Wang, Zhen; Yuan, Jinduo; Li, Qiang; Zhang, Yan

    2013-01-01

    Trithorax group proteins methylate lysine 4 of histone 3 (H3K4) at active gene promoters. MLL5 protein, a member of the Trithorax protein family, has been implicated in the control of the cell cycle progression; however, the underlying molecular mechanism(s) have not been fully determined. In this study, we found that the MLL5 protein can associate with the cell cycle regulator “host cell factor” (HCF-1). The interaction between MLL5 and HCF-1 is mediated by the “HCF-1 binding motif” (HBM) of the MLL5 protein and the Kelch domain of the HCF-1 protein. Confocal microscopy showed that the MLL5 protein largely colocalized with HCF-1 in the nucleus. Knockdown of MLL5 resulted in reduced cell proliferation and cell cycle arrest in the G1 phase. Moreover, down-regulation of E2F1 target gene expression and decreased H3K4me3 levels at E2F1-responsive promoters were observed in MLL5 knockdown cells. Additionally, the core subunits, including ASH2L, RBBP5, and WDR5, that are necessary for effective H3K4 methyltransferase activities of the Trithorax protein complexes, were absent in the MLL5 complex, suggesting that a distinct mechanism may be used by MLL5 for exerting its H3K4 methyltransferase activity. Together, our findings demonstrate that MLL5 could associate with HCF-1 and then be recruited to E2F1-responsive promoters to stimulate H3K4 trimethylation and transcriptional activation, thereby facilitating the cell cycle G1 to S phase transition. PMID:23629655

  19. Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein-Protein Interaction between Menin and Mixed Lineage Leukemia (MLL).

    PubMed

    Borkin, Dmitry; Pollock, Jonathan; Kempinska, Katarzyna; Purohit, Trupta; Li, Xiaoqin; Wen, Bo; Zhao, Ting; Miao, Hongzhi; Shukla, Shirish; He, Miao; Sun, Duxin; Cierpicki, Tomasz; Grembecka, Jolanta

    2016-02-11

    Development of potent small molecule inhibitors of protein-protein interactions with optimized druglike properties represents a challenging task in lead optimization process. Here, we report synthesis and structure-based optimization of new thienopyrimidine class of compounds, which block the protein-protein interaction between menin and MLL fusion proteins that plays an important role in acute leukemias with MLL translocations. We performed simultaneous optimization of both activity and druglike properties through systematic exploration of substituents introduced to the indole ring of lead compound 1 (MI-136) to identify compounds suitable for in vivo studies in mice. This work resulted in the identification of compound 27 (MI-538), which showed significantly increased activity, selectivity, polarity, and pharmacokinetic profile over 1 and demonstrated a pronounced effect in a mouse model of MLL leukemia. This study, which reports detailed structure-activity and structure-property relationships for the menin-MLL inhibitors, demonstrates challenges in optimizing inhibitors of protein-protein interactions for potential therapeutic applications. PMID:26744767

  20. Systematic Classification of Mixed-Lineage Leukemia Fusion Partners Predicts Additional Cancer Pathways

    PubMed Central

    2016-01-01

    Chromosomal translocations of the human mixed-lineage leukemia (MLL) gene have been analyzed for more than 20 yr at the molecular level. So far, we have collected about 80 direct MLL fusions (MLL-X alleles) and about 120 reciprocal MLL fusions (X-MLL alleles). The reason for the higher amount of reciprocal MLL fusions is that the excess is caused by 3-way translocations with known direct fusion partners. This review is aiming to propose a solution for an obvious problem, namely why so many and completely different MLL fusion alleles are always leading to the same leukemia phenotypes (ALL, AML, or MLL). This review is aiming to explain the molecular consequences of MLL translocations, and secondly, the contribution of the different fusion partners. A new hypothesis will be posed that can be used for future research, aiming to find new avenues for the treatment of this particular leukemia entity. PMID:26709255

  1. 11q23 Translocations split the [open quotes]AT-hook[close quotes] cruciform DNA-binding region and the transcriptional repression domain from the activation domain of the mixed-lineage leukemia (MLL) gene

    SciTech Connect

    Zeleznik-Le, N.J.; Harden, A.M.; Rowley, J.D. )

    1994-10-25

    Translocations involving chromosome band 11q23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene encodes a putative transcription factor with homology to the zinc fingers and other domains of the Drosophila trithorax gene product and to the [open quotes]AT-hook[close quotes] motif of high mobility group proteins. To map potential transcriptional activation or repression domains of the MLL protein, yeast GAL4 DNA-binding domain and MLL hybrid protein-expressing plasmids were cotransfected with chloramphenicol acetyltransferase reporter plasmids in a transient transfection system. We found that MLL contains a strong activation domain and a repression domain. The former, located telomeric (3[prime]) to the breakpoint region, activated transcription 18-fold to >200-fold, depending on the promoter and cell line used for transfection. A repression domain that repressed transcription 4-fold was located centromeric (5[prime]) to the breakpoint region of MLL. The MLL AT-hook domain protein was expressed in bacteria and was utilized in a gel mobility shift assay to assess DNA-binding activity. The MLL AT-hook domain could bind cruciform DNA, recognizing structure rather than sequence of the target DNA. In translocations involving MLL, loss of an activation domain with retention of a repression domain and a DNA-binding domain on the der(11) chromosome could alter the expression of downstream target genes, suggesting a potential mechanism of action for MLL in leukemia. 35 refs., 5 figs., 1 tab.

  2. Acute leukemias of different lineages have similar MLL gene fusions encoding related chimeric proteins resulting from chromosomal translocation

    SciTech Connect

    Corral, J.; Forster, A.; Thompson, S.; Rabbitts, T.H. ); Lampert, F. ); Kaneko, Y. ); Slater, R.; Kroes, W.G. ); Van Der Schoot, C.E. ); Ludwig, W.D. ); Karpas, A. ); Pocock, C.; Cotter, F. )

    1993-09-15

    The MLL gene, on human chromosome 11q23, undergoes chromosomal translocation in acute leukemias, resulting in gene fusion with AF4 (chromosome 4) and ENL (chromosome 19). The authors report here translocation of MLL with nine different chromosomes and two paracentric chromosome 11 deletions in early B cell, B- or T-cell lineage, or nonlymphocytic acute leukemias. The mRNA translocation junction from 22t(4;11) patients, including six adult leukemias, and nine t(11;19) tumors reveals a remarkable conservation of breakpoints within MLL, AF4, or ENL genes, irrespective of tumor phenotype. Typically, the breakpoints are upstream of the zinc-finger region of MLL, and deletion of this region can accompany translocation, supporting the der(11) chromosome as the important component in leukemogenesis. Partial sequence of a fusion between MLL and the AFX1 gene from chromosome X shows the latter to be rich in Ser/Pro codons, like the ENL mRNA. These data suggest that the heterogeneous 11q23 abnormalities might cause attachment of Ser/Pro-rich segments to the NH[sub 2] terminus of MLL, lacking the zinc-finger region, and that translocation occurs in early hematopoietic cells, before commitment to distinct lineages. 36 refs., 2 figs.

  3. Pharmacologic inhibition of the menin-MLL interaction blocks progression of MLL leukemia in vivo

    PubMed Central

    Borkin, Dmitry; He, Shihan; Miao, Hongzhi; Kempinska, Katarzyna; Pollock, Jonathan; Chase, Jennifer; Purohit, Trupta; Malik, Bhavna; Zhao, Ting; Wang, Jingya; Wen, Bo; Zong, Hongliang; Jones, Morgan; Danet-Desnoyers, Gwenn; Guzman, Monica L.; Talpaz, Moshe; Bixby, Dale L.; Sun, Duxin; Hess, Jay L.; Muntean, Andrew G.; Maillard, Ivan; Cierpicki, Tomasz; Grembecka, Jolanta

    2015-01-01

    Summary Chromosomal translocations affecting Mixed Lineage Leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report development of highly potent and orally bioavailable small molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification. PMID:25817203

  4. The PHD fingers of MLL block MLL fusion protein–mediated transformation

    PubMed Central

    Muntean, Andrew G.; Giannola, Diane; Udager, Aaron M.

    2008-01-01

    Chromosomal translocations involving the mixed lineage leukemia (MLL) gene are associated with aggressive acute lymphoid and myeloid leukemias. These translocations are restricted to an 8.3-kb breakpoint region resulting in fusion of amino terminal MLL sequences in frame to 1 of more than 60 different translocation partners. The translocations consistently delete the plant homeodomain (PHD) fingers and more carboxyl terminal MLL sequences. The function of the PHD fingers is obscure and their specific role in transformation has not been explored. Here we show that inclusion of the PHD fingers in the MLL fusion protein MLL-AF9 blocked immortalization of hematopoietic progenitors. Inclusion of 2 or more PHD fingers reduced association with the Hoxa9 locus and suppressed Hoxa9 up-regulation in hematopoietic progenitors. These data provide an explanation for why MLL translocation breakpoints exclude the PHD fingers and suggest a possible role for these domains in regulating the function of wild-type MLL. PMID:18796627

  5. Structure of WDR5 bound to Mixed Lineage Leukemia protein-1 peptide

    SciTech Connect

    Patel, A.; Dharmarajan, V; Cosgrove, M

    2008-01-01

    The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic discs-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine containing motif, called the 'Win' or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-A crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodynamic binding experiments show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymatic activity of the MLL1 core complex.

  6. MLL1 and MLL1 fusion proteins have distinct functions in regulating leukemic transcription program

    PubMed Central

    Xu, Jing; Li, Li; Xiong, Jie; denDekker, Aaron; Ye, Andrew; Karatas, Hacer; Liu, Liu; Wang, He; Qin, Zhaohui S; Wang, Shaomeng; Dou, Yali

    2016-01-01

    Mixed lineage leukemia protein-1 (MLL1) has a critical role in human MLL1 rearranged leukemia (MLLr) and is a validated therapeutic target. However, its role in regulating global gene expression in MLLr cells, as well as its interplay with MLL1 fusion proteins remains unclear. Here we show that despite shared DNA-binding and cofactor interacting domains at the N terminus, MLL1 and MLL-AF9 are recruited to distinct chromatin regions and have divergent functions in regulating the leukemic transcription program. We demonstrate that MLL1, probably through C-terminal interaction with WDR5, is recruited to regulatory enhancers that are enriched for binding sites of E-twenty-six (ETS) family transcription factors, whereas MLL-AF9 binds to chromatin regions that have no H3K4me1 enrichment. Transcriptome-wide changes induced by different small molecule inhibitors also highlight the distinct functions of MLL1 and MLL-AF9. Taken together, our studies provide novel insights on how MLL1 and MLL fusion proteins contribute to leukemic gene expression, which have implications for developing effective therapies in the future.

  7. HoxBlinc RNA Recruits Set1/MLL Complexes to Activate Hox Gene Expression Patterns and Mesoderm Lineage Development.

    PubMed

    Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Naohiro; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

    2016-01-01

    Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1(+) mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1(+) precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1(+) precursors and differentiation of Flk1(+) cells into hematopoietic lineages. PMID:26725110

  8. HoxBlinc RNA recruits Set1/MLL complexes to activate Hox gene expression patterns and mesoderm lineage development

    PubMed Central

    Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Nao; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

    2015-01-01

    Summary Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1+ mesoderm and then promotes hematopoietic differentiation through regulating hoxb gene pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated KD or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb gene expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1+ precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1+ precursors and differentiation of Flk1+ cells into hematopoietic lineages. PMID:26725110

  9. Downregulation of RUNX1/CBFβ by MLL fusion proteins enhances hematopoietic stem cell self-renewal

    PubMed Central

    Zhao, Xinghui; Chen, Aili; Yan, Xiaomei; Zhang, Yue; He, Fuhong; Hayashi, Yoshihiro; Dong, Yunzhu; Rao, Yalan; Li, Bo; Conway, Rajeana M.; Maiques-Diaz, Alba; Elf, Shannon E.; Huang, Nuomin; Zuber, Johannes; Xiao, Zhijian; Tse, William; Tenen, Daniel G.; Wang, Qianfei; Chen, Wei; Mulloy, James C.; Nimer, Stephen D.

    2014-01-01

    RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9–mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs. PMID:24449215

  10. Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization

    PubMed Central

    Popovic, Relja; Riesbeck, Laurie E.; Velu, Chinavenmeni S.; Chaubey, Aditya; Zhang, Jiwang; Achille, Nicholas J.; Erfurth, Frank E.; Eaton, Katherine; Lu, Jun; Grimes, H. Leighton; Chen, Jianjun; Rowley, Janet D.

    2009-01-01

    Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster, in a pattern similar to that of the surrounding 5′ Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196–specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore, overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. PMID:19188669

  11. Monitoring mixed lineage leukemia expression may help identify patients with mixed lineage leukemia--rearranged acute leukemia who are at high risk of relapse after allogeneic hematopoietic stem cell transplantation.

    PubMed

    Liu, Jing; Wang, Yu; Xu, Lan-Ping; Liu, Dai-Hong; Qin, Ya-Zhen; Chang, Ying-Jun; Liu, Kai-Yan; Huang, Xiao-Jun

    2014-07-01

    To evaluate the prognostic value of the expression of the mixed lineage leukemia (MLL) gene for predicting the relapse of patients with MLL-rearranged acute leukemia (AL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the levels of MLL transcripts in bone marrow (BM) specimens were monitored serially by real-time quantitative polymerase chain reaction (RQ-PCR) at predetermined time points in 40 patients with MLL-rearranged AL who were treated with allo-HSCT. These patients were followed for a median of 24.5 months (range, 8 to 60 months). A total of 236 BM samples were collected and analyzed. Of these, 230 were monitored concurrently for minimal residual disease (MRD) by flow cytometry (FCM) for leukemia-associated aberrant immune phenotypes and by RQ-PCR for the expression of the Wilms tumor (WT1) gene. The 3-year cumulative incidence of relapse in patients who experienced MLL-positive patients (MLL > .0000%) (n = 9) after HSCT was 93.5% (95% confidence interval [CI], 87% to 100%) compared with 12.5% (95% CI, 5.6% to 19.4%) for MLL-negative patients (n = 31) (P < .001). For these 2 patient groups, the 3-year overall survival (OS) was 12.5% (95% CI, .8% to 24.2%) and 77.8% (95% CI, 68.4% to 87.2%) (P < .001), respectively, and the 3-year leukemia-free survival (LFS) was 0% and 72.2% (95% CI, 61.1% to 83.3%), respectively (P < .001). MLL positivity was associated with a higher rate of relapse (hazard ratio [HR], 18.643; 95% CI, 3.449 to 57.025; P = .001), lower LFS (HR, 7.267; 95% CI, 2.038 to 25.916; P = .002), and lower OS (HR, 8.259; 95% CI, 2.109 to 32.336; P = .002), as determined by Cox multivariate analysis. The expression of the MLL gene had a higher specificity and sensitivity than WT1 or MRD monitored by FCM for predicting the relapse of the patients with MLL + AL. Our results suggest that monitoring the expression of the MLL gene may help to identify patients with MLL + AL who are at high risk of relapse after allo-HSCT and may

  12. MLL-SEPTIN gene fusions in hematological malignancies.

    PubMed

    Cerveira, Nuno; Bizarro, Susana; Teixeira, Manuel R

    2011-08-01

    The mixed lineage leukemia (MLL) locus is involved in more than 60 different rearrangements with a remarkably diverse group of fusion partners in approximately 10% of human leukemias. MLL rearrangements include chromosomal translocations, gene internal duplications, chromosome 11q deletions or inversions and MLL gene insertions into other chromosomes, or vice versa. MLL fusion partners can be classified into four distinct categories: nuclear proteins, cytoplasmatic proteins, histone acetyltransferases and septins. Five different septin genes (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) have been identified as MLL fusion partners, giving rise to chimeric fusion proteins in which the N terminus of MLL is fused, in frame, to almost the entire open reading frame of the septin partner gene. The rearranged alleles result from heterogeneous breaks in distinct introns of both MLL and its septin fusion partner, originating distinct gene fusion variants. MLL-SEPTIN rearrangements have been repeatedly identified in de novo and therapy related myeloid neoplasia in both children and adults, and some clinicopathogenetic associations are being uncovered. The fundamental roles of septins in cytokinesis, membrane remodeling and compartmentalization can provide some clues on how abnormalities in the septin cytoskeleton and MLL deregulation could be involved in the pathogenesis of hematological malignancies. PMID:21714766

  13. Mixed lineage leukaemia histone methylases 1 collaborate with ERα to regulate HOXA10 expression in AML

    PubMed Central

    Yao, Jie; Fang, Li-Chao; Yang, Zai-Lin; Huang, Hui; Li, Yan; Deng, Jun; Zheng, Junsong

    2014-01-01

    HOXA10, a homeobox-containing gene involved in definitive haematopoiesis, which implicated in the pathogenesis of AML (acute myeloid leukaemia), has been studied extensively. But the regulatory mechanism that drives HOXA10 expression is still unclear. In the present paper, HOXA10 regulated by MLL1 (mixed lineage leukaemia histone methylase 1) with an epigenetic way has been demonstrated. The HOXA10 promoter contains several EREs (oestrogen response elements), including ERE1 and ERE2, which are close to the transcription start site, and are associated with E2-mediated activation of HOXA10. It has been shown that knockdown of the ERα (oestrogen receptor α) suppresses E2-mediated activation of HOXA10. Similarly, knockdown of MLL1 suppresses activation of HOXA10 and is bound to the ERE of HOXA10 promoter in an E2-dependent manner by forming complex with ERα. Knockdown of ERα affects the E2-dependent binding of MLL1 into HOXA10 EREs, suggesting critical roles of ERα in recruiting MLL on the HOXA10 promoter. More interestingly, the methylation status of histone protein H3K4 (H3 at lysine 4) with E2 is much higher than without E2 treatment in leukaemia cell. On the contrary, the methylation status of HOXA10 promoter with E2 treatment is much lower, which elevate the HOXA10 expression. Moreover, with ERα knockdown, the H3K4 methylation level is also decrease in myeloid cell. Overall, it has been clearly demonstrated that HOXA10 is transcriptionally regulated by MLL1, which, in coordination with ERα, plays a critical role in this process with epigenetic way and suggests a potential anti-E2 treatment of AML. PMID:25307539

  14. Structure of the MLL CXXC domain – DNA complex and its functional role in MLL-AF9 leukemia

    PubMed Central

    Cierpicki, Tomasz; Risner, Laurie E.; Grembecka, Jolanta; Lukasik, Stephen M.; Popovic, Relja; Omonkowska, Monika; Shultis, David S.; Zeleznik-Le, Nancy J.; Bushweller, John H.

    2010-01-01

    MLL (Mixed Lineage Leukemia) is the target of chromosomal translocations which cause leukemias with poor prognosis. All leukemogenic MLL fusion proteins retain the CXXC domain which binds to nonmethylated CpG DNA. We present the solution structure of the MLL CXXC domain in complex with DNA, showing for the first time how the CXXC domain distinguishes nonmethylated from methylated CpG DNA. Based on the structure, we designed point mutations which disrupt DNA binding. Introduction of these mutations into MLL-AF9 results in increased DNA methylation of specific CpG nucleotides in Hoxa9, increased H3K9 methylation, decreased expression of Hoxa9 locus transcripts, loss of immortalization potential, and inability to induce leukemia in mice. These results establish that DNA binding by the CXXC domain and protection against DNA methylation is essential for MLL fusion leukemia. They also provide support for this interaction as a potential target for therapeutic intervention. PMID:20010842

  15. Linking MLL and the HGF-MET signaling pathway in liver cancer.

    PubMed

    Marquardt, Jens U; Thorgeirsson, Snorri S

    2013-07-01

    Mixed-lineage leukemia (MLL; also known as myeloid/lymphoid), the human homolog of trithorax in Drosophila, is a transcriptional coactivator that plays an essential role during early development and hematopoiesis. Furthermore, MLL is critically involved in the epigenetic regulation of cell cycle, senescence, DNA damage, and stem cell self-renewal. Chromosomal aberrations of MLL in acute leukemias are well documented, but the role of this gene in solid malignancies remains unclear. In this issue of the JCI, Takeda et al. describe a novel epigenetic link between MLL and the HGF-MET signaling pathway conferring invasive and metastatic properties to hepatocellular carcinoma cells. PMID:23934122

  16. The MLL recombinome of acute leukemias in 2013

    PubMed Central

    Meyer, C; Hofmann, J; Burmeister, T; Gröger, D; Park, T S; Emerenciano, M; Pombo de Oliveira, M; Renneville, A; Villarese, P; Macintyre, E; Cavé, H; Clappier, E; Mass-Malo, K; Zuna, J; Trka, J; De Braekeleer, E; De Braekeleer, M; Oh, S H; Tsaur, G; Fechina, L; van der Velden, V H J; van Dongen, J J M; Delabesse, E; Binato, R; Silva, M L M; Kustanovich, A; Aleinikova, O; Harris, M H; Lund-Aho, T; Juvonen, V; Heidenreich, O; Vormoor, J; Choi, W W L; Jarosova, M; Kolenova, A; Bueno, C; Menendez, P; Wehner, S; Eckert, C; Talmant, P; Tondeur, S; Lippert, E; Launay, E; Henry, C; Ballerini, P; Lapillone, H; Callanan, M B; Cayuela, J M; Herbaux, C; Cazzaniga, G; Kakadiya, P M; Bohlander, S; Ahlmann, M; Choi, J R; Gameiro, P; Lee, D S; Krauter, J; Cornillet-Lefebvre, P; Te Kronnie, G; Schäfer, B W; Kubetzko, S; Alonso, C N; zur Stadt, U; Sutton, R; Venn, N C; Izraeli, S; Trakhtenbrot, L; Madsen, H O; Archer, P; Hancock, J; Cerveira, N; Teixeira, M R; Lo Nigro, L; Möricke, A; Stanulla, M; Schrappe, M; Sedék, L; Szczepański, T; Zwaan, C M; Coenen, E A; van den Heuvel-Eibrink, M M; Strehl, S; Dworzak, M; Panzer-Grümayer, R; Dingermann, T; Klingebiel, T; Marschalek, R

    2013-01-01

    Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (∼90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements. PMID:23628958

  17. MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein

    PubMed Central

    Xia, Zhen-Biao; Anderson, Melanie; Diaz, Manuel O.; Zeleznik-Le, Nancy J.

    2003-01-01

    The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. Histone deacetylase 1 interacts with the MLL repression domain, partially mediating its activity; binding of Cyp33 to the adjacent MLL-PHD domain potentiates this binding. Because the MLL repression domain activity was only partially relieved with the histone deacetylase inhibitor trichostatin A, we explored other protein interactions with this domain. Polycomb group proteins HPC2 and BMI-1 and the corepressor C-terminal-binding protein also bind the MLL repression domain. Expression of exogenous BMI-1 potentiates MLL repression domain activity. Functional antagonism between Mll and Bmi-1 has been shown genetically in murine knockout models for Mll and Bmi-1. Our new data suggest a model whereby recruitment of BMI-1 to the MLL protein may be able to modulate its function. Furthermore, repression mediated by histone deacetylases and that mediated by polycomb group proteins may act either independently or together for MLL function in vivo. PMID:12829790

  18. A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex.

    PubMed

    Patel, Anamika; Vought, Valarie E; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S

    2008-11-21

    The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro. PMID:18829457

  19. Requirement for CDK6 in MLL-rearranged acute myeloid leukemia

    PubMed Central

    Placke, Theresa; Faber, Katrin; Nonami, Atsushi; Putwain, Sarah L.; Salih, Helmut R.; Heidel, Florian H.; Krämer, Alwin; Root, David E.; Barbie, David A.; Krivtsov, Andrei V.; Armstrong, Scott A.; Hahn, William C.; Huntly, Brian J.; Sykes, Stephen M.; Milsom, Michael D.; Scholl, Claudia

    2014-01-01

    Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9–driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. PMID:24764564

  20. Requirement for CDK6 in MLL-rearranged acute myeloid leukemia.

    PubMed

    Placke, Theresa; Faber, Katrin; Nonami, Atsushi; Putwain, Sarah L; Salih, Helmut R; Heidel, Florian H; Krämer, Alwin; Root, David E; Barbie, David A; Krivtsov, Andrei V; Armstrong, Scott A; Hahn, William C; Huntly, Brian J; Sykes, Stephen M; Milsom, Michael D; Scholl, Claudia; Fröhling, Stefan

    2014-07-01

    Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. PMID:24764564

  1. Expression of HOXB genes is significantly different in acute myeloid leukemia with a partial tandem duplication of MLL vs. a MLL translocation: a cross-laboratory study.

    PubMed

    Liu, Hsi-Che; Shih, Lee-Yung; May Chen, Mei-Ju; Wang, Chien-Chih; Yeh, Ting-Chi; Lin, Tung-Huei; Chen, Chien-Yu; Lin, Chih-Jen; Liang, Der-Cherng

    2011-05-01

    In acute myeloid leukemia (AML), the mixed lineage leukemia (MLL) gene may be rearranged to generate a partial tandem duplication (PTD), or fused to partner genes through a chromosomal translocation (tMLL). In this study, we first explored the differentially expressed genes between MLL-PTD and tMLL using gene expression profiling of our cohort (15 MLL-PTD and 10 tMLL) and one published data set. The top 250 probes were chosen from each set, resulting in 29 common probes (21 unique genes) to both sets. The selected genes include four HOXB genes, HOXB2, B3, B5, and B6. The expression values of these HOXB genes significantly differ between MLL-PTD and tMLL cases. Clustering and classification analyses were thoroughly conducted to support our gene selection results. Second, as MLL-PTD, FLT3-ITD, and NPM1 mutations are identified in AML with normal karyotypes, we briefly studied their impact on the HOXB genes. Another contribution of this study is to demonstrate that using public data from other studies enriches samples for analysis and yields more conclusive results. PMID:21665178

  2. Granulocytic Sarcoma in MLL-Positive Infant Acute Myelogenous Leukemia

    PubMed Central

    Park, Kyoung Un; Lee, Dong Soon; Lee, Hye Seung; Kim, Chong Jai; Cho, Han Ik

    2001-01-01

    Granulocytic sarcoma is considered to be rare and its frequent occurrence is associated with specific genetic changes such as t(8;21). To investigate an association between MLL (mixed lineage leukemia or myeloid-lymphoid leukemia) rearrangement and granulocytic sarcoma, we applied fluorescence in situ hybridization for detection of the 11q23/MLL rearrangements on the bone marrow cells of 40 patients with childhood acute myelogenous leukemia (AML). Nine (22.5%) of 40 patients exhibited MLL rearrangements. Three (33.3%) of these nine patients had granulocytic sarcoma and were younger than 12 months of age. Of these three patients one presented as granulocytic sarcoma of both testes with cerebrospinal fluid involvement, the second case presented in the form of an abdominal mass, and the third as a periorbital granulocytic sarcoma. On the other hand, no granulocytic sarcomas were found among MLL-negative patients. It is likely that MLL-positive infant AML may predispose granulocytic sarcoma. Regarding the findings of our study and those of other reports, we would guess that the incidence of granulocytic sarcoma in pediatric MLL-positive AML may be equal to or greater than the 18 to 24% described in AML with t(8;21). Further investigations designed to identify 11q23/MLL abnormalities of leukemic cells or extramedullary tumor may be helpful for the precise diagnosis of granulocytic sarcoma. PMID:11733351

  3. An Mll-dependent Hox program drives hematopoietic progenitor expansion.

    PubMed

    Ernst, Patricia; Mabon, Meghann; Davidson, Alan J; Zon, Leonard I; Korsmeyer, Stanley J

    2004-11-23

    Chromosomal translocations disrupting the Mixed lineage leukemia (Mll) gene result in leukemia, with aberrant expression of some native Mll target genes (reviewed in). The Mll gene encodes a Trithorax-group chromatin regulator that is essential for the development of hematopoietic stem cells (HSCs) during embryogenesis. Like Trithorax, MLL positively regulates clustered homeodomain or Hox genes, yet the role of Hox genes collectively in the development of the mammalian hematopoietic system has been difficult to ascertain because of redundancy among Hox paralogs. Here, we show that in the absence of MLL, early hematopoietic progenitors develop despite reduced expression of HoxA, HoxB, and HoxC genes. However, these progenitors exhibit a marked reduction in their ability to generate hematopoietic colonies, a subsequent process requiring cell division and differentiation. Reactivation of a subset of Hox genes or, remarkably, reexpression of a single Hox gene in Mll-deficient progenitors rescued hematopoietic-colony frequency and growth. In contrast, expression of other MLL target genes such as Pitx2 or expression of anti-apoptotic BCL-2 failed to rescue hematopoietic-colony frequency. Furthermore, our results highlight a shared function of Hox proteins at this point in the development of the hematopoietic system. PMID:15556871

  4. Musashi2 sustains the mixed-lineage leukemia–driven stem cell regulatory program

    PubMed Central

    Park, Sun-Mi; Gönen, Mithat; Vu, Ly; Minuesa, Gerard; Tivnan, Patrick; Barlowe, Trevor S.; Taggart, James; Lu, Yuheng; Deering, Raquel P.; Hacohen, Nir; Figueroa, Maria E.; Paietta, Elisabeth; Fernandez, Hugo F.; Tallman, Martin S.; Melnick, Ari; Levine, Ross; Leslie, Christina; Lengner, Christopher J.; Kharas, Michael G.

    2015-01-01

    Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic alterations. Overexpression of the RNA-binding protein Musashi2 (MSI2) has been previously shown to predict poor survival in leukemia. Here, we demonstrated that conditional deletion of Msi2 in the hematopoietic compartment results in delayed leukemogenesis, reduced disease burden, and a loss of LSC function in a murine leukemia model. Gene expression profiling of these Msi2-deficient animals revealed a loss of the hematopoietic/leukemic stem cell self-renewal program and an increase in the differentiation program. In acute myeloid leukemia patients, the presence of a gene signature that was similar to that observed in Msi2-deficent murine LSCs correlated with improved survival. We determined that MSI2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc, and Ikzf2 mRNAs. Moreover, depletion of MLL target Ikzf2 in LSCs reduced colony formation, decreased proliferation, and increased apoptosis. Our data provide evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and suggest MSI2 as a potential therapeutic target for myeloid leukemia. PMID:25664853

  5. Design and synthesis of benzylpiperidine inhibitors targeting the menin-MLL1 interface.

    PubMed

    Ren, Jing; Xu, Wei; Tang, Le; Su, Minbo; Chen, Danqi; Chen, Yue-Lei; Zang, Yi; Li, Jia; Shen, Jingkang; Zhou, Yubo; Xiong, Bing

    2016-09-15

    Menin is an essential oncogenic cofactor for mixed lineage leukemia (MLL)-mediated leukemogenesis, functioning through its direct interaction with MLL1 protein. Therefore, targeting the menin-MLL1 protein-protein interface represents a promising strategy to block MLL-mediated leukemogenesis. On the basis of co-crystal structure analysis, starting from thienopyrimidine chemotype, we have investigated the detailed structure-activity relationship of the piperazinyl-dihydrothiazole moiety. Several compounds were found with potent inhibitory activity against menin and better activities in cell-based experiments than MI-2-2. Molecular docking analysis revealed a less explored subpocket, which could be used for the design of new menin-MLL1 inhibitors. PMID:27528435

  6. High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction

    SciTech Connect

    Karatas, Hacer; Townsend, Elizabeth C; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng

    2013-02-12

    Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein–protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH–, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with Ki < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein–protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.

  7. Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4.

    PubMed

    Dhar, Shilpa S; Lee, Sung-Hun; Kan, Pu-Yeh; Voigt, Philipp; Ma, Li; Shi, Xiaobing; Reinberg, Danny; Lee, Min Gyu

    2012-12-15

    Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD(4-6)) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4's nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD(4-6) reduce PHD(4-6)'s binding ability and MLL4's catalytic activity. PHD(4-6)'s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s's interference with PHD(4-6)'s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation. PMID:23249737

  8. Development of Five Dual-Color, Double-Fusion Fluorescence in Situ Hybridization Assays for the Detection of Common MLL Translocation Partners

    PubMed Central

    Keefe, Jeannette G.; Sukov, William R.; Knudson, Ryan A.; Nguyen, Lai P.; Williamson, Cynthia; Sinnwell, Jason P.; Ketterling, Rhett P.

    2010-01-01

    Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene at 11q23 are frequent in adult and childhood acute leukemia and have been associated with an unfavorable prognosis. Recent evidence suggests that MLL gene partners may influence prognosis. Five translocations account for ∼80% of MLL rearrangements: t(4;11)(q21;q23), AFF1/MLL; t(6;11)(q27;q23), MLLT4/MLL; t(9;11)(p22;q23), MLLT3/MLL; t(11;19)(q23;p13.1), MLL/ELL; and t(11;19)(q23;p13.3), MLL/MLLT1. We have designed dual-color, double-fusion fluorescence in situ hybridization (D-FISH) probe sets to identify these translocations. A blinded study was performed for each probe set using 25 normal bone marrow samples, 25 t(4;11), 20 t(6;11), 20 t(9;11), 18 t(11;19p13.1), and 20 t(11;19p13.3) leukemia specimens as defined by chromosome analysis. The findings demonstrated abnormal D-FISH results for 24 of 25 AFF1/MLL, 19 of 20 MLLT4/MLL, all 20 MLLT3/MLL, all 18 MLL/ELL, and all 20 MLL/MLLT1 samples, confirming the efficacy of these D-FISH assays in detecting these common MLL/partner translocations. Our D-FISH assays were more accurate than chromosome analysis at distinguishing disruption of 19p13.1/ELL from that of 19p13.3/MLLT1. We also demonstrated a statistically significant increase in complex/unbalanced MLL/partner translocations occurring in pediatric patients versus adult patients (P = 0.02). A normal cutoff of 0.6% was established, suggesting an application for these assays in minimal residual disease detection and disease monitoring. PMID:20539022

  9. HGF-MET signals via the MLL-ETS2 complex in hepatocellular carcinoma.

    PubMed

    Takeda, Shugaku; Liu, Han; Sasagawa, Satoru; Dong, Yiyu; Trainor, Paul A; Cheng, Emily H; Hsieh, James J

    2013-07-01

    HGF signals through its cognate receptor, MET, to orchestrate diverse biological processes, including cell proliferation, cell fate specification, organogenesis, and epithelial-mesenchymal transition. Mixed-lineage leukemia (MLL), an epigenetic regulator, plays critical roles in cell fate, stem cell, and cell cycle decisions. Here, we describe a role for MLL in the HGF-MET signaling pathway. We found a shared phenotype among Mll(-/-), Hgf(-/-), and Met(-/-) mice with common cranial nerve XII (CNXII) outgrowth and myoblast migration defects. Phenotypic analysis demonstrated that MLL was required for HGF-induced invasion and metastatic growth of hepatocellular carcinoma cell lines. HGF-MET signaling resulted in the accumulation of ETS2, which interacted with MLL to transactivate MMP1 and MMP3. ChIP assays demonstrated that activation of the HGF-MET pathway resulted in increased occupancy of the MLL-ETS2 complex on MMP1 and MMP3 promoters, where MLL trimethylated histone H3 lysine 4 (H3K4), activating transcription. Our results present an epigenetic link between MLL and the HGF-MET signaling pathway, which may suggest new strategies for therapeutic intervention. PMID:23934123

  10. MLL3 Is a Haploinsufficient 7q Tumor Suppressor in Acute Myeloid Leukemia

    PubMed Central

    Chen, Chong; Liu, Yu; Rappaport, Amy R.; Kitzing, Thomas; Schultz, Nikolaus; Zhao, Zhen; Shroff, Aditya S.; Dickins, Ross A.; Vakoc, Christopher R.; Bradner, James E.; Stock, Wendy; LeBeau, Michelle M.; Shannon, Kevin M.; Kogan, Scott; Zuber, Johannes; Lowe, Scott W.

    2014-01-01

    SUMMARY Recurring deletions of chromosome 7 and 7q [−7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are associated with poor prognosis. However, the identity of functionally relevant tumor suppressors on 7q remains unclear. Using RNAi and CRISPR/Cas9 approaches, we show that an ~50% reduction in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in −7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3-suppressed leukemias, like human −7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor and suggests a therapeutic option for this aggressive disease. PMID:24794707

  11. Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis.

    PubMed

    Arcipowski, Kelly M; Bulic, Marinka; Gurbuxani, Sandeep; Licht, Jonathan D

    2016-01-01

    Two of the most common myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), are associated with exceedingly low survival rates despite recent therapeutic advances. While their etiology is not completely understood, evidence suggests that certain chromosomal abnormalities contribute to MDS and AML progression. Among the most frequent chromosomal abnormalities in these disorders are alterations of chromosome 7: either complete loss of one copy of chromosome 7 (-7) or partial deletion of 7q (del(7q)), both of which increase the risk of progression from MDS to AML and are associated with chemoresistance. Notably, 7q36.1, a critical minimally deleted region in 7q, includes the gene encoding the histone methyltransferase mixed-lineage leukemia 3 (MLL3), which is also mutated in a small percentage of AML patients. However, the mechanisms by which MLL3 loss contributes to malignancy are unknown. Using an engineered mouse model expressing a catalytically inactive form of Mll3, we found a significant shift in hematopoiesis toward the granulocyte/macrophage lineage, correlating with myeloid infiltration and enlargement of secondary lymphoid organs. Therefore, we propose that MLL3 loss in patients may contribute to the progression of MDS and AML by promoting myelopoiesis. PMID:27610619

  12. Mll partial tandem duplication induces aberrant Hox expression in vivo via specific epigenetic alterations

    PubMed Central

    Dorrance, Adrienne M.; Liu, Shujun; Yuan, Weifeng; Becknell, Brian; Arnoczky, Kristy J.; Guimond, Martin; Strout, Matthew P.; Feng, Lan; Nakamura, Tatsuya; Yu, Li; Rush, Laura J.; Weinstein, Michael; Leone, Gustavo; Wu, Lizhao; Ferketich, Amy; Whitman, Susan P.; Marcucci, Guido; Caligiuri, Michael A.

    2006-01-01

    We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%–7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. MllPTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. MllPTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML. PMID:16981007

  13. A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

    PubMed

    Starnes, Linda M; Su, Dan; Pikkupeura, Laura M; Weinert, Brian T; Santos, Margarida A; Mund, Andreas; Soria, Rebeca; Cho, Young-Wook; Pozdnyakova, Irina; Kubec Højfeldt, Martina; Vala, Andrea; Yang, Wenjing; López-Méndez, Blanca; Lee, Ji-Eun; Peng, Weiqun; Yuan, Joan; Ge, Kai; Montoya, Guillermo; Nussenzweig, André; Choudhary, Chunaram; Daniel, Jeremy A

    2016-01-15

    Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities. PMID:26744420

  14. A PTIP–PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex

    PubMed Central

    Starnes, Linda M.; Su, Dan; Pikkupeura, Laura M.; Weinert, Brian T.; Santos, Margarida A.; Mund, Andreas; Soria, Rebeca; Cho, Young-Wook; Pozdnyakova, Irina; Kubec Højfeldt, Martina; Vala, Andrea; Yang, Wenjing; López-Méndez, Blanca; Lee, Ji-Eun; Peng, Weiqun; Yuan, Joan; Ge, Kai; Montoya, Guillermo; Nussenzweig, André; Choudhary, Chunaram; Daniel, Jeremy A.

    2016-01-01

    Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP–PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities. PMID:26744420

  15. The same site on the integrase-binding domain of lens epithelium–derived growth factor is a therapeutic target for MLL leukemia and HIV

    PubMed Central

    Murai, Marcelo J.; Pollock, Jonathan; He, Shihan; Miao, Hongzhi; Purohit, Trupta; Yokom, Adam; Hess, Jay L.; Muntean, Andrew G.; Grembecka, Jolanta

    2014-01-01

    Lens epithelium-derived growth factor (LEDGF) is a chromatin-associated protein implicated in leukemia and HIV type 1 infection. LEDGF associates with mixed-lineage leukemia (MLL) fusion proteins and menin and is required for leukemic transformation. To better understand the molecular mechanism underlying the LEDGF integrase-binding domain (IBD) interaction with MLL fusion proteins in leukemia, we determined the solution structure of the MLL-IBD complex. We found a novel MLL motif, integrase domain binding motif 2 (IBM2), which binds to a well-defined site on IBD. Point mutations within IBM2 abolished leukemogenic transformation by MLL-AF9, validating that this newly identified motif is essential for the oncogenic activity of MLL fusion proteins. Interestingly, the IBM2 binding site on IBD overlaps with the binding site for the HIV integrase (IN), and IN was capable of efficiently sequestering IBD from the menin-MLL complex. A short IBM2 peptide binds to IBD directly and inhibits both the IBD-MLL/menin and IBD-IN interactions. Our findings show that the same site on IBD is involved in binding to MLL and HIV-IN, revealing an attractive approach to simultaneously target LEDGF in leukemia and HIV. PMID:25305204

  16. SEPT2 is a new fusion partner of MLL in acute myeloid leukemia with t(2;11)(q37;q23).

    PubMed

    Cerveira, N; Correia, C; Bizarro, S; Pinto, C; Lisboa, S; Mariz, J M; Marques, M; Teixeira, M R

    2006-10-01

    We have identified a new mixed lineage leukemia (MLL) gene fusion partner in a patient with treatment-related acute myeloid leukemia (AML) presenting a t(2;11)(q37;q23) as the only cytogenetic abnormality. Fluorescence in situ hybridization demonstrated a rearrangement of the MLL gene and molecular genetic analyses identified a septin family gene, SEPT2, located on chromosome 2q37, as the fusion partner of MLL. RNA and DNA analyses showed the existence of an in-frame fusion of MLL exon 7 with SEPT2 exon 3, with the genomic breakpoints located in intron 7 and 2 of MLL and SEPT2, respectively. Search for DNA sequence motifs revealed the existence of two sequences with 94.4% homology with the topoisomerase II consensus cleavage site in MLL intron 7 and SEPT2 intron 2. SEPT2 is the fifth septin family gene fused with MLL, making this gene family the most frequently involved in MLL-related AML (about 10% of all known fusion partners). The protein encoded by SEPT2 is highly homologous to septins 1, 4 and 5 and is involved in the coordination of several key steps of mitosis. Further studies are warranted to understand why the septin protein family is particularly involved in the pathogenesis of MLL-associated leukemia. PMID:16682951

  17. RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

    PubMed Central

    Wilkinson, Adam C.; Ballabio, Erica; Geng, Huimin; North, Phillip; Tapia, Marta; Kerry, Jon; Biswas, Debabrata; Roeder, Robert G.; Allis, C. David; Melnick, Ari; de Bruijn, Marella F.T.R.; Milne, Thomas A.

    2013-01-01

    Summary The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product. PMID:23352661

  18. Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis

    PubMed Central

    Ballabio, Erica; Milne, Thomas A.

    2012-01-01

    Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don’t alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL) protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis. PMID:24213472

  19. Epigenetic control of gene expression in leukemogenesis: Cooperation between wild type MLL and MLL fusion proteins

    PubMed Central

    Ballabio, Erica; Milne, Thomas A

    2014-01-01

    Although there has been great progress in the treatment of human cancers, especially leukemias, many remain resistant to treatment. A major current focus is the development of so-called epigenetic drugs. Epigenetic states are stable enough to persist through multiple cell divisions, but by their very nature are reversible and thus are amenable to therapeutic manipulation. Exciting work in this area has produced a new breed of highly specific small molecules designed to inhibit epigenetic proteins, some of which have entered clinical trials. The current and future development of epigenetic drugs is greatly aided by highly detailed information about normal and aberrant epigenetic changes at the molecular level. In this review we focus on a class of aggressive acute leukemias caused by mutations in the Mixed Lineage Leukemia (MLL) gene. We provide an overview of how detailed molecular analysis of MLL leukemias has provided several early-stage epigenetic drugs and propose that further study of MLL leukemogenesis may continue to provide molecular details that potentially have a wider range of applications in human cancers. PMID:27308325

  20. MLL leukemia induction by genome editing of human CD34+ hematopoietic cells

    PubMed Central

    Buechele, Corina; Breese, Erin H.; Schneidawind, Dominik; Lin, Chiou-Hong; Jeong, Johan; Duque-Afonso, Jesus; Wong, Stephen H. K.; Smith, Kevin S.; Negrin, Robert S.; Porteus, Matthew

    2015-01-01

    Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease–mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia. PMID:26311362

  1. A non-active-site SET domain surface crucial for the interaction of MLL1 and the RbBP5/Ash2L heterodimer within MLL family core complexes.

    PubMed

    Shinsky, Stephen A; Hu, Michael; Vought, Valarie E; Ng, Sarah B; Bamshad, Michael J; Shendure, Jay; Cosgrove, Michael S

    2014-06-12

    The mixed lineage leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1-MLL4 and SETd1a,b. Dimethylation of H3K4 requires a sub-complex including WRAD (WDR5, RbBP5, Ash2L, and DPY-30), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active-site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5/Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or KIS, are required for formation of a second active site within SET1 family core complexes. PMID:24680668

  2. Crosstalk between leukemia-associated proteins MOZ and MLL regulates HOX gene expression in human cord blood CD34+ cells.

    PubMed

    Paggetti, J; Largeot, A; Aucagne, R; Jacquel, A; Lagrange, B; Yang, X-J; Solary, E; Bastie, J-N; Delva, L

    2010-09-01

    MOZ and MLL, encoding a histone acetyltransferase (HAT) and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. In MOZ (MOnocytic leukemia Zinc-finger protein)/CBP- or mixed lineage leukemia (MLL)-rearranged leukemias, abnormal levels of HOX transcription factors have been found to be critical for leukemogenesis. We show that MOZ and MLL cooperate to regulate these key genes in human cord blood CD34+ cells. These chromatin-modifying enzymes interact, colocalize and functionally cooperate, and both are recruited to multiple HOX promoters. We also found that WDR5, an adaptor protein essential for lysine 4 trimethylation of histone H3 (H3K4me3) by MLL, colocalizes and interacts with MOZ. We detected the binding of the HAT MOZ to H3K4me3, thus linking histone methylation to acetylation. In CD34+ cells, depletion of MLL causes release of MOZ from HOX promoters, which is correlated to defective histone activation marks, leading to repression of HOX gene expression and alteration of commitment of CD34+ cells into myeloid progenitors. Thus, our results unveil the role of the interaction between MOZ and MLL in CD34+ cells in which both proteins have a critical role in hematopoietic cell-fate decision, suggesting a new molecular mechanism by which MOZ or MLL deregulation leads to leukemogenesis. PMID:20581860

  3. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

    SciTech Connect

    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  4. The CDK9 Inhibitor Dinaciclib Exerts Potent Apoptotic and Antitumor Effects in Preclinical Models of MLL-Rearranged Acute Myeloid Leukemia.

    PubMed

    Baker, Adele; Gregory, Gareth P; Verbrugge, Inge; Kats, Lev; Hilton, Joshua J; Vidacs, Eva; Lee, Erwin M; Lock, Richard B; Zuber, Johannes; Shortt, Jake; Johnstone, Ricky W

    2016-03-01

    Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins. PMID:26627013

  5. A non-active site SET domain surface crucial for the interaction of MLL1 and the RbBP5-ASH2L heterodimer within MLL family core complexes

    PubMed Central

    Shinsky, Stephen A.; Hu, Michael; Vought, Valarie E.; Ng, Sarah B.; Bamshad, Michael J.; Shendure, Jay; Cosgrove, Michael S.

    2014-01-01

    The Mixed Lineage Leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1–4, and SET1d1a,b. Dimethylation of H3K4 requires a sub-complex including WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome (KS) missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5-Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or (KIS), are required for formation of a second active site within SET1 family core complexes. PMID:24680668

  6. Discovery of MLL1 binding units, their localization to CpG Islands, and their potential function in mitotic chromatin

    PubMed Central

    2013-01-01

    Background Mixed Lineage Leukemia 1 (MLL1) is a mammalian ortholog of the Drosophila Trithorax. In Drosophila, Trithorax complexes transmit the memory of active genes to daughter cells through interactions with Trithorax Response Elements (TREs). However, despite their functional importance, nothing is known about sequence features that may act as TREs in mammalian genomic DNA. Results By analyzing results of reported DNA binding assays, we identified several CpG rich motifs as potential MLL1 binding units (defined as morphemes). We find that these morphemes are dispersed within a relatively large collection of human promoter sequences and appear densely packed near transcription start sites of protein-coding genes. Genome wide analyses localized frequent morpheme occurrences to CpG islands. In the human HOX loci, the morphemes are spread across CpG islands and in some cases tail into the surrounding shores and shelves of the islands. By analyzing results of chromatin immunoprecipitation assays, we found a connection between morpheme occurrences, CpG islands, and chromatin segments reported to be associated with MLL1. Furthermore, we found a correspondence of reported MLL1-driven “bookmarked” regions in chromatin to frequent occurrences of MLL1 morphemes in CpG islands. Conclusion Our results implicate the MLL1 morphemes in sequence-features that define the mammalian TREs and provide a novel function for CpG islands. Apparently, our findings offer the first evidence for existence of potential TREs in mammalian genomic DNA and the first evidence for a connection between CpG islands and gene-bookmarking by MLL1 to transmit the memory of highly active genes during mitosis. Our results further suggest a role for overlapping morphemes in producing closely packed and multiple MLL1 binding events in genomic DNA so that MLL1 molecules could interact and reside simultaneously on extended potential transcriptional maintenance elements in human chromosomes to transmit the

  7. Novel Cryptic Rearrangements in Adult B-Cell Precursor Acute Lymphoblastic Leukemia Involving the MLL Gene.

    PubMed

    Othman, Moneeb A K; Grygalewicz, Beata; Pienkowska-Grela, Barbara; Rincic, Martina; Rittscher, Katharina; Melo, Joana B; Carreira, Isabel M; Meyer, Britta; Marzena, Watek; Liehr, Thomas

    2015-05-01

    MLL (mixed-lineage-leukemia) gene rearrangements are typical for acute leukemia and are associated with an aggressive course of disease, with a worse outcome than comparable case, and thus require intensified treatment. Here we describe a 69-year-old female with adult B cell precursor acute lymphoblastic leukemia (BCP-ALL) with hyperleukocytosis and immunophenotype CD10- and CD19+ with cryptic MLL rearrangements. G-banding at the time of diagnosis showed a normal karyotype: 46,XX. Molecular cytogenetics using multitude multicolor banding (mMCB) revealed a complex rearrangement of the two copies of chromosome 11. However, a locus-specific probe additionally identified that the MLL gene at 11q23.3 was disrupted, and that the 5' region was inserted into the chromosomal sub-band 4q21; thus the aberration involved three chromosomes and five break events. Unfortunately, the patient died six months after the initial diagnosis from serious infections and severe complications. Overall, the present findings confirm that, by far not all MLL aberrations are seen by routine chromosome banding techniques and that fluorescence in situ hybridization (FISH) should be regarded as standard tool to access MLL rearrangements in patients with BCP-ALL. PMID:25699572

  8. Essential role of PR-domain protein MDS1-EVI1 in MLL-AF9 leukemia

    PubMed Central

    Owens, Kristina; Hatem, Layla; Glass, Carolyn H.; Karuppaiah, Kannan; Camargo, Fernando

    2013-01-01

    A subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9–induced transformation both in vitro and in vivo. And, although Nup98-HOXA9, MEIS1-HOXA9, and E2A-Hlf could transform ME-deficient cells, both MLL-AF9 and MLL-ENL were ineffective, indicating that the ME requirement is specific to MLL fusion leukemia. Further, we show that the PR domain is essential for MFP-induced transformation. These studies clearly indicate an essential role of PR-domain protein ME in MFP leukemia, suggesting that ME may be a novel target for therapeutic intervention for this group of leukemias. PMID:24021671

  9. Aberrant overexpression and function of the miR-17-92 cluster in MLL-rearranged acute leukemia

    PubMed Central

    Mi, Shuangli; Li, Zejuan; Chen, Ping; He, Chunjiang; Cao, Donglin; Elkahloun, Abdel; Lu, Jun; Pelloso, Luis A.; Wunderlich, Mark; Huang, Hao; Luo, Roger T.; Sun, Miao; He, Miao; Neilly, Mary Beth; Zeleznik-Le, Nancy J.; Thirman, Michael J.; Mulloy, James C.; Liu, Paul P.; Rowley, Janet D.; Chen, Jianjun

    2010-01-01

    MicroRNA (miRNA)-17-92 cluster (miR-17-92), containing seven individual miRNAs, is frequently amplified and overexpressed in lymphomas and various solid tumors. We have found that it is also frequently amplified and the miRNAs are aberrantly overexpressed in mixed lineage leukemia (MLL)-rearranged acute leukemias. Furthermore, we show that MLL fusions exhibit a much stronger direct binding to the locus of this miRNA cluster than does wild-type MLL; these changes are associated with elevated levels of histone H3 acetylation and H3K4 trimethylation and an up-regulation of these miRNAs. We further observe that forced expression of this miRNA cluster increases proliferation and inhibits apoptosis of human cells. More importantly, we show that this miRNA cluster can significantly increase colony-forming capacity of normal mouse bone marrow progenitor cells alone and, particularly, in cooperation with MLL fusions. Finally, through combinatorial analysis of miRNA and mRNA arrays of mouse bone marrow progenitor cells transfected with this miRNA cluster and/or MLL fusion gene, we identified 363 potential miR-17-92 target genes that exhibited a significant inverse correlation of expression with the miRNAs. Remarkably, these potential target genes are significantly enriched (P < 0.01; >2-fold) in cell differentiation, hematopoiesis, cell cycle, and apoptosis. Taken together, our studies suggest that overexpression of miR-17-92 cluster in MLL-rearranged leukemias is likely attributed to both DNA copy number amplification and direct up-regulation by MLL fusions, and that the miRNAs in this cluster may play an essential role in the development of MLL-associated leukemias through inhibiting cell differentiation and apoptosis, while promoting cell proliferation, by regulating relevant target genes. PMID:20133587

  10. Hypomethylation and expression of BEX2, IGSF4 and TIMP3 indicative of MLL translocations in Acute Myeloid Leukemia

    PubMed Central

    Röhrs, Sonja; Dirks, Wilhelm G; Meyer, Claus; Marschalek, Rolf; Scherr, Michaela; Slany, Robert; Wallace, Andrew; Drexler, Hans G; Quentmeier, Hilmar

    2009-01-01

    Background Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines. Results Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLLmu AML cells, while 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu AML cell lines. MLLwt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion These results suggest

  11. NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1

    PubMed Central

    Aguilar-Arnal, Lorena; Katada, Sayako; Orozco-Solis, Ricardo

    2015-01-01

    The circadian clock controls the transcription of hundred genes through specific chromatin remodeling events. The histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) coordinates recruitment of CLOCK–BMAL1 activator complexes to chromatin, an event associated to cyclic H3K4 tri-methylation at circadian promoters. Remarkably, in mouse liver circadian H3K4me3 is modulated by SIRT1, a NAD+ dependent deacetylase involved in clock control. We show that mammalian MLL1 is acetylated at two conserved residues, K1130 and K1133. Notably, MLL1 acetylation is cyclic, controlled by the clock and by SIRT1, and impacts the methyltransferase activity of MLL1. Moreover, H3K4 methylation at clock-controlled gene promoters is influenced by pharmacological or genetic inactivation of SIRT1. Finally, MLL1 acetylation and H3K4me3 levels at circadian gene promoters depend on NAD+ circadian levels. These findings reveal a previously unappreciated regulatory pathway between energy metabolism and histone methylation. PMID:25751424

  12. Reprogramming of the Epigenome by MLL1 Links Early-Life Environmental Exposures to Prostate Cancer Risk.

    PubMed

    Wang, Quan; Trevino, Lindsey S; Wong, Rebecca Lee Yean; Medvedovic, Mario; Chen, Jing; Ho, Shuk-Mei; Shen, Jianjun; Foulds, Charles E; Coarfa, Cristian; O'Malley, Bert W; Shilatifard, Ali; Walker, Cheryl L

    2016-08-01

    Tissue and organ development is a time of exquisite sensitivity to environmental exposures, which can reprogram developing tissues to increase susceptibility to adult diseases, including cancer. In the developing prostate, even brief exposure to endocrine-disrupting chemicals (EDCs) can increase risk for developing cancer in adulthood, with disruption of the epigenome thought to play a key role in this developmental reprogramming. We find that EDC-induced nongenomic phosphoinositide 3-kinase; (PI3K) signaling engages the histone methyltransferase mixed-lineage leukemia 1 (MLL1), responsible for the histone H3 lysine 4 trimethylation (H3K4me3) active epigenetic mark, to increase cleavage and formation of active MLL1 dimers. In the developing prostate, EDC-induced MLL1 activation increased H3K4me3 at genes associated with prostate cancer, with increased H3K4me3 and elevated basal and hormone-induced expression of reprogrammed genes persisting into adulthood. These data identify a mechanism for MLL1 activation that is vulnerable to disruption by environmental exposures, and link MLL1 activation by EDCs to developmental reprogramming of genes involved in prostate cancer. PMID:27219490

  13. Neuronal Kmt2a/Mll1 histone methyltransferase is essential for prefrontal synaptic plasticity and working memory.

    PubMed

    Jakovcevski, Mira; Ruan, Hongyu; Shen, Erica Y; Dincer, Aslihan; Javidfar, Behnam; Ma, Qi; Peter, Cyril J; Cheung, Iris; Mitchell, Amanda C; Jiang, Yan; Lin, Cong L; Pothula, Venu; Stewart, A Francis; Ernst, Patricia; Yao, Wei-Dong; Akbarian, Schahram

    2015-04-01

    Neuronal histone H3-lysine 4 methylation landscapes are defined by sharp peaks at gene promoters and other cis-regulatory sequences, but molecular and cellular phenotypes after neuron-specific deletion of H3K4 methyl-regulators remain largely unexplored. We report that neuronal ablation of the H3K4-specific methyltransferase, Kmt2a/Mixed-lineage leukemia 1 (Mll1), in mouse postnatal forebrain and adult prefrontal cortex (PFC) is associated with increased anxiety and robust cognitive deficits without locomotor dysfunction. In contrast, only mild behavioral phenotypes were observed after ablation of the Mll1 ortholog Kmt2b/Mll2 in PFC. Impaired working memory after Kmt2a/Mll1 ablation in PFC neurons was associated with loss of training-induced transient waves of Arc immediate early gene expression critical for synaptic plasticity. Medial prefrontal layer V pyramidal neurons, a major output relay of the cortex, demonstrated severely impaired synaptic facilitation and temporal summation, two forms of short-term plasticity essential for working memory. Chromatin immunoprecipitation followed by deep sequencing in Mll1-deficient cortical neurons revealed downregulated expression and loss of the transcriptional mark, trimethyl-H3K4, at <50 loci, including the homeodomain transcription factor Meis2. Small RNA-mediated Meis2 knockdown in PFC was associated with working memory defects similar to those elicited by Mll1 deletion. Therefore, mature prefrontal neurons critically depend on maintenance of Mll1-regulated H3K4 methylation at a subset of genes with an essential role in cognition and emotion. PMID:25834037

  14. MLL5 Orchestrates a Cancer Self-Renewal State by Repressing the Histone Variant H3.3 and Globally Reorganizing Chromatin.

    PubMed

    Gallo, Marco; Coutinho, Fiona J; Vanner, Robert J; Gayden, Tenzin; Mack, Stephen C; Murison, Alex; Remke, Marc; Li, Ren; Takayama, Naoya; Desai, Kinjal; Lee, Lilian; Lan, Xiaoyang; Park, Nicole I; Barsyte-Lovejoy, Dalia; Smil, David; Sturm, Dominik; Kushida, Michelle M; Head, Renee; Cusimano, Michael D; Bernstein, Mark; Clarke, Ian D; Dick, John E; Pfister, Stefan M; Rich, Jeremy N; Arrowsmith, Cheryl H; Taylor, Michael D; Jabado, Nada; Bazett-Jones, David P; Lupien, Mathieu; Dirks, Peter B

    2015-12-14

    Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM. PMID:26626085

  15. Molecular mechanism of MLL PHD3 and RNA recognition by the Cyp33 RRM domain

    PubMed Central

    Hom, Robert A.; Chang, Pei-Yun; Roy, Siddhartha; Musselman, Catherine A.; Glass, Karen C.; Selezneva, Anna I.; Gozani, Or; Ismagilov, Rustem F.; Cleary, Michael L.; Kutateladze, Tatiana G.

    2010-01-01

    Summary The nuclear protein Cyclophilin 33 (Cyp33) is a peptidyl-prolyl cis-trans isomerase that catalyzes cis-trans isomerization of the peptide bond preceding a proline and promotes folding and conformational changes in folded and unfolded proteins. The N-terminal RRM domain of Cyp33 has been found to associate with the third plant homeodomain (PHD3) finger of the Mixed Lineage Leukemia (MLL) proto-oncoprotein and a poly-A RNA sequence. Here, we report a 1.9 Å resolution crystal structure of the RRM domain of Cyp33 and describe the molecular mechanism of PHD3 and RNA recognition. The Cyp33 RRM domain folds into a five-stranded antiparallel β-sheet and two α-helices. The RRM domain but not the catalytic module of Cyp33 binds strongly to PHD3, exhibiting a 2 μM affinity as measured by Isothermal Titration Calorimetry (ITC). NMR chemical shift perturbation (CSP) analysis and dynamics data reveal that the β strands and the β2–β3 loop of the RRM domain are involved in the interaction with PHD3. Mutations in the PHD3-binding site or deletions in the β 2/β 3 loop lead to a significantly reduced affinity or abrogation of the interaction. The RNA-binding pocket of the Cyp33 RRM domain, mapped based on NMR CSP and mutagenesis, partially overlaps with the PHD3-binding site, and RNA association is abolished in the presence of MLL PHD3. Full-length Cyp33 acts as a negative regulator of MLL-induced transcription and reduces the expression levels of MLL target genes MEIS1 and HOXA9. Together, these in vitro and in vivo data provide insight into the multiple functions of Cyp33 RRM and suggest a Cyp33-dependent mechanism for regulating the transcriptional activity of MLL. PMID:20460131

  16. miR-196b directly targets both HOXA9/MEIS1 oncogenes and FAS tumour suppressor in MLL-rearranged leukaemia

    PubMed Central

    Li, Zejuan; Huang, Hao; Chen, Ping; He, Miao; Li, Yuanyuan; Arnovitz, Stephen; Jiang, Xi; He, Chunjiang; Hyjek, Elizabeth; Zhang, Jun; Zhang, Zhiyu; Elkahloun, Abdel; Cao, Donglin; Shen, Chen; Wunderlich, Mark; Wang, Yungui; Neilly, Mary Beth; Jin, Jie; Wei, Minjie; Lu, Jun; Valk, Peter J.M.; Delwel, Ruud; Lowenberg, Bob; Le Beau, Michelle M.; Vardiman, James; Mulloy, James C.; Zeleznik-Le, Nancy J.; Liu, Paul P.; Zhang, Jiwang; Chen, Jianjun

    2012-01-01

    HOXA9, and MEIS1 have essential oncogenic roles in mixed lineage leukaemia (MLL)-rearranged leukaemia. Here we show that they are direct targets of miRNA-196b, a microRNA (miRNA) located adjacent to and co-expressed with HOXA9, in MLL-rearranged leukaemic cells. Forced expression of miR-196b significantly delays MLL-fusion-mediated leukemogenesis in primary bone marrow transplantation through suppressing Hoxa9/Meis1 expression. However, ectopic expression of miR-196b results in more aggressive leukaemic phenotypes and causes much faster leukemogenesis in secondary transplantation than MLL fusion alone, likely through the further repression of Fas expression, a proapoptotic gene downregulated in MLL-rearranged leukaemia. Overexpression of FAS significantly inhibits leukemogenesis and reverses miR-196b-mediated phenotypes. Targeting Hoxa9/Meis1 and Fas by miR-196b is probably also important for normal haematopoiesis. Thus, our results uncover a previously unappreciated miRNA-regulation mechanism by which a single miRNA may target both oncogenes and tumour suppressors, simultaneously, or, sequentially, in tumourigenesis and normal development per cell differentiation, indicating that miRNA regulation is much more complex than previously thought. PMID:22353710

  17. The miR-17∼92 cluster contributes to MLL leukemia through the repression of MEIS1 competitor PKNOX1.

    PubMed

    Mian, Yousaf A; Zeleznik-Le, Nancy J

    2016-07-01

    Mixed lineage leukemias have a relatively poor prognosis and arise as a result of translocations between the MLL(KMT2A) gene and one of multiple partner genes. Downstream targets of MLL are aberrantly upregulated and include the developmentally important HOX genes and MEIS1, as well as multiple microRNAs (miRNAs), including the miR-17∼92 cluster. Here we examined the contribution of specific miRNAs to MLL leukemias through knockdown studies utilizing custom anti-microRNA oligonucleotides. Combinatorial treatment against miR-17-5p and miR-19a-3p of the miR-17∼92 cluster dramatically reduces colony forming ability of MLL-fusion containing cell lines relative to non-MLL acute myeloid leukemia (AML) controls. To determine the mechanism by which these miRNAs contribute to leukemia, we validated PKNOX1 as a target of both miR-17-5p and miR-19a-3p. MEIS1 and PKNOX1 are TALE domain proteins that participate in ternary complexes with HOX and PBX partners. Here we establish the competitive relationship between PKNOX1 and MEIS1 in PBX-containing complex formation and determine the antagonistic role of PKNOX1 to leukemia in a murine MLL-AF9 model. These data implicate the miR-17∼92 cluster as part of a regulatory mechanism necessary to maintain MEIS1/HOXA9 -mediated transformation in MLL leukemia, indicating that targeting multiple non-homologous miRNAs may be utilized as a novel therapeutic regimen. PMID:27123834

  18. Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia.

    PubMed

    Grebien, Florian; Vedadi, Masoud; Getlik, Matthäus; Giambruno, Roberto; Grover, Amit; Avellino, Roberto; Skucha, Anna; Vittori, Sarah; Kuznetsova, Ekaterina; Smil, David; Barsyte-Lovejoy, Dalia; Li, Fengling; Poda, Gennadiy; Schapira, Matthieu; Wu, Hong; Dong, Aiping; Senisterra, Guillermo; Stukalov, Alexey; Huber, Kilian V M; Schönegger, Andreas; Marcellus, Richard; Bilban, Martin; Bock, Christoph; Brown, Peter J; Zuber, Johannes; Bennett, Keiryn L; Al-Awar, Rima; Delwel, Ruud; Nerlov, Claus; Arrowsmith, Cheryl H; Superti-Furga, Giulio

    2015-08-01

    The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML. PMID:26167872

  19. Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia.

    PubMed

    Sanjuan-Pla, Alejandra; Bueno, Clara; Prieto, Cristina; Acha, Pamela; Stam, Ronald W; Marschalek, Rolf; Menéndez, Pablo

    2015-12-17

    Infant B-cell acute lymphoblastic leukemia (B-ALL) accounts for 10% of childhood ALL. The genetic hallmark of most infant B-ALL is chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene. Despite improvement in the clinical management and survival (∼85-90%) of childhood B-ALL, the outcome of infants with MLL-rearranged (MLL-r) B-ALL remains dismal, with overall survival <35%. Among MLL-r infant B-ALL, t(4;11)+ patients harboring the fusion MLL-AF4 (MA4) display a particularly poor prognosis and a pro-B/mixed phenotype. Studies in monozygotic twins and archived blood spots have provided compelling evidence of a single cell of prenatal origin as the target for MA4 fusion, explaining the brief leukemia latency. Despite its aggressiveness and short latency, current progress on its etiology, pathogenesis, and cellular origin is limited as evidenced by the lack of mouse/human models recapitulating the disease phenotype/latency. We propose this is because infant cancer is from an etiologic and pathogenesis standpoint distinct from adult cancer and should be seen as a developmental disease. This is supported by whole-genome sequencing studies suggesting that opposite to the view of cancer as a "multiple-and-sequential-hit" model, t(4;11) alone might be sufficient to spawn leukemia. The stable genome of these patients suggests that, in infant developmental cancer, one "big-hit" might be sufficient for overt disease and supports a key contribution of epigenetics and a prenatal cell of origin during a critical developmental window of stem cell vulnerability in the leukemia pathogenesis. Here, we revisit the biology of t(4;11)+ infant B-ALL with an emphasis on its origin, genetics, and disease models. PMID:26463423

  20. Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia

    PubMed Central

    Bueno, Clara; Prieto, Cristina; Acha, Pamela; Stam, Ronald W.; Marschalek, Rolf; Menéndez, Pablo

    2015-01-01

    Infant B-cell acute lymphoblastic leukemia (B-ALL) accounts for 10% of childhood ALL. The genetic hallmark of most infant B-ALL is chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene. Despite improvement in the clinical management and survival (∼85-90%) of childhood B-ALL, the outcome of infants with MLL-rearranged (MLL-r) B-ALL remains dismal, with overall survival <35%. Among MLL-r infant B-ALL, t(4;11)+ patients harboring the fusion MLL-AF4 (MA4) display a particularly poor prognosis and a pro-B/mixed phenotype. Studies in monozygotic twins and archived blood spots have provided compelling evidence of a single cell of prenatal origin as the target for MA4 fusion, explaining the brief leukemia latency. Despite its aggressiveness and short latency, current progress on its etiology, pathogenesis, and cellular origin is limited as evidenced by the lack of mouse/human models recapitulating the disease phenotype/latency. We propose this is because infant cancer is from an etiologic and pathogenesis standpoint distinct from adult cancer and should be seen as a developmental disease. This is supported by whole-genome sequencing studies suggesting that opposite to the view of cancer as a “multiple-and-sequential-hit” model, t(4;11) alone might be sufficient to spawn leukemia. The stable genome of these patients suggests that, in infant developmental cancer, one “big-hit” might be sufficient for overt disease and supports a key contribution of epigenetics and a prenatal cell of origin during a critical developmental window of stem cell vulnerability in the leukemia pathogenesis. Here, we revisit the biology of t(4;11)+ infant B-ALL with an emphasis on its origin, genetics, and disease models. PMID:26463423

  1. p27kip1 Maintains a Subset of Leukemia Stem Cells in the Quiescent State in Murine MLL-Leukemia

    PubMed Central

    Zhang, Jun; Seet, Christopher; Sun, Clare; Li, Jing; You, Dewen; Volk, Andrew; Breslin, Peter; Li, Xingyu; Wei, Wei; Qian, Zhijian; Zeleznik-Le, Nancy J.; Zhang, Zhou; Zhang, Jiwang

    2013-01-01

    MLL (mixed-lineage leukemia)-fusion genes induce the development of leukemia through deregulation of normal MLL target genes, such as HOXA9 and MEIS1. Both HOXA9 and MEIS1 are required for MLL-fusion gene-induced leukemogenesis. Co-expression of HOXA9 and MEIS1 induces acute myeloid leukemia (AML) similar to that seen in mice in which MLL-fusion genes are over-expressed. p27kip1 (p27 hereafter), a negative regulator of the cell cycle, has also been defined as an MLL target, the expression of which is up-regulated in MLL leukemic cells (LCs). To investigate whether p27 plays a role in the pathogenesis of MLL-leukemia, we examined the effects of p27 deletion (p27-/-) on MLL-AF9 (MA9)-induced murine AML development. HOXA9/MEIS1 (H/M)-induced, p27 wild-type (p27+/+) and p27-/- AML were studied in parallel as controls. We found that LCs from both MA9-AML and H/M-AML can be separated into three fractions, a CD117-CD11bhi differentiated fraction as well as CD117+CD11bhi and CD117+CD11blo, two less differentiated fractions. The CD117+CD11blo fraction, comprising only 1-3% of total LCs, expresses higher levels of early hematopoietic progenitor markers but lower levels of mature myeloid cell markers compared to other populations of LCs. p27 is expressed and is required for maintaining the quiescent and drug-resistant states of the CD117+CD11blo fraction of MA9-LCs but not of H/M-LCs. p27 deletion significantly compromises the leukemogenic capacity of CD117+CD11blo MA9-LCs by reducing the frequency of leukemic stem cells (LSCs) but does not do so in H/M-LCs. In addition, we found that p27 is highly expressed and required for cell cycle arrest in the CD117-CD11bhi fraction in both types of LCs. Furthermore, we found that c-Myc expression is required for maintaining LCs in an undifferentiated state independently of proliferation. We concluded that p27 represses the proliferation of LCs, which is specifically required for maintaining the quiescent and drug-resistant states of a

  2. Menin critically links MLL proteins with LEDGF on cancer-associated target genes

    PubMed Central

    Yokoyama, Akihiko; Cleary, Michael L.

    2009-01-01

    Summary Menin displays the unique ability to either promote oncogenic function in the hematopoietic lineage or suppress tumorigenesis in the endocrine lineage, however its molecular mechanism of action has not been defined. We demonstrate here that these discordant functions are unified by menin's ability to serve as a molecular adaptor that physically links the MLL histone methyltransferase with LEDGF, a chromatin-associated protein previously implicated in leukemia, auto-immunity and HIV-1 pathogenesis. LEDGF is required for both MLL-dependent transcription and leukemic transformation. Conversely, a subset of menin mutations in MEN1 patients abrogates interaction with LEDGF while preserving MLL interaction, but nevertheless compromises MLL/menin-dependent functions. Thus, LEDGF critically associates with MLL and menin at the nexus of transcriptional pathways that are recurrently targeted in diverse diseases. PMID:18598942

  3. High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis

    PubMed Central

    Kao, Hsiao-Wen; Liang, Der-Cherng; Kuo, Ming-Chung; Wu, Jin-Hou; Dunn, Po; Wang, Po-Nan; Lin, Tung-Liang; Shih, Yu-Shu; Liang, Sung-Tzu; Lin, Tung-Huei; Lai, Chen-Yu; Lin, Chun-Hui; Shih, Lee-Yung

    2015-01-01

    The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML. PMID:26375248

  4. The promiscuous MLL gene links chromosomal translocations to cellular differentiation and tumour tropism.

    PubMed

    Collins, Emma C; Rabbitts, Terence H

    2002-09-01

    MLL is a promiscuous gene involved in a diversity of chromosomal fusions in haematological malignancies, usually resulting from chromosomal translocations. MLL-associated chromosomal rearrangements usually occur in tumours of specific haematological lineages, suggesting a crucial role for the MLL fusion partner in determining disease phenotype (or tumour tropism). The MLL gene is homologous to Drosophila trithorax, and is likewise involved in embryo pattern formation. Common themes linking several of the MLL partners include a possible involvement in embryo patterning via Hox gene regulation and chromatin remodelling. These findings reinforce the link between developmental regulation and chromosomal translocations, and indicate the role of chromosomal translocation in activating genes capable of determining tumour phenotype in leukaemias and sarcomas. PMID:12223315

  5. MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199

    PubMed Central

    Benito, Juliana M.; Godfrey, Laura; Kojima, Kensuke; Hogdal, Leah; Wunderlich, Mark; Geng, Huimin; Marzo, Isabel; Harutyunyan, Karine G.; Golfman, Leonard; North, Phillip; Kerry, Jon; Ballabio, Erica; Chonghaile, Triona Ní; Gonzalo, Oscar; Qiu, Yihua; Jeremias, Irmela; Debose, LaKiesha; O’Brien, Eric; Ma, Helen; Zhou, Ping; Jacamo, Rodrigo; Park, Eugene; Coombes, Kevin R.; Zhang, Nianxiang; Thomas, Deborah A.; O’Brien, Susan; Kantarjian, Hagop M.; Leverson, Joel D.; Kornblau, Steven M.; Andreeff, Michael; Müschen, Markus; Zweidler-McKay, Patrick A.; Mulloy, James C.; Letai, Anthony; Milne, Thomas A.; Konopleva, Marina

    2015-01-01

    Summary Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias. PMID:26711339

  6. MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199.

    PubMed

    Benito, Juliana M; Godfrey, Laura; Kojima, Kensuke; Hogdal, Leah; Wunderlich, Mark; Geng, Huimin; Marzo, Isabel; Harutyunyan, Karine G; Golfman, Leonard; North, Phillip; Kerry, Jon; Ballabio, Erica; Chonghaile, Triona Ní; Gonzalo, Oscar; Qiu, Yihua; Jeremias, Irmela; Debose, LaKiesha; O'Brien, Eric; Ma, Helen; Zhou, Ping; Jacamo, Rodrigo; Park, Eugene; Coombes, Kevin R; Zhang, Nianxiang; Thomas, Deborah A; O'Brien, Susan; Kantarjian, Hagop M; Leverson, Joel D; Kornblau, Steven M; Andreeff, Michael; Müschen, Markus; Zweidler-McKay, Patrick A; Mulloy, James C; Letai, Anthony; Milne, Thomas A; Konopleva, Marina

    2015-12-29

    Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias. PMID:26711339

  7. Clinical multiplexed exome sequencing distinguishes adult oligodendroglial neoplasms from astrocytic and mixed lineage gliomas

    PubMed Central

    Cryan, Jane B.; Haidar, Sam; Ramkissoon, Lori A.; Bi, Wenya Linda; Knoff, David S.; Schultz, Nikolaus; Abedalthagafi, Malak; Brown, Loreal; Wen, Patrick Y.; Reardon, David A.; Dunn, Ian F.; Folkerth, Rebecca D.; Santagata, Sandro; Lindeman, Neal I.; Ligon, Azra H.; Beroukhim, Rameen; Hornick, Jason L.; Alexander, Brian M.; Ligon, Keith L.; Ramkissoon, Shakti H.

    2014-01-01

    Classifying adult gliomas remains largely a histologic diagnosis based on morphology; however astrocytic, oligodendroglial and mixed lineage tumors can display overlapping histologic features. We used multiplexed exome sequencing (OncoPanel) on 108 primary or recurrent adult gliomas, comprising 65 oligodendrogliomas, 28 astrocytomas and 15 mixed oligoastrocytomas to identify lesions that could enhance lineage classification. Mutations in TP53 (20/28, 71%) and ATRX (15/28, 54%) were enriched in astrocytic tumors compared to oligodendroglial tumors of which 4/65 (6%) had mutations in TP53 and 2/65 (3%) had ATRX mutations. We found that oligoastrocytomas harbored mutations in TP53 (80%, 12/15) and ATRX (60%, 9/15) at frequencies similar to pure astrocytic tumors, suggesting that oligoastrocytomas and astrocytomas may represent a single genetic or biological entity. p53 protein expression correlated with mutation status and showed significant increases in astrocytomas and oligoastrocytomas compared to oligodendrogliomas, a finding that also may facilitate accurate classification. Furthermore our OncoPanel analysis revealed that 15% of IDH1/2 mutant gliomas would not be detected by traditional IDH1 (p.R132H) antibody testing, supporting the use of genomic technologies in providing clinically relevant data. In all, our results demonstrate that multiplexed exome sequencing can support evaluation and classification of adult low-grade gliomas with a single clinical test. PMID:25257301

  8. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy.

    PubMed

    Gardner, Rebecca; Wu, David; Cherian, Sindhu; Fang, Min; Hanafi, Laïla-Aïcha; Finney, Olivia; Smithers, Hannah; Jensen, Michael C; Riddell, Stanley R; Maloney, David G; Turtle, Cameron J

    2016-05-19

    Administration of lymphodepletion chemotherapy followed by CD19-specific chimeric antigen receptor (CAR)-modified T cells is a remarkably effective approach to treating patients with relapsed and refractory CD19(+) B-cell malignancies. We treated 7 patients with B-cell acute lymphoblastic leukemia (B-ALL) harboring rearrangement of the mixed lineage leukemia (MLL) gene with CD19 CAR-T cells. All patients achieved complete remission (CR) in the bone marrow by flow cytometry after CD19 CAR-T-cell therapy; however, within 1 month of CAR-T-cell infusion, 2 of the patients developed acute myeloid leukemia (AML) that was clonally related to their B-ALL, a novel mechanism of CD19-negative immune escape. These reports have implications for the management of patients with relapsed and refractory MLL-B-ALL who receive CD19 CAR-T-cell therapy. PMID:26907630

  9. Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

    PubMed

    Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

    2016-09-01

    Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. PMID:27259981

  10. Impact of loss of BH3-only proteins on the development and treatment of MLL-fusion gene-driven AML in mice.

    PubMed

    Bilardi, Rebecca A; Anstee, Natasha S; Glaser, Stefan P; Robati, Mikara; Vandenberg, Cassandra J; Cory, Suzanne

    2016-01-01

    Inhibition of the apoptosis pathway controlled by opposing members of the Bcl-2 protein family plays a central role in cancer development and resistance to therapy. To investigate how pro-apoptotic Bcl-2 homology domain 3 (BH3)-only proteins impact on acute myeloid leukemia (AML), we generated mixed lineage leukemia (MLL)-AF9 and MLL-ENL AMLs from BH3-only gene knockout mice. Disease development was not accelerated by loss of Bim, Puma, Noxa, Bmf, or combinations thereof; hence these BH3-only proteins are apparently ineffectual as tumor suppressors in this model. We tested the sensitivity of MLL-AF9 AMLs of each genotype in vitro to standard chemotherapeutic drugs and to the proteasome inhibitor bortezomib, with or without the BH3 mimetic ABT-737. Loss of Puma and/or Noxa increased resistance to cytarabine, daunorubicin and etoposide, while loss of Bim protected against cytarabine and loss of Bmf had no impact. ABT-737 increased sensitivity to the genotoxic drugs but was not dependent on any BH3-only protein tested. The AML lines were very sensitive to bortezomib and loss of Noxa conveyed significant resistance. In vivo, several MLL-AF9 AMLs responded well to daunorubicin and this response was highly dependent on Puma and Noxa but not Bim. Combination therapy with ABT-737 provided little added benefit at the daunorubicin dose trialed. Bortezomib also extended survival of AML-bearing mice, albeit less than daunorubicin. In summary, our genetic studies reveal the importance of Puma and Noxa for the action of genotoxics currently used to treat MLL-driven AML and suggest that, while addition of ABT-737-like BH3 mimetics might enhance their efficacy, new Noxa-like BH3 mimetics targeting Mcl-1 might have greater potential. PMID:27584789

  11. Chlorpyrifos Induces MLL Translocations Through Caspase 3-Dependent Genomic Instability and Topoisomerase II Inhibition in Human Fetal Liver Hematopoietic Stem Cells.

    PubMed

    Lu, Chengquan; Liu, Xiaohui; Liu, Chang; Wang, Jian; Li, Chunna; Liu, Qi; Li, Yachen; Li, Shuangyue; Sun, Shu; Yan, Jinsong; Shao, Jing

    2015-10-01

    Household pesticide exposure during pregnancy has been associated with a more than 2-fold increased risk in infant leukemia, and chlorpyrifos (CPF) is among the most frequently applied insecticides. During early fetal development, liver is a hematopoietic organ with majority of cells being CD34(+) hematopoietic stem cells (CD34(+)HSC). The in utero injury to CD34(+)HSC has been known to underlie the pathogenesis of several blood disorders, often involving rearrangements of the mixed-lineage leukemia (MLL) gene on 11q23. In this study, we evaluated the leukemogenic potential of CPF in human fetal liver-derived CD34(+)HSC. Specifically, exposure to 10 μM CPF led to decrease in viability, inhibition in proliferation and induction of DNA double-strand breaks (DSBs) and occurrence of MLL(+) rearrangements. In particular, we observed CPF-mediated cell cycle disturbance as shown by G0/G1 arrest, in contrast to etoposide (VP-16), an anticancer drug used as a positive control and known to induce G2/M arrest. Further study on mechanisms underlying DNA DSBs and MLL(+) rearrangements revealed that CPF might act as topoisomerase II poison, a mechanism of action similar to VP-16. On the other hand, CPF was also shown to induce early apoptosis through active caspase-3 activation, a pathway known to underlie DNA DSBs and MLL(+) translocations. Our data indicate that in utero injury of CD34(+)HSC by CPF may contribute to the increased risk of infant leukemia. Future work will elucidate the mechanism and the type of CPF-induced MLL(+) translocations in HSC. PMID:26198043

  12. The E3 ligase CHIP mediates ubiquitination and degradation of mixed-lineage kinase 3.

    PubMed

    Blessing, Natalya A; Brockman, April L; Chadee, Deborah N

    2014-08-01

    Mixed-lineage kinase 3 (MLK3) activates mitogen-activated protein kinase (MAPK) signaling pathways and has important functions in migration, invasion, proliferation, tumorigenesis, and apoptosis. We investigated the role of the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) in the regulation of MLK3 protein levels. We show that CHIP interacts with MLK3 and, together with the E2 ubiquitin-conjugating enzyme UbcH5 (UbcH5a, -b, -c, or -d), ubiquitinates MLK3 in vitro. CHIP or Hsp70 overexpression promoted endogenous MLK3 ubiquitination and induced a decline in MLK3 protein levels in cells with Hsp90 inhibition. Furthermore, CHIP overexpression caused a proteasome-dependent reduction in exogenous MLK3 protein. Geldanamycin (GA), heat shock, and osmotic shock treatments also reduced the level of MLK3 protein via a CHIP-dependent mechanism. In addition, CHIP depletion in ovarian cancer SKOV3 cells increased cell invasion, and the enhancement of invasiveness was abrogated by small interfering RNA (siRNA)-mediated knockdown of MLK3. Thus, CHIP modulates MLK3 protein levels in response to GA and stress stimuli, and CHIP-dependent regulation of MLK3 is required for suppression of SKOV3 ovarian cancer cell invasion. PMID:24912674

  13. Discovery, Synthesis and Characterization of an Orally Bioavailable, Brain Penetrant Inhibitor of Mixed Lineage Kinase 3

    PubMed Central

    Goodfellow, Val S.; Loweth, Colin J.; Ravula, Satheesh B.; Wiemann, Torsten; Nguyen, Thong; Xu, Yang; Todd, Daniel E.; Sheppard, David; Pollack, Scott; Polesskaya, Oksana; Marker, Daniel F.; Dewhurst, Stephen; Gelbard, Harris A.

    2014-01-01

    Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson’s Disease and HIV-1 Associated Neurocognitive Disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes, and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development. PMID:24044867

  14. Regulation of mixed-lineage kinase activation in JNK-dependent morphogenesis

    PubMed Central

    Garlena, Rebecca A.; Gonda, Rebecca L.; Green, Alyssa B.; Pileggi, Rachel M.; Stronach, Beth

    2010-01-01

    Normal cells respond appropriately to various signals, while sustaining proper developmental programs and tissue homeostasis. Inappropriate signal reception, response or attenuation, can upset the normal balance of signaling within cells, leading to dysfunction or tissue malformation. To understand the molecular mechanisms that regulate protein-kinase-based signaling in the context of tissue morphogenesis, we analyzed the domain requirements of Drosophila Slpr, a mixed-lineage kinase (MLK), for Jun N-terminal kinase (JNK) signaling. The N-terminal half of Slpr is involved in regulated signaling whereas the C-terminal half promotes cortical protein localization. The SH3 domain negatively regulates Slpr activity consistent with autoinhibition via a conserved proline motif. Also, like many kinases, conserved residues in the activation segment of the catalytic domain regulate Slpr. Threonine 295, in particular, is essential for function. Slpr activation requires dual input from the MAP4K Misshapen (Msn), through its C-terminal regulatory domain, and the GTPase Rac, which both bind to the LZ–CRIB region of Slpr in vitro. Although Rac is sufficient to activate JNK signaling, our results indicate that there are Slpr-independent functions for Rac in dorsal closure. Finally, expression of various Slpr constructs alone or with upstream activators reveals a wide-ranging response at the cell and tissue level. PMID:20736302

  15. Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors

    PubMed Central

    Marusiak, Anna A.; Edwards, Zoe C.; Hugo, Willy; Trotter, Eleanor W.; Girotti, Maria R.; Stephenson, Natalie L.; Kong, Xiangju; Gartside, Michael G.; Fawdar, Shameem; Hudson, Andrew; Breitwieser, Wolfgang; Hayward, Nicholas K.; Marais, Richard; Lo, Roger S.; Brognard, John

    2014-01-01

    RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2–18 months. Here we demonstrate that the mixed lineage kinases (MLK1–4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1–4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors. PMID:24849047

  16. Lessons Learned: HIV Points the Way Towards Precision Treatment of Mixed-Lineage Leukemia.

    PubMed

    Cermakova, Katerina; Weydert, Caroline; Christ, Frauke; De Rijck, Jan; Debyser, Zeger

    2016-08-01

    Protein-protein interactions are involved in most if not all pathogenic and pathophysiological processes and represent attractive therapeutic targets. Extensive biological and clinical research efforts have led to the identification and validation of several cellular hubs that are crucially involved in disease pathogenesis. An interesting example of such a hub is the lens epithelium-derived growth factor (LEDGF/p75), a protein that tethers multiple unrelated proteins and protein complexes to the chromatin. Its chromatin-tethering ability is linked to at least two unrelated diseases-HIV infection and MLL-rearranged acute leukemia. In this review we discuss recent progress in our understanding of the interaction of LEDGF/p75 with its binding partners and focus on the first steps towards therapies targeting protein-protein interactions of LEDGF/p75. PMID:27290878

  17. Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

    SciTech Connect

    Zhan, Yu; Abi Saab, Widian F.; Modi, Nidhi; Stewart, Amanda M.; Liu, Jinsong; Chadee, Deborah N.

    2012-08-15

    Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells. -- Highlights: Black-Right-Pointing-Pointer Ovarian cancer cell lines have high levels of total and phosphorylated MLK3. Black-Right-Pointing-Pointer MLK3 is required for MMP expression and activity in ovarian cancer cells. Black-Right-Pointing-Pointer MLK3 is required for invasion of SKOV3 and HEY1B ovarian cancer cells. Black-Right-Pointing-Pointer MLK3-dependent regulation of MMP-2 and MMP-9 activities requires ERK and JNK.

  18. Mll5 Is Required for Normal Spermatogenesis

    PubMed Central

    Yap, Damian B.; Walker, David C.; Prentice, Leah M.; McKinney, Steven; Turashvili, Gulisa; Mooslehner-Allen, Katrin; de Algara, Teresa Ruiz; Fee, John; de Tassigny, Xavier d'Anglemont; Colledge, William H.; Aparicio, Samuel

    2011-01-01

    Background Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis. Methodology/Principal Findings Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated. Conclusions/Significance Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects. PMID:22069496

  19. Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice

    PubMed Central

    Schröter, David; Matthews, Helen C.; Bogani, Debora; Moir, Lee; Long, Anna; Church, Christopher; Hugill, Alison; Anstee, Quentin M.; Goldin, Rob; Thursz, Mark; Hollfelder, Florian; Cox, Roger D.

    2013-01-01

    We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. PMID:23826075

  20. The mixed lineage kinase-3 inhibitor URMC-099 improves therapeutic outcomes for long-acting antiretroviral therapy.

    PubMed

    Zhang, Gang; Guo, Dongwei; Dash, Prasanta K; Araínga, Mariluz; Wiederin, Jayme L; Haverland, Nicole A; Knibbe-Hollinger, Jaclyn; Martinez-Skinner, Andrea; Ciborowski, Pawel; Goodfellow, Val S; Wysocki, Tadeusz A; Wysocki, Beata J; Poluektova, Larisa Y; Liu, Xin-Ming; McMillan, JoEllyn M; Gorantla, Santhi; Gelbard, Harris A; Gendelman, Howard E

    2016-01-01

    During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the mixed lineage kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2Rγc-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance. From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the mixed lineage kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future. PMID:26472049

  1. Mixed acinar-neuroendocrine-ductal carcinoma of the pancreas: a tale of three lineages.

    PubMed

    Anderson, Mark J; Kwong, Christina A; Atieh, Mohammed; Pappas, Sam G

    2016-01-01

    Most pancreatic cancers arise from a single cell type, although mixed pancreatic carcinomas represent a rare exception. The rarity of these aggressive malignancies and the limitations of fine-needle aspiration (FNA) pose significant barriers to diagnosis and appropriate management. We report a case of a 54-year-old man presenting with abdominal pain, jaundice and a hypodense lesion within the uncinate process on CT. FNA suggested poorly differentiated adenocarcinoma, which was subsequently resected via pancreaticoduodenectomy. Pathological analysis yielded diagnosis of invasive mixed acinar-neuroendocrine-ductal pancreatic carcinoma. Given the rare and deadly nature of these tumours, clinicians must be aware of their pathophysiology and do practice with a high degree of clinical suspicion, when appropriate. Surgical resection and thorough pathological analysis with immunohistochemical staining and electron microscopy remain the standards of care for mixed pancreatic tumours without gross evidence of metastasis. Diligent characterisation of the presentation and histological findings associated with these neoplasms should continue in order to promote optimal diagnostic and therapeutic strategies. PMID:27257019

  2. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.

    PubMed

    Wang, Huayi; Sun, Liming; Su, Lijing; Rizo, Josep; Liu, Lei; Wang, Li-Feng; Wang, Fu-Sheng; Wang, Xiaodong

    2014-04-10

    Programmed necrotic cell death induced by the tumor necrosis factor alpha (TNF-α) family of cytokines is dependent on a kinase cascade consisting of receptor-interacting kinases RIP1 and RIP3. How these kinase activities cause cells to die by necrosis is not known. The mixed lineage kinase domain-like protein MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. RIP3 phosphorylates MLKL at the T357 and S358 sites. Reported here is the development of a monoclonal antibody that specifically recognizes phosphorylated MLKL in cells dying of this pathway and in human liver biopsy samples from patients suffering from drug-induced liver injury. The phosphorylated MLKL forms an oligomer that binds to phosphatidylinositol lipids and cardiolipin. This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death. PMID:24703947

  3. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

    PubMed Central

    Chen, Xin; Li, Wenjuan; Ren, Junming; Huang, Deli; He, Wan-ting; Song, Yunlong; Yang, Chao; Li, Wanyun; Zheng, Xinru; Chen, Pengda; Han, Jiahuai

    2014-01-01

    Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture. PMID:24366341

  4. Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid.

    PubMed Central

    Rasmussen, R K; Rusak, J; Price, G; Robinson, P J; Simpson, R J; Dorow, D S

    1998-01-01

    Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin. PMID:9742220

  5. MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome

    PubMed Central

    Wang, Qian-fei; Wu, George; Mi, Shuangli; He, Fuhong; Wu, Jun; Dong, Jingfang; Luo, Roger T.; Mattison, Ryan; Kaberlein, Joseph J.; Prabhakar, Shyam; Ji, Hongkai

    2011-01-01

    MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations result in the formation of chimeric MLL fusion proteins that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of MLL fusions. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL model, MLL fusion protein binding and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL-ENL–bound genes, only 12 demonstrate a significant increase in mRNA expression on induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1, which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes. PMID:21518926

  6. Loss of MLL PHD-finger-3 is necessary for MLL-ENL-induced hematopoietic stem cell immortalization

    PubMed Central

    Chen, Jing; Santillan, Donna A.; Koonce, Mark; Wei, Wei; Luo, Roger; Thirman, Michael J.; Zeleznik-Le, Nancy J.; Diaz, Manuel O.

    2009-01-01

    Reciprocal chromosomal translocations at the MLL gene locus result in expression of novel fusion proteins such as MLL-ENL associated with leukemia. The three PHD finger cassette, one of the highly conserved domains in MLL, is absent in all fusion proteins. This domain has been shown to interact with Cyp33, a cyclophilin which enhances the recruitment of HDACs to the MLL repression domain and mediates HOX gene repression. Insertion of the third PHD finger of MLL, into MLL-ENL allows the recruitment of Cyp33, and subsequently HDAC1, to the fusion protein. Furthermore, expression of the fusion protein with the PHD finger insertion mediates the down-regulation of the HOXC8 gene expression in a Cyp33 dependent manner. Finally, the addition of the PHD finger domain or the 3rd PHD finger alone, into MLL-ENL, blocks the hematopoietic-stem-cell immortalization potential of the fusion protein in serial plating colony assays. Insertion of only the 1st and 2nd PHD fingers has no such effect. These data support the hypothesis that the binding of Cyp33 to the MLL 3rd PHD finger switches the MLL function from trans-activation to repression. In the immortalizing MLL fusion protein, the loss of the PHD fingers, in combination with the gain of the activation domain of ENL, or of other partner proteins, makes the fusion protein a constitutive trans-activator. This leads to constitutive over expression of MLL target genes that block stem cell commitment and promote stem cell renewal, probably the first step in MLL-related leukemogenesis. PMID:18676843

  7. Performance of the MLL-IonCatcher

    NASA Astrophysics Data System (ADS)

    Neumayr, J. B.; Thirolf, P. G.; Habs, D.; Heinz, S.; Kolhinen, V. S.; Sewtz, M.; Szerypo, J.

    2006-06-01

    The MLL-IonCatcher is a new setup for the thermalization of fusion-evaporation reaction products in highly pure helium with subsequent extraction of the stopped, singly charged ions. The setup consists of a buffer-gas stopping cell and a radio frequency quadrupole (RFQ)-based extraction system and is placed at the Tandem accelerator facility of the MLL in Garching. Test experiments were performed using the α emitter Er152 with total kinetic energies of around 180keV/u, produced via the reaction Sb121(Cl35,4n). During the on-line measurements the number of ions entering the stopping chamber through the entrance window was determined via the detection of their specific α-decay energy. After being thermalized in the helium buffer gas at 40-140mbar the ions were guided by a combination of electric rf and dc fields towards the extraction nozzle where the ion transport was taken over by the gas flow. Subsequent to the extraction by a supersonic gas jet the ions were separated from the buffer gas and guided by the extraction RFQ towards a Si detector, where the specific α-decay energy was detected. Depending on the electric field strength and the pressure of the buffer gas an overall efficiency including stopping and extraction between 10% and 16% has been achieved.

  8. The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression

    PubMed Central

    Xia, Zhen-Biao; Popovic, Relja; Chen, Jing; Theisler, Catherine; Stuart, Tara; Santillan, Donna A.; Erfurth, Frank; Diaz, Manuel O.; Zeleznik-Le, Nancy J.

    2005-01-01

    MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27kip1) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias. PMID:16169901

  9. Evolving Catalytic Properties of the MLL Family SET Domain

    PubMed Central

    Zhang, Ying; Mittal, Anshumali; Reid, James; Reich, Stephanie; Gamblin, Steven J.; Wilson, Jon R.

    2015-01-01

    Summary Methylation of histone H3 lysine-4 is a hallmark of chromatin associated with active gene expression. The activity of H3K4-specific modification enzymes, in higher eukaryotes the MLL (or KMT2) family, is tightly regulated. The MLL family has six members, each with a specialized function. All contain a catalytic SET domain that associates with a core multiprotein complex for activation. These SET domains segregate into three classes that correlate with the arrangement of targeting domains that populate the rest of the protein. Here we show that, unlike MLL1, the MLL4 SET domain retains significant activity without the core complex. We also present the crystal structure of an inactive MLL4-tagged SET domain construct and describe conformational changes that account for MLL4 intrinsic activity. Finally, our structure explains how the MLL SET domains are able to add multiple methyl groups to the target lysine, despite having the sequence characteristics of a classical monomethylase. PMID:26320581

  10. B-cell acute lymphoblastic leukemia with mature phenotype and MLL rearrangement: report of five new cases and review of the literature.

    PubMed

    Sajaroff, Elisa Olga; Mansini, Adrian; Rubio, Patricia; Alonso, Cristina Noemí; Gallego, Marta S; Coccé, Mariela C; Eandi-Eberle, Silvia; Bernasconi, Andrea Raquel; Ampatzidou, Maria; Paterakis, George; Papadhimitriou, Stefanos I; Petrikkos, Loizos; Papadakis, Vassilios; Polychronopoulou, Sophia; Rossi, Jorge G; Felice, Maria Sara

    2016-10-01

    The association between mature-B phenotype and MLL abnormalities in acute lymphoblastic leukemia (ALL) is a very unusual finding; only 14 pediatric cases have been reported so far. We describe the clinical and biological characteristics and outcome of five pediatric cases of newly diagnosed B lineage ALL with MLL abnormalities and mature immunophenotype based on light chain restriction and surface Ig expression. Blasts showed variable expression of CD10/CD34/TdT. MLL abnormalities with no MYC involvement were detected in all patients by G-banding, FISH, and/or RT-PCR. Three patients were treated according to Interfant protocol, one to ALLIC-09, and one received B-NHL-BFM-2004. All patients achieved complete remission and three of them relapsed. Despite the small cohort size, it could be postulated that B lineage ALL with MLL abnormalities and mature phenotype is a distinct entity that differs both from the typical Pro B ALL observed in infants and mature B-ALL with high MYC expression. PMID:26857438

  11. Rebuilding Synaptic Architecture in HIV-1 Associated Neurocognitive Disease (HAND) – A Therapeutic Strategy Based on Modulation of Mixed Lineage Kinase

    PubMed Central

    Gelbard, Harris A.; Dewhurst, Stephen; Maggirwar, Sanjay B.; Kiebala, Michelle; Polesskaya, Oksana; Gendelman, Howard E.

    2010-01-01

    Work from our laboratories has validated mixed lineage kinase type 3 (MLK3) as an enzyme pathologically activated in the central nervous system (CNS) by HIV-1 neurotoxins. In this review, we discuss MLK3 activation in the context of the neuropathogenesis of HIV-1 associated neurocognitive deficits (HAND). We use findings from the literature to substantiate the neuropathologic relevance of MLK3 to neurodegenerative disease, with an emphasis on Parkinson’s disease (PD) that shares a number of important phenotypic and neuropathologic characteristics with HAND. We discuss signal transduction pathways downstream from MLK3 activation, with an emphasis on their involvement in microglia and neurons in preclinical models of HAND. Finally, we make a case for pharmacologic intervention targeted at inhibition of MLK3 as a strategy to reverse HAND in light of the fact that combination antiretroviral therapy, despite successfully managing systemic infection of HIV-1, has been largely unsuccessful in eradicating HAND. PMID:20880503

  12. Chromodomain-helicase-DNA binding protein 5, 7 and pronecrotic mixed lineage kinase domain-like protein serve as potential prognostic biomarkers in patients with resected pancreatic adenocarcinomas

    PubMed Central

    Seldon, Crystal S; Colbert, Lauren E; Hall, William A; Fisher, Sarah B; Yu, David S; Landry, Jerome C

    2016-01-01

    Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomain-helicase-DNA binding protein 5, chromodomain-helicase-DNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation, and should be validated in future studies. PMID:27096031

  13. Identification of MLL-fusion/MYC⊣miR-26⊣TET1 signaling circuit in MLL-rearranged leukemia.

    PubMed

    Huang, Hao; Jiang, Xi; Wang, Jinhua; Li, Yuanyuan; Song, Chun-Xiao; Chen, Ping; Li, Shenglai; Gurbuxani, Sandeep; Arnovitz, Stephen; Wang, Yungui; Weng, Hengyou; Neilly, Mary Beth; He, Chuan; Li, Zejuan; Chen, Jianjun

    2016-03-28

    Expression of functionally important genes is often tightly regulated at both transcriptional and post-transcriptional levels. We reported previously that TET1, the founding member of the TET methylcytosine dioxygenase family, plays an essential oncogenic role in MLL-rearranged acute myeloid leukemia (AML), where it is overexpressed owing to MLL-fusion-mediated direct up-regulation at the transcriptional level. Here we show that the overexpression of TET1 in MLL-rearranged AML also relies on the down-regulation of miR-26a, which directly negatively regulates TET1 expression at the post-transcriptional level. Through inhibiting expression of TET1 and its downstream targets, forced expression of miR-26a significantly suppresses the growth/viability of human MLL-rearranged AML cells, and substantially inhibits MLL-fusion-mediated mouse hematopoietic cell transformation and leukemogenesis. Moreover, c-Myc, an oncogenic transcription factor up-regulated in MLL-rearranged AML, mediates the suppression of miR-26a expression at the transcriptional level. Collectively, our data reveal a previously unappreciated signaling pathway involving the MLL-fusion/MYC⊣miR-26a⊣TET1 signaling circuit, in which miR-26a functions as an essential tumor-suppressor mediator and its transcriptional repression is required for the overexpression and oncogenic function of TET1 in MLL-rearranged AML. Thus, restoration of miR-26a expression/function holds therapeutic potential to treat MLL-rearranged AML. PMID:26791235

  14. Mll5 contributes to hematopoietic stem cell fitness and homeostasis

    PubMed Central

    Zhang, Yan; Wong, Jasmine; Klinger, Mark; Tran, Mary T.; Shannon, Kevin M.

    2009-01-01

    MLL5 is a novel trithorax group gene and a candidate tumor suppressor gene located within a 2.5-Mb interval of chromosome band 7q22 that frequently is deleted in human myeloid malignancy. Here we show that inactivation of the Mll5 gene in mice results in a 30% reduction in the average representation of hematopoietic stem cells and in functional impairment of long-term hematopoietic repopulation potential under competitive conditions. Bone marrow cells from Mll5-deficient mice were defective in spleen colony-forming assays, and the mutant mice showed enhanced susceptibility to 5-fluorouracil–induced myelosuppression. Heterozygous and homozygous Mll5 mutant mice did not spontaneously develop hematologic cancers, and loss of Mll5 did not alter the phenotype of a fatal myeloproliferative disorder induced by oncogenic Kras in vivo. Collectively, the data reveal an important role for Mll5 in HSC homeostasis and provide a basis for further studies to explore its role in leukemogenesis. PMID:18818388

  15. Molecular characterization of a rare MLL-AF4 (MLL-AFF1) fusion rearrangement in infant leukemia.

    PubMed

    Bizarro, Susana; Cerveira, Nuno; Correia, Cecília; Lisboa, Susana; Peixoto, Ana; Norton, Lucília; Teixeira, Manuel R

    2007-10-01

    The t(4;11)(q21;q23) involving the genes MLL and AF4 (alias for AFF1) is detected in 50-70% of infant leukemia. We characterize at both the DNA and RNA level a rare MLL-AF4 fusion transcript identified in a 15-month-old girl with acute lymphoblastic leukemia. Direct sequence analysis of the reverse transcriptase-polymerase chain reaction product showed an in-frame fusion between MLL exon 9 and AF4 exon 6. We further demonstrated that the genomic breakpoints were located 1,553 bp downstream of MLL exon 9 and 1,239 bp upstream of AF4 exon 6. Four Alu repeats were detected in MLL intron 9 and two Alu repeats and one LINE1 repetitive element were identified downstream of AF4 exon 5. Finally, a 9-bp polypurine (A) tract and an 8-bp polypyrimidine (T) tract were found flanking the translocation breakpoint. In summary, we have characterized at both the RNA and the DNA level a rare MLL-AF4 fusion variant that was presumably mediated by Alu repeats or polypurine and polypyrimidine tracts located in the vicinity of genomic breakpoints. PMID:17889710

  16. Collagen-GAG Scaffold Biophysical Properties Bias MSC Lineage Choice in the Presence of Mixed Soluble Signals

    PubMed Central

    Caliari, Steven R.

    2014-01-01

    Biomaterial strategies for regenerating multitissue structures require unique approaches. One strategy is to design scaffolds so that their local biophysical properties can enhance site-specific effects of an otherwise heterogeneous biomolecular environment. This investigation examined the role of biomaterial physical properties (relative density, mineral content) on the human mesenchymal stem cell phenotype in the presence of mixed soluble signals to drive osteogenesis or chondrogenesis. We tested a series of three-dimensional collagen–glycosaminoglycan scaffolds with properties inspired by extracellular matrix characteristics across the osteotendinous interface (tendon, cartilage, and bone). We found that selective scaffold mineralization induced a depressed chondrogenic response compared with nonmineralized groups as demonstrated by gene expression and histological analyses. Interestingly, the greatest chondrogenic response was found in a higher density, nonmineralized scaffold variant despite increased contraction and cellular condensation in lower density nonmineralized scaffolds. In fact, the lower density scaffolds demonstrated a significantly higher expression of osteogenic transcripts as well as ample mineralization after 21 days of culture. This effect may be due to local stiffening of the scaffold microenvironment as the scaffold contracts, leading to increased cell density, accelerated differentiation, and possible endochondral ossification as evidenced by a transition from a glycosaminoglycan (GAG)-rich milieu to higher mineralization at later culture times. These findings will help shape the design rules for graded biomaterials to regenerate distinct fibrillar, fibrocartilagenous, and mineralized regions of orthopedic interfaces. PMID:24568607

  17. The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF).

    PubMed

    Daviau, Alex; Di Fruscio, Marco; Blouin, Richard

    2009-04-01

    Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling. PMID:19146952

  18. Identification of MLL partner genes in 27 patients with acute leukemia from a single cytogenetic laboratory.

    PubMed

    De Braekeleer, Etienne; Meyer, Claus; Douet-Guilbert, Nathalie; Basinko, Audrey; Le Bris, Marie-Josée; Morel, Frédéric; Berthou, Christian; Marschalek, Rolf; Férec, Claude; De Braekeleer, Marc

    2011-12-01

    Chromosomal rearrangements involving the MLL gene have been associated with many different types of hematological malignancies. Fluorescent in situ hybridization with a panel of probes coupled with long distance inverse-PCR was used to identify chromosomal rearrangements involving the MLL gene. Between 1995 and 2010, 27 patients with an acute leukemia were found to have a fusion gene involving MLL. All seven ALL patients with B cell acute lymphoblastic leukemia were characterized by the MLL/AFF1 fusion gene resulting from a translocation (5 patients) or an insertion (2 patients). In the 19 AML patients with acute myeloblastic leukemia, 31.6% of all characterized MLL fusion genes were MLL/MLLT3, 21.1% MLL/ELL, 10.5% MLL/MLLT6 and 10.5% MLL/EPS15. Two patients had rare or undescribed fusion genes, MLL/KIAA0284 and MLL/FLNA. Seven patients (26%) had a complex chromosomal rearrangement (three-way translocations, insertions, deletions) involving the MLL gene. Splicing fusion genes were found in three patients, leading to a MLL/EPS15 fusion in two and a MLL/ELL fusion in a third patient. This study showed that fusion involving the MLL gene can be generated through various chromosomal rearrangements such as translocations, insertions and deletions, some being complex or cryptic. A systematic approach should be used in all cases of acute leukemia starting with FISH analyses using a commercially available MLL split signal probe. Then, the analysis has to be completed, if necessary, by further molecular cytogenetic and genomic PCR methods. PMID:21900057

  19. The Polycomb complex PRC2 supports aberrant self-renewal in a mouse model of MLL-AF9;NrasG12D acute myeloid leukemia

    PubMed Central

    Shi, Junwei; Wang, Eric; Zuber, Johannes; Rappaport, Amy; Taylor, Meredith; Johns, Christopher

    2014-01-01

    The Trithorax and Polycomb groups of chromatin regulators are critical for cell-lineage specification during normal development; functions that often become deregulated during tumorigenesis. As an example, oncogenic fusions of the Trithorax-related protein MLL can initiate aggressive leukemias by altering the transcriptional circuitry governing hematopoietic cell differentiation, a process that is known to require additional epigenetic pathways to implement. Here we used shRNA screening to identify chromatin regulators uniquely required in a mouse model of MLL-fusion acute myeloid leukemia, which revealed a role for the Polycomb Repressive Complex 2 (PRC2) in maintenance of this disease. shRNA-mediated suppression of PRC2 subunits Eed, Suz12, or Ezh1/Ezh2 led to proliferation-arrest and differentiation of leukemia cells, with a minimal impact on growth of several non-transformed hematopoietic cell lines. The requirement for PRC2 in leukemia is partly due to its role in direct transcriptional repression of genes that limit the self-renewal potential of hematopoietic cells, including Cdkn2a. In addition to implicating a role for PRC2 in the pathogenesis of MLL-fusion leukemia, our results suggest, more generally, that Trithorax and Polycomb group proteins can cooperate with one another to maintain aberrant lineage programs in cancer. PMID:22469984

  20. The MLL3/MLL4 Branches of the COMPASS Family Function as Major Histone H3K4 Monomethylases at Enhancers

    PubMed Central

    Hu, Deqing; Gao, Xin; Morgan, Marc A.; Herz, Hans-Martin; Smith, Edwin R.

    2013-01-01

    Histone H3 lysine 4 (H3K4) can be mono-, di-, and trimethylated by members of the COMPASS (complex of proteins associated with Set1) family from Saccharomyces cerevisiae to humans, and these modifications can be found at distinct regions of the genome. Monomethylation of histone H3K4 (H3K4me1) is relatively more enriched at metazoan enhancer regions compared to trimethylated histone H3K4 (H3K4me3), which is enriched at transcription start sites in all eukaryotes. Our recent studies of Drosophila melanogaster demonstrated that the Trithorax-related (Trr) branch of the COMPASS family regulates enhancer activity and is responsible for the implementation of H3K4me1 at these regions. There are six COMPASS family members in mammals, two of which, MLL3 (GeneID 58508) and MLL4 (GeneID 8085), are most closely related to Drosophila Trr. Here, we use chromatin immunoprecipitation-sequencing (ChIP-seq) of this class of COMPASS family members in both human HCT116 cells and mouse embryonic stem cells and find that MLL4 is preferentially found at enhancer regions. MLL3 and MLL4 are frequently mutated in cancer, and indeed, the widely used HCT116 cancer cell line contains inactivating mutations in the MLL3 gene. Using HCT116 cells in which MLL4 has also been knocked out, we demonstrate that MLL3 and MLL4 are major regulators of H3K4me1 in these cells, with the greatest loss of monomethylation at enhancer regions. Moreover, we find a redundant role between Mll3 (GeneID 231051) and Mll4 (GeneID 381022) in enhancer H3K4 monomethylation in mouse embryonic fibroblast (MEF) cells. These findings suggest that mammalian MLL3 and MLL4 function in the regulation of enhancer activity and that mutations of MLL3 and MLL4 that are found in cancers could exert their properties through malfunction of these Trr/MLL3/MLL4-specific (Trrific) enhancers. PMID:24081332

  1. Estrogen treatment induces MLL aberrations in human lymphoblastoid cells

    PubMed Central

    Schnyder, Sabine; Du, Nga T.; Le, Hongan B.; Singh, Sheetal; Loredo, Grace A.; Vaughan, Andrew T.

    2009-01-01

    Epidemiological data indicates increased risk of infant acute leukemia involving MLL gene aberrations with use of oral contraceptives. To determine whether estrogens might be implicated, we examined the effect of estradiol (E2) or 4-OH-E2 in an in vitro model of translocation susceptibility. Genomic DNA from the TK6 human lymphoblastoid cell line was screened by ligation mediated PCR and inverse PCR at a rearrangement hot spot within the MLL breakpoint cluster region to detect DNA aberrations. An increase in DNA double strand breaks was observed within this region after exposure to either E2 or 4-OH-E2. An increase in the frequency of MLL translocations was only found after exposure to E2. Induction of cleavage due to increased activation of apoptotic nucleases was excluded by pre-treatment with the pancaspase inhibitor, zVAD.fmk. We conclude that concentrations of E2 and 4-OH-E2 that may occur during pregnancy, or during use of oral contraceptives, can cause aberrations of the MLL gene and could thus be a factor in the early events of leukemogenesis occurring in utero. PMID:19264358

  2. Genetic and clinical characterization of 45 acute leukemia patients with MLL gene rearrangements from a single institution.

    PubMed

    Cerveira, Nuno; Lisboa, Susana; Correia, Cecília; Bizarro, Susana; Santos, Joana; Torres, Lurdes; Vieira, Joana; Barros-Silva, João D; Pereira, Dulcineia; Moreira, Cláudia; Meyer, Claus; Oliva, Tereza; Moreira, Ilídia; Martins, Ângelo; Viterbo, Luísa; Costa, Vítor; Marschalek, Rolf; Pinto, Armando; Mariz, José M; Teixeira, Manuel R

    2012-10-01

    Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant. PMID:22846743

  3. Structural and Biochemical Insights into MLL1 Core Complex Assembly

    SciTech Connect

    Avdic, Vanja; Zhang, Pamela; Lanouette, Sylvain; Groulx, Adam; Tremblay, Véronique; Brunzelle, Joseph; Couture, Jean-François

    2012-05-02

    Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5s {beta}-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.

  4. CDKN2 Gene Deletion as Poor Prognosis Predictor Involved in the Progression of Adult B-Lineage Acute Lymphoblastic Leukemia Patients

    PubMed Central

    Xu, Na; Li, Yu-ling; Zhou, Xuan; Cao, Rui; Li, Huan; Lu, Qi-si; Li, Lin; Lu, Zi-yuan; Huang, Ji-xian; Sun, Jing; Liu, Qi-fa; Du, Qing-feng; Liu, Xiao-li

    2015-01-01

    Deletion of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) is well known in many hematologic malignancies, but only few reports have investigated this deletion effect on clinical prognosis. This study performed analysis of the CDKN2 deletion in 215 adult B- lineage acute lymphoblastic leukemia (B-ALL) patients, and related cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; Mixed Lineage Leukemia (MLL) rearrangement; MYC, Immunoglobulin heavy locus (IGH) translocation). The prevalence of CDKN2 deletions in all study populations was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells (WBC) count, BM blast percentage, extra infiltration and induction complete remission (CR) rate. Analysis in relapse patients revealed that the distribution of CDKN2 deletion is higher in relapse patients (44.6%) than all patients (28.4%, P=0.006). Deletion of CDKN2 was significantly associated with poor outcomes including decreased overall survival (OS) (P<0.001), lower disease free-survival (DFS) (P<0.001), and increased cumulative incidence of relapse (P=0.002); Also, CDKN2 deletion was strongly associated with IGH translocation (P=0.021); and had an adverse effect on patients with BCR-ABL fusion gene or with MLL rearrangement. Patients with CDKN2 gene deletion benefited from allogenic hematopoietic stem cell transplantation (Allo-HSCT). Deletion of CDKN2 gene was commonly observed through leukemia progression and was poor prognostic marker in long-term outcomes. PMID:26516359

  5. Histone H3 Lysine 79 Methyltransferase Dot1 Is Required for Immortalization by MLL Oncogenes

    PubMed Central

    Chang, Ming-Jin; Wu, Hongyu; Achille, Nicholas J.; Reisenauer, Mary Rose; Chou, Chau-Wen; Zeleznik-Le, Nancy J.; Hemenway, Charles S.; Zhang, Wenzheng

    2011-01-01

    Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein–mediated leukemogenesis and implicate Dot1 as a potential therapeutic target. PMID:21159644

  6. Transient potential receptor melastatin-2 (Trpm2) does not influence murine MLL-AF9-driven AML leukemogenesis or in vitro response to chemotherapy.

    PubMed

    Haladyna, Jessica N; Pastuer, Taylor; Riedel, Simone S; Perraud, Anne-Laure; Bernt, Kathrin M

    2016-07-01

    Transient potential receptor melastatin-2 (TRPM2) is a nonselective cationic, Ca(2+)-permeable transmembrane pore that is preferentially expressed in cells of the myeloid lineage and modulates signaling pathways converging into NF-kB. This is of potential interest for acute myeloid leukemia (AML) therapy, as NF-κB signaling is emerging as a key pathway, mediating drug resistance and leukemia-initiating cell survival in AML. Inhibition of NF-κB signaling has been found to be synergistic with chemotherapy. TRPM2 is overexpressed in AML compared with normal bone marrow, with the highest levels in the FAB M3-6 subtypes. To determine the effect of TRPM2 depletions in a defined genetic model, we established MLL-AF9-driven AML on a Trpm2(-/-) genetic background. Trpm2(-/-) MLL-AF9 leukemias displayed reduced NF-κB phosphorylation as well as nuclear translocation. In vivo, primary and secondary recipients of Trpm2(-/-) MLL-AF9 leukemias exhibit increased latency compared with recipients of wild-type leukemia cells. However, the difference in latency was small and was lost in tertiary transplants. The effect of loss of Trpm2 in a BCR-ABL/NUP98-HOXA9 fusion model was even smaller. Given reports that loss or inhibition of TRPM2 enhanced killing by DNA-damaging agents in neuroblastoma, breast cancer, and prostate cancer cell lines, we exposed Trpm2(-/-) and Trpm2(wt) primary MLL-AF9 leukemias to doxorubicin, cytarabine, and etoposide, but found no difference in IC50 values. The in vitro response to decitabine was also unaffected. In summary, Trpm2 does not seem to play a major role in myeloid leukemogenesis. Additionally, loss of Trpm2 does not augment the cytotoxicity of standard AML chemotherapeutic agents. PMID:27033163

  7. MLL5 maintains spindle bipolarity by preventing aberrant cytosolic aggregation of PLK1.

    PubMed

    Zhao, Wei; Liu, Jie; Zhang, Xiaoming; Deng, Lih-Wen

    2016-03-28

    Faithful chromosome segregation with bipolar spindle formation is critical for the maintenance of genomic stability. Perturbation of this process often leads to severe mitotic failure, contributing to tumorigenesis. MLL5 has been demonstrated to play vital roles in cell cycle progression and the maintenance of genomic stability. Here, we identify a novel interaction between MLL5 and PLK1 in the cytosol that is crucial for sustaining spindle bipolarity during mitosis. Knockdown of MLL5 caused aberrant PLK1 aggregation that led to acentrosomal microtubule-organizing center (aMTOC) formation and subsequent spindle multipolarity. Further molecular studies revealed that the polo-box domain (PBD) of PLK1 interacted with a binding motif on MLL5 (Thr887-Ser888-Thr889), and this interaction was essential for spindle bipolarity. Overexpression of wild-type MLL5 was able to rescue PLK1 mislocalization and aMTOC formation in MLL5-KD cells, whereas MLL5 mutants incapable of interacting with the PBD failed to do so. We thus propose that MLL5 preserves spindle bipolarity through maintaining cytosolic PLK1 in a nonaggregated form. PMID:27002166

  8. Initiation of MLL-rearranged AML is dependent on C/EBPα

    PubMed Central

    Ohlsson, Ewa; Hasemann, Marie Sigurd; Willer, Anton; Lauridsen, Felicia Kathrine Bratt; Rapin, Nicolas; Jendholm, Johan

    2014-01-01

    MLL-fusion proteins are potent inducers of oncogenic transformation, and their expression is considered to be the main oncogenic driving force in ∼10% of human acute myeloid leukemia (AML) patients. These oncogenic fusion proteins are responsible for the initiation of a downstream transcriptional program leading to the expression of factors such as MEIS1 and HOXA9, which in turn can replace MLL-fusion proteins in overexpression experiments. To what extent MLL fusion proteins act on their own during tumor initiation, or if they collaborate with other transcriptional regulators, is unclear. Here, we have compared gene expression profiles from human MLL-rearranged AML to normal progenitors and identified the myeloid tumor suppressor C/EBPα as a putative collaborator in MLL-rearranged AML. Interestingly, we find that deletion of Cebpa rendered murine hematopoietic progenitors completely resistant to MLL-ENL–induced leukemic transformation, whereas C/EBPα was dispensable in already established AMLs. Furthermore, we show that Cebpa-deficient granulocytic-monocytic progenitors were equally resistant to transformation and that C/EBPα collaborates with MLL-ENL in the induction of a transcriptional program, which is also apparent in human AML. Thus, our studies demonstrate a key role of C/EBPα in MLL fusion–driven transformation and find that it sharply demarcates tumor initiation and maintenance. PMID:24367003

  9. Mutation of cancer driver MLL2 results in transcription stress and genome instability.

    PubMed

    Kantidakis, Theodoros; Saponaro, Marco; Mitter, Richard; Horswell, Stuart; Kranz, Andrea; Boeing, Stefan; Aygün, Ozan; Kelly, Gavin P; Matthews, Nik; Stewart, Aengus; Stewart, A Francis; Svejstrup, Jesper Q

    2016-02-15

    Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis. PMID:26883360

  10. Mutation of cancer driver MLL2 results in transcription stress and genome instability

    PubMed Central

    Kantidakis, Theodoros; Saponaro, Marco; Mitter, Richard; Horswell, Stuart; Kranz, Andrea; Boeing, Stefan; Aygün, Ozan; Kelly, Gavin P.; Matthews, Nik; Stewart, Aengus; Stewart, A. Francis; Svejstrup, Jesper Q.

    2016-01-01

    Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis. PMID:26883360

  11. Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells.

    PubMed

    Largeot, Anne; Paggetti, Jérôme; Broséus, Julien; Aucagne, Romain; Lagrange, Brice; Martin, Romain Z; Berthelet, Jean; Quéré, Ronan; Lucchi, Géraldine; Ducoroy, Patrick; Bastie, Jean-Noël; Delva, Laurent

    2013-12-01

    MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of the HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto the HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin. PMID:23994619

  12. The potential of clofarabine in MLL-rearranged infant acute lymphoblastic leukaemia.

    PubMed

    Stumpel, Dominique J P M; Schneider, Pauline; Pieters, Rob; Stam, Ronald W

    2015-09-01

    MLL-rearranged acute lymphoblastic leukaemia (ALL) in infants is the most difficult-to-treat type of childhood ALL, displaying a chemotherapy-resistant phenotype, and unique histone modifications, gene expression signatures and DNA methylation patterns. MLL-rearranged infant ALL responds remarkably well to nucleoside analogue drugs in vitro, such as cytarabine and cladribine, and to the demethylating agents decitabine and zebularine as measured by cytotoxicity assays. These observations led to the inclusion of cytarabine into the treatment regimens currently used for infants with ALL. However, survival chances for infants with MLL-rearranged ALL do still not exceed 30-40%. Here we explored the in vitro potential of the novel nucleoside analogue clofarabine for MLL-rearranged infant ALL. Therefore we used both cell line models as well as primary patient cells. Compared with other nucleoside analogues, clofarabine effectively targeted primary MLL-rearranged infant ALL cells at the lowest concentrations, with median LC50 values of ∼25 nM. Interestingly, clofarabine displayed synergistic cytotoxic effects in combination with cytarabine. Furthermore, at concentrations of 5-10nM clofarabine induced demethylation of the promoter region of the tumour suppressor gene FHIT (Fragile Histidine Triad), a gene typically hypermethylated in MLL-rearranged ALL. Demethylation of the FHIT promoter region was accompanied by subtle re-expression of this gene both at the mRNA and protein level. We conclude that clofarabine is an interesting candidate for further studies in MLL-rearranged ALL in infants. PMID:26188848

  13. Mixed heterolobosean and novel gregarine lineage genes from culture ATCC 50646: Long-branch artefacts, not lateral gene transfer, distort α-tubulin phylogeny.

    PubMed

    Cavalier-Smith, Thomas

    2015-04-01

    Contradictory and confusing results can arise if sequenced 'monoprotist' samples really contain DNA of very different species. Eukaryote-wide phylogenetic analyses using five genes from the amoeboflagellate culture ATCC 50646 previously implied it was an undescribed percolozoan related to percolatean flagellates (Stephanopogon, Percolomonas). Contrastingly, three phylogenetic analyses of 18S rRNA alone, did not place it within Percolozoa, but as an isolated deep-branching excavate. I resolve that contradiction by sequence phylogenies for all five genes individually, using up to 652 taxa. Its 18S rRNA sequence (GQ377652) is near-identical to one from stained-glass windows, somewhat more distant from one from cooling-tower water, all three related to terrestrial actinocephalid gregarines Hoplorhynchus and Pyxinia. All four protein-gene sequences (Hsp90; α-tubulin; β-tubulin; actin) are from an amoeboflagellate heterolobosean percolozoan, not especially deeply branching. Contrary to previous conclusions from trees combining protein and rRNA sequences or rDNA trees including Eozoa only, this culture does not represent a major novel deep-branching eukaryote lineage distinct from Heterolobosea, and thus lacks special significance for deep eukaryote phylogeny, though the rDNA sequence is important for gregarine phylogeny. α-Tubulin trees for over 250 eukaryotes refute earlier suggestions of lateral gene transfer within eukaryotes, being largely congruent with morphology and other gene trees. PMID:25769111

  14. Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

    PubMed Central

    Hubert, Amy; Henderson, Jordana M.; Ross, Kelly G.; Cowles, Martis W.; Torres, Jessica; Zayas, Ricardo M.

    2013-01-01

    Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5–1 and mll5–2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5–2 are required for regeneration and that set1, trr-1 and mll5–2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo. PMID:23235145

  15. Expression pattern of the septin gene family in acute myeloid leukemias with and without MLL-SEPT fusion genes.

    PubMed

    Santos, Joana; Cerveira, Nuno; Bizarro, Susana; Ribeiro, Franclim R; Correia, Cecília; Torres, Lurdes; Lisboa, Susana; Vieira, Joana; Mariz, José M; Norton, Lucília; Snijder, Simone; Mellink, Clemens H; Buijs, Arjan; Shih, Lee-Yung; Strehl, Sabine; Micci, Francesca; Heim, Sverre; Teixeira, Manuel R

    2010-05-01

    Septins are proteins associated with crucial steps in cell division and cellular integrity. In humans, 14 septin genes have been identified, of which five (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) are known to participate in reciprocal translocations with the MLL gene in myeloid neoplasias. We have recently shown a significant down-regulation of both SEPT2 and MLL in myeloid neoplasias with the MLL-SEPT2 fusion gene. In this study, we examined the expression pattern of the other 13 known septin genes in altogether 67 cases of myeloid neoplasia, including three patients with the MLL-SEPT2 fusion gene, four with MLL-SEPT6 fusion, and three patients with the MLL-SEPT9 fusion gene. When compared with normal controls, a statistically significant down-regulation was observed for the expression of both MLL (6.4-fold; p=0.008) and SEPT6 (1.7-fold; p=0.002) in MLL-SEPT6 leukemia. Significant down-regulation of MLL was also found in MLL-MLLT3 leukemias. In addition, there was a trend for SEPT9 down-regulation in MLL-SEPT9 leukemias (4.6-fold; p=0.077). Using hierarchical clustering analysis to compare acute myeloid leukemia genetic subgroups based on their similarity of septin expression changes, we found that MLL-SEPT2 and MLL-SEPT6 neoplasias cluster together apart from the remaining subgroups and that PML-RARA leukemia presents under-expression of most septin family genes. PMID:19748670

  16. Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia

    PubMed Central

    Strick, Reiner; Strissel, Pamela L.; Borgers, Susanne; Smith, Steve L.; Rowley, Janet D.

    2000-01-01

    Chromosomal translocations involving the MLL gene occur in about 80% of infant leukemia. In the search for possible agents inducing infant leukemia, we identified bioflavonoids, natural substances in food as well as in dietary supplements, that cause site-specific DNA cleavage in the MLL breakpoint cluster region (BCR) in vivo. The MLL BCR DNA cleavage was shown in primary progenitor hematopoietic cells from healthy newborns and adults as well as in cell lines; it colocalized with the MLL BCR cleavage site induced by chemotherapeutic agents, such as etoposide (VP16) and doxorubicin (Dox). Both in vivo and additional in vitro experiments demonstrated topoisomerase II (topo II) as the target of bioflavonoids similar to VP16 and Dox. Based on 20 bioflavonoids tested, we identified a common structure essential for topo II-induced DNA cleavage. Reversibility experiments demonstrated a religation of the bioflavonoid as well as the VP16-induced MLL cleavage site. Our observations support a two-stage model of cellular processing of topo II inhibitors: The first and reversible stage of topo II-induced DNA cleavage results in DNA repair, but also rarely in chromosome translocations; whereas the second, nonreversible stage leads to cell death because of an accumulation of DNA damage. These results suggest that maternal ingestion of bioflavonoids may induce MLL breaks and potentially translocations in utero leading to infant and early childhood leukemia. PMID:10758153

  17. Structure-based design and synthesis of small molecular inhibitors disturbing the interaction of MLL1-WDR5.

    PubMed

    Li, Dong-Dong; Chen, Wei-Lin; Xu, Xiao-Li; Jiang, Fen; Wang, Lei; Xie, Yi-Yue; Zhang, Xiao-Jin; Guo, Xiao-Ke; You, Qi-Dong; Sun, Hao-Peng

    2016-08-01

    MLL1 complex catalyzes the methylation of H3K4, and plays important roles in the development of acute leukemia harboring MLL fusion proteins. Targeting MLL1-WDR5 protein-protein interaction (PPI) to inhibit the activity of histone methyltransferase of MLL1 complex is a novel strategy for treating of acute leukemia. WDR5-47 (IC50 = 0.3 μM) was defined as a potent small molecule to disturb the interaction of MLL1-WDR5. Here, we described structure-based design and synthesis of small molecular inhibitors to block MLL1-WDR5 PPI. Especially, compound 23 (IC50 = 104 nM) was the most potent small molecular, and about 3-times more potent than WDR5-47. We also discussed the SAR of these series of compounds with docking study, which may stimulate more potent compounds. PMID:27116709

  18. Unraveling the Activation Mechanism of Taspase1 which Controls the Oncogenic AF4-MLL Fusion Protein.

    PubMed

    Sabiani, Samaneh; Geppert, Tim; Engelbrecht, Christian; Kowarz, Eric; Schneider, Gisbert; Marschalek, Rolf

    2015-05-01

    We have recently demonstrated that Taspase1-mediated cleavage of the AF4-MLL oncoprotein results in the formation of a stable multiprotein complex which forms the key event for the onset of acute proB leukemia in mice. Therefore, Taspase1 represents a conditional oncoprotein in the context of t(4;11) leukemia. In this report, we used site-directed mutagenesis to unravel the molecular events by which Taspase1 becomes sequentially activated. Monomeric pro-enzymes form dimers which are autocatalytically processed into the enzymatically active form of Taspase1 (αββα). The active enzyme cleaves only very few target proteins, e.g., MLL, MLL4 and TFIIA at their corresponding consensus cleavage sites (CSTasp1) as well as AF4-MLL in the case of leukemogenic translocation. This knowledge was translated into the design of a dominant-negative mutant of Taspase1 (dnTASP1). As expected, simultaneous expression of the leukemogenic AF4-MLL and dnTASP1 causes the disappearance of the leukemogenic oncoprotein, because the uncleaved AF4-MLL protein (328 kDa) is subject to proteasomal degradation, while the cleaved AF4-MLL forms a stable oncogenic multi-protein complex with a very long half-life. Moreover, coexpression of dnTASP1 with a BFP-CSTasp1-GFP FRET biosensor effectively inhibits cleavage. The impact of our findings on future drug development and potential treatment options for t(4;11) leukemia will be discussed. PMID:26137584

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

    PubMed

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

    2016-01-01

    Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias. PMID:27625305

  20. Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4

    PubMed Central

    Herz, Hans-Martin; Mohan, Man; Garruss, Alexander S.; Liang, Kaiwei; Takahashi, Yoh-hei; Mickey, Kristen; Voets, Olaf; Verrijzer, C. Peter; Shilatifard, Ali

    2012-01-01

    Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers. PMID:23166019

  1. A novel MLL2 gene mutation in a Korean patient with Kabuki syndrome

    PubMed Central

    Kim, Soo Jin; Cho, Sung Yoon; Maeng, Se Hyun; Sohn, Young Bae; Kim, Su-Jin; Ki, Chang-Seok

    2013-01-01

    Kabuki syndrome (KS) is a rare genetic disease with a distinctive dysmorphic face, intellectual disability, and multiple congenital abnormalities. KS is inherited in an autosomal dominant manner. As the primary cause of KS, MLL2 mutations have been identified in 56-76% of affected individuals who have been tested, suggesting that there may be additional genes associated with KS. Recently, a few KS individuals have been found to have de novo partial or complete deletions of an X chromosome gene, KDM6A, which encodes a histone demethylase that interacts with MLL2. Nevertheless, mutations in MLL2 are the major cause of KS. Although there are a few reports of KS patients in Korea, none of these had been confirmed by genetic analysis. Here, we report a case of a Korean patient with clinical features of KS. Using direct sequencing, we identified a frameshift heterozygous mutation for MLL2: (c.5256_5257delGA;p.Lys1753Alafs*34). Clinically, the patient presented with typical facial features, and diagnosis of KS was based on the diagnostic criteria. While KS is a rare disease, other malformations that overlap with those found in individuals with KS are common. Hence, the diagnosis of KS by mutational analysis can be a valuable method for patients with KS-like syndromes. Furthermore, in the near future, other genes could be identified in patients with KS without a detectable MLL2 mutation. PMID:24019847

  2. Phylogenetic lineages in Entomophthoromycota

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Entomophthoromycota Humber is one of five major phylogenetic lineages among the former phylum Zygomycota. These early terrestrial fungi share evolutionarily ancestral characters such as coenocytic mycelium and gametangiogamy as a sexual process resulting in zygospore formation. Previous molecular st...

  3. An insertional mutagenesis screen identifies genes that cooperate with Mll-AF9 in a murine leukemogenesis model

    PubMed Central

    Bergerson, Rachel J.; Collier, Lara S.; Sarver, Aaron L.; Been, Raha A.; Lugthart, Sanne; Diers, Miechaleen D.; Zuber, Johannes; Rappaport, Amy R.; Nixon, Molly J.; Silverstein, Kevin A. T.; Fan, Danhua; Lamblin, Anne-Francoise J.; Wolff, Linda; Kersey, John H.; Delwel, Ruud; Lowe, Scott W.; O'Sullivan, M. Gerard; Kogan, Scott C.; Adams, David J.

    2012-01-01

    Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes. PMID:22427200

  4. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

    PubMed

    Bigenzahn, Johannes W; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K; Scorzoni, Stefania; Vladimer, Gregory I; Müller, André C; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L; Superti-Furga, Giulio

    2016-03-01

    Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression. PMID:26933192

  5. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification

    PubMed Central

    Mueller, Dorothee; Bach, Christian; Zeisig, Deniz; Garcia-Cuellar, Maria-Paz; Monroe, Sara; Sreekumar, Arun; Zhou, Rong; Nesvizhskii, Alexey; Chinnaiyan, Arul; Hess, Jay L.

    2007-01-01

    Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79–specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation. PMID:17855633

  6. An Evolutionary Conserved Epigenetic Mark of Polycomb Response Elements Implemented by Trx/MLL/COMPASS.

    PubMed

    Rickels, Ryan; Hu, Deqing; Collings, Clayton K; Woodfin, Ashley R; Piunti, Andrea; Mohan, Man; Herz, Hans-Martin; Kvon, Evgeny; Shilatifard, Ali

    2016-07-21

    Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. Here we demonstrate that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homolog, MLL1, deposits H3K4me2 at CpG-dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb repressive complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, we can rescue expression of these loci, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, our study provides rules for identifying cell-type-specific functional mammalian PREs within the human genome. PMID:27447986

  7. MEIS1 regulates an HLF–oxidative stress axis in MLL-fusion gene leukemia

    PubMed Central

    Roychoudhury, Jayeeta; Clark, Jason P.; Gracia-Maldonado, Gabriel; Unnisa, Zeenath; Wunderlich, Mark; Link, Kevin A.; Dasgupta, Nupur; Aronow, Bruce; Huang, Gang; Mulloy, James C.

    2015-01-01

    Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment. PMID:25740828

  8. Novel variants in MLL confer to bladder cancer recurrence identified by whole-exome sequencing

    PubMed Central

    Wang, Yongqiang; Huang, Yi; Liu, Huan; Li, Feida; He, Luyun; Sun, Da; Yu, Yuan; Li, Qiaoling; Huang, Peide; Zhang, Meng; Zhao, Xin; Bi, Tengteng; Zhuang, Xuehan; Zhang, Liyan; Lu, Jingxiao; Sun, Xiaojuan; Zhou, Fangjian; Liu, Chunxiao; Yang, Guosheng; Hou, Yong; Fan, Zusen; Cai, Zhiming

    2016-01-01

    Bladder cancer (BC) is distinguished by high rate of recurrence after surgery, but the underlying mechanisms remain poorly understood. Here we performed the whole-exome sequencing of 37 BC individuals including 20 primary and 17 recurrent samples in which the primary and recurrent samples were not from the same patient. We uncovered that MLL, EP400, PRDM2, ANK3 and CHD5 exclusively altered in recurrent BCs. Specifically, the recurrent BCs and bladder cancer cells with MLL mutation displayed increased histone H3 tri-methyl K4 (H3K4me3) modification in tissue and cell levels and showed enhanced expression of GATA4 and ETS1 downstream. What's more, MLL mutated bladder cancer cells obtained with CRISPR/Cas9 showed increased ability of drug-resistance to epirubicin (a chemotherapy drug for bladder cancer) than wild type cells. Additionally, the BC patients with high expression of GATA4 and ETS1 significantly displayed shorter lifespan than patients with low expression. Our study provided an overview of the genetic basis of recrudescent bladder cancer and discovered that genetic alterations of MLL were involved in BC relapse. The increased modification of H3K4me3 and expression of GATA4 and ETS1 would be the promising targets for the diagnosis and therapy of relapsed bladder cancer. PMID:26625313

  9. Hematopoietic Lineage Diversification, Simplified.

    PubMed

    Drissen, Roy; Nerlov, Claus

    2016-08-01

    Hematopoiesis is a complex process that requires a high degree of transcriptional diversification during lineage commitment and differentiation. de Graaf et al. (2016) have now generated a comprehensive gene expression dataset that allows cell-type-specific genes as well as associated transcription factor expression patterns to be readily identified. PMID:27494670

  10. Differential role of Id1 in MLL-AF9-driven leukemia based on cell of origin.

    PubMed

    Man, Na; Sun, Xiao-Jian; Tan, Yurong; García-Cao, Marta; Liu, Fan; Cheng, Guoyan; Hatlen, Megan; Xu, Haiming; Shah, Ronit; Chastain, Nolan; Liu, Na; Huang, Gang; Zhou, Yuan; Sheng, Mengyao; Song, Junhong; Yang, Feng-Chun; Benezra, Robert; Nimer, Stephen D; Wang, Lan

    2016-05-12

    Inhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemogenesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1(-/-), p21(-/-), or Id1(-/-)p21(-/-) mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9-transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP(+) leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP(+) AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9-driven leukemia. PMID:26944543

  11. Bronchial isomerism in a Kabuki syndrome patient with a novel mutation in MLL2 gene

    PubMed Central

    2014-01-01

    Background Kabuki syndrome (KS) is a rare, multiple congenital anomalies/intellectual disability syndrome caused by mutations of MLL2 gene, which codifies for a histone methyltrasferase that regulates the embryogenesis and the tissue development. Left-bronchial isomerism is a rare congenital abnormality that can be defined as the absence of the normal lateralizing features which distinguish right and left-sides in the lungs. To date, this is the first report of left-bronchial isomerism in association with KS. Case presentation A one-month-old Caucasian male patient underwent our attention for microcephaly, dysmorphic features (long palpebral fissures, eyebrows with sparse lateral third, everted lower eyelids, blue sclerae, large dysplastic ears, lower lip pits), persistent fetal fingertip pads, short stature, heart defects (interventricular defect and aortic coarctation), unilateral cryptorchidism, hypotonia and delay in gross motor skills. These features suggested a diagnosis of KS and a molecular analysis confirmed a novel frame-shift mutation in the exon 11 of MLL2 gene. Subsequently, given recurrent respiratory infections with a normal immunological status, he underwent a chest CT scan that showed a left bronchial isomerism. Conclusion We report a patient affected by KS, with a novel MLL2 mutation and an atypical phenotype characterized by left-side bronchial isomerism. Interestingly, genes involved in the heterotaxia/isomerism such as ROCK2 and SHROOM3 are known to interact with MLL2 gene. In order to achieve a correct diagnosis and an appropriate therapy, the presence of pulmonary anatomical variations should be investigated in KS patients with respiratory signs not associated to immunological deficiency. Finally, our findings support the hypothesis that the mutations leading to a complete loss of function of MLL2 gene is often associated with complex visceral malformations. PMID:24472332

  12. Direct somatic lineage conversion.

    PubMed

    Tanabe, Koji; Haag, Daniel; Wernig, Marius

    2015-10-19

    The predominant view of embryonic development and cell differentiation has been that rigid and even irreversible epigenetic marks are laid down along the path of cell specialization ensuring the proper silencing of unrelated lineage programmes. This model made the prediction that specialized cell types are stable and cannot be redirected into other lineages. Accordingly, early attempts to change the identity of somatic cells had little success and was limited to conversions between closely related cell types. Nuclear transplantation experiments demonstrated, however, that specialized cells even from adult mammals can be reprogrammed into a totipotent state. The discovery that a small combination of transcription factors can reprogramme cells to pluripotency without the need of oocytes further supported the view that these epigenetic barriers can be overcome much easier than assumed, but the extent of this flexibility was still unclear. When we showed that a differentiated mesodermal cell can be directly converted to a differentiated ectodermal cell without a pluripotent intermediate, it was suggested that in principle any cell type could be converted into any other cell type. Indeed, the work of several groups in recent years has provided many more examples of direct somatic lineage conversions. Today, the question is not anymore whether a specific cell type can be generated by direct reprogramming but how it can be induced. PMID:26416679

  13. AF4 uses the SL1 components of RNAP1 machinery to initiate MLL fusion- and AEP-dependent transcription.

    PubMed

    Okuda, Hiroshi; Kanai, Akinori; Ito, Shinji; Matsui, Hirotaka; Yokoyama, Akihiko

    2015-01-01

    Gene rearrangements generate MLL fusion genes, which can lead to aggressive leukemia. In most cases, MLL fuses with a gene encoding a component of the AEP (AF4 family/ENL family/P-TEFb) coactivator complex. MLL-AEP fusion proteins constitutively activate their target genes to immortalize haematopoietic progenitors. Here we show that AEP and MLL-AEP fusion proteins activate transcription through selectivity factor 1 (SL1), a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). The pSER domain of AF4 family proteins associates with SL1 on chromatin and loads TATA-binding protein (TBP) onto the promoter to initiate RNA polymerase II (RNAP2)-dependent transcription. These results reveal a previously unknown transcription initiation mechanism involving AEP and a role for SL1 as a TBP-loading factor in RNAP2-dependent gene activation. PMID:26593443

  14. A novel spliced fusion of MLL with CT45A2 in a pediatric biphenotypic acute leukemia

    PubMed Central

    2010-01-01

    Background Abnormalities of 11q23 involving the MLL gene are found in approximately 10% of human leukemias. To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level. In this work we present the identification of a novel MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia. Methods Cytogenetics, fluorescence in situ hybridization (FISH), molecular studies (RT-PCR and LDI-PCR), and bioinformatic sequence analysis were used to characterize the CT45A2 gene as novel MLL fusion partner in pediatric acute leukemia. Results Fluorescence in situ hybridization of bone marrow G-banded metaphases demonstrated a cryptic insertion of 11q23 in Xq26.3 involving the MLL gene. Breakpoint fusion analysis revealed that a DNA fragment of 653 kb from 11q23, containing MLL exons 1-9 in addition to 16 other 11q23 genes, was inserted into the upstream region of the CT45A2 gene located at Xq26.3. In addition, a deletion at Xq26.3 encompassing the 3' region of the DDX26B gene (exons 9-16) and the entire CT45A1 gene was identified. RNA analysis revealed the presence of a novel MLL-CT45A2 fusion transcript in which the first 9 exons of the MLL gene were fused in-frame to exon 2 of the CT45A2 gene, resulting in a spliced MLL fusion transcript with an intact open reading frame. The resulting chimeric transcript predicts a fusion protein where the N-terminus of MLL is fused to the entire open reading frame of CT45A2. Finally, we demonstrate that all breakpoint regions are rich in long repetitive motifs, namely LINE/L1 and SINE/Alu sequences, but all breakpoints were exclusively identified outside these repetitive DNA sequences. Conclusion We have identified CT45A2 as a novel spliced MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia, as a result of a cryptic insertion of 11q23 in Xq26.3. Since CT45A2 is the first

  15. Instrument for nm-scale spatial resolution x-ray microscopy using MLL optics

    NASA Astrophysics Data System (ADS)

    Nazaretski, Evgeny; Yan, Hanfei; Lauer, Kenneth; Mullany, Brian; Kuhne, Dennis; Xu, Weihe; Huang, Xiaojing; Gofron, Kazimierz; Kalbfleisch, Sebastian; Yan, Hui; Shu, Deming; Bouet, Nathalie; Zhou, Juan; Conley, Raymond; Chu, Yong

    2015-03-01

    The Hard X-ray Nanoprobe (HXN) beamline at the NSLS-II has been designed and constructed to address challenges related to nanoscale science and technology. HXN will provide a suite of experimental capabilities which include scanning fluorescence, diffraction, differential phase contrast and ptychography utilizing Multilayer Laue Lenses (MLL) and zoneplate (ZP) as nanofocusing optics. To provide more versatility and explore the phase space in materials research studies, the instrument is equipped with a temperature regulation system capable of varying specimen temperature between 100 K and 1000 K. During this presentation, different phases of the instrument development process will be reviewed. Various prototype systems designed and constructed will be discussed. Preliminary data demonstrating 2D sub-20 nm imaging resolution using MLL optics will be presented. Some of the early science applications will be covered emphasizing strengths of the developed instrument.

  16. MLL1 and DOT1L cooperate with meningioma-1 to induce acute myeloid leukemia.

    PubMed

    Riedel, Simone S; Haladyna, Jessica N; Bezzant, Matthew; Stevens, Brett; Pollyea, Daniel A; Sinha, Amit U; Armstrong, Scott A; Wei, Qi; Pollock, Roy M; Daigle, Scott R; Jordan, Craig T; Ernst, Patricia; Neff, Tobias; Bernt, Kathrin M

    2016-04-01

    Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML. PMID:26927674

  17. MLL1 and DOT1L cooperate with meningioma-1 to induce acute myeloid leukemia

    PubMed Central

    Riedel, Simone S.; Haladyna, Jessica N.; Bezzant, Matthew; Stevens, Brett; Pollyea, Daniel A.; Sinha, Amit U.; Armstrong, Scott A.; Wei, Qi; Pollock, Roy M.; Daigle, Scott R.; Jordan, Craig T.; Ernst, Patricia; Bernt, Kathrin M.

    2016-01-01

    Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin–derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML. PMID:26927674

  18. Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia

    PubMed Central

    Giambruno, Roberto; Grover, Amit; Avellino, Roberto; Skucha, Anna; Vittori, Sarah; Kuznetsova, Ekaterina; Smil, David; Barsyte-Lovejoy, Dalia; Li, Fengling; Poda, Gennadiy; Schapira, Matthieu; Wu, Hong; Dong, Aiping; Senisterra, Guillermo; Stukalov, Alexey; Huber, Kilian V. M.; Schönegger, Andreas; Marcellus, Richard; Bilban, Martin; Bock, Christoph; Brown, Peter J.; Zuber, Johannes; Bennett, Keiryn L.; Al-awar, Rima; Delwel, Ruud; Nerlov, Claus

    2015-01-01

    The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short 30 kDa C/EBPα translational isoform, termed p30, represents the most common type of CEBPA mutations in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as its down-regulation inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a novel small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30-cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML. PMID:26167872

  19. MSF (MLL septin-like fusion), a fusion partner gene of MLL, in a therapy-related acute myeloid leukemia with a t(11;17)(q23;q25)

    PubMed Central

    Osaka, Mitsuhiko; Rowley, Janet D.; Zeleznik-Le, Nancy J.

    1999-01-01

    MLL (ALL1, Htrx, HRX), which is located on chromosome band 11q23, frequently is rearranged in patients with therapy-related acute myeloid leukemia who previously were treated with DNA topoisomerase II inhibitors. In this study, we have identified a fusion partner of MLL in a 10-year-old female who developed therapy-related acute myeloid leukemia 17 months after treatment for Hodgkin’s disease. Leukemia cells of this patient had a t(11;17)(q23;q25), which involved MLL as demonstrated by Southern blot analysis. The partner gene was cloned from cDNA of the leukemia cells by use of a combination of adapter reverse transcriptase–PCR, rapid amplification of 5′ cDNA ends, and blast database analysis to identify expressed sequence tags. The full-length cDNA of 2.8 kb was found to be an additional member of the septin family, therefore it was named MSF (MLL septin-like fusion). Members of the septin family conserve the GTP binding domain, localize in the cytoplasm, and interact with cytoskeletal filaments. A major 4-kb transcript of MSF was expressed ubiquitously; a 1.7-kb transcript was found in most tissues. An additional 3-kb transcript was found only in hematopoietic tissues. By amplification with MLL exon 5 forward primer and reverse primers in MSF, the appropriately sized products were obtained. MSF is highly homologous to hCDCrel-1, which is a partner gene of MLL in leukemias with a t(11;22)(q23;q11.2). Further analysis of MSF may help to delineate the function of MLL partner genes in leukemia, particularly in therapy-related leukemia. PMID:10339604

  20. Unraveling the Activation Mechanism of Taspase1 which Controls the Oncogenic AF4–MLL Fusion Protein

    PubMed Central

    Sabiani, Samaneh; Geppert, Tim; Engelbrecht, Christian; Kowarz, Eric; Schneider, Gisbert; Marschalek, Rolf

    2015-01-01

    We have recently demonstrated that Taspase1-mediated cleavage of the AF4–MLL oncoprotein results in the formation of a stable multiprotein complex which forms the key event for the onset of acute proB leukemia in mice. Therefore, Taspase1 represents a conditional oncoprotein in the context of t(4;11) leukemia. In this report, we used site-directed mutagenesis to unravel the molecular events by which Taspase1 becomes sequentially activated. Monomeric pro-enzymes form dimers which are autocatalytically processed into the enzymatically active form of Taspase1 (αββα). The active enzyme cleaves only very few target proteins, e.g., MLL, MLL4 and TFIIA at their corresponding consensus cleavage sites (CSTasp1) as well as AF4–MLL in the case of leukemogenic translocation. This knowledge was translated into the design of a dominant-negative mutant of Taspase1 (dnTASP1). As expected, simultaneous expression of the leukemogenic AF4–MLL and dnTASP1 causes the disappearance of the leukemogenic oncoprotein, because the uncleaved AF4–MLL protein (328 kDa) is subject to proteasomal degradation, while the cleaved AF4–MLL forms a stable oncogenic multi-protein complex with a very long half-life. Moreover, coexpression of dnTASP1 with a BFP-CSTasp1-GFP FRET biosensor effectively inhibits cleavage. The impact of our findings on future drug development and potential treatment options for t(4;11) leukemia will be discussed. PMID:26137584

  1. Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification.

    PubMed

    Matutes, Estella; Pickl, Winfried F; Van't Veer, Mars; Morilla, Ricardo; Swansbury, John; Strobl, Herbert; Attarbaschi, Andishe; Hopfinger, Georg; Ashley, Sue; Bene, Marie Christine; Porwit, Anna; Orfao, Alberto; Lemez, Petr; Schabath, Richard; Ludwig, Wolf-Dieter

    2011-03-17

    The features of 100 mixed-phenotype acute leukemias (MPALs), fulfilling WHO 2008 criteria, are documented. Myeloid and T-lineage features were demonstrated by cytoplasmic myeloperoxidase and CD3; B-lineage features were demonstrated by at least 2 B-lymphoid markers. There were 62 men and 38 women; 68% were adults. Morphology was consistent with acute lymphoblastic leukemia (ALL; 43%), acute myeloid leukemia (AML; 42%), or inconclusive (15%). Immunophenotyping disclosed B + myeloid (59%), T + myeloid (35%), B + T (4%), or trilineage (2%) combinations. Cytogenetics evidenced t(9;22)/(Ph(+)) (20%), 11q23/MLL rearrangements (8%), complex (32%), aberrant (27%), or normal (13%) karyotypes. There was no correlation between age, morphology, immunophenotype, or cytogenetics. Response to treatment and outcome were available for 67 and 70 patients, respectively; 27 received ALL, 34 AML, 5 a combination of ALL + AML therapy, and 1 imatinib. ALL treatment induced a response in 85%, AML therapy in 41%; 3 of 5 patients responded to the combination therapy. Forty (58%) patients died, 33 of resistant disease. Overall median survival was 18 months and 37% of patients are alive at 5 years. Age, Ph(+), and AML therapy were predictors for poor outcome (P < .001; P = .002; P = .003). MPAL is confirmed to be a poor-risk disease. Adults and Ph(+) patients should be considered for transplantation in first remission. PMID:21228332

  2. DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia

    PubMed Central

    Chen, C.W.; Koche, R.P.; Sinha, A.U.; Deshpande, A.J.; Zhu, N.; Eng, R.; Doench, J.G.; Xu, H.; Chu, S.H.; Qi, J.; Wang, X.; Delaney, C.; Bernt, K.M.; Root, D.E.; Hahn, W.C.; Bradner, J.E.; Armstrong, S.A.

    2015-01-01

    MLL -rearrangements generate MLL-fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL-fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL-fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling activation and repression of gene expression could provide novel opportunities for combination therapy. PMID:25822366

  3. T-Cell Lineage Determination

    PubMed Central

    Yang, Qi; Bell, J. Jeremiah; Bhandoola, Avinash

    2010-01-01

    Summary T cells originate from hematopoietic stem cells (HSCs) in the bone marrow but complete their development in the thymus. HSCs give rise to a variety of non-renewing hematopoietic progenitors, among which a rare subset migrates to the thymus via the bloodstream. The earliest T-cell progenitors identified in the thymus are not T-lineage restricted but possess the ability to give rise to cells of many different lineages. Alternative lineage potentials are gradually lost as progenitors progress towards later developmental stages. Here, we review the early developmental events that might be involved in T-cell lineage fate determination, including the properties of possible thymus settling progenitors, their homing into the thymus, and their T-cell lineage specification and commitment. PMID:20969581

  4. Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissue-inducer cell lineage.

    PubMed

    Ishizuka, Isabel E; Chea, Sylvestre; Gudjonson, Herman; Constantinides, Michael G; Dinner, Aaron R; Bendelac, Albert; Golub, Rachel

    2016-03-01

    The precise lineage relationship between innate lymphoid cells (ILCs) and lymphoid tissue-inducer (LTi) cells is poorly understood. Using single-cell multiplex transcriptional analysis of 100 lymphoid genes and single-cell cultures of fetal liver precursor cells, we identified the common proximal precursor to these lineages and found that its bifurcation was marked by differential induction of the transcription factors PLZF and TCF1. Acquisition of individual effector programs specific to the ILC subsets ILC1, ILC2 and ILC3 was initiated later, at the common ILC precursor stage, by transient expression of mixed ILC1, ILC2 and ILC3 transcriptional patterns, whereas, in contrast, the development of LTi cells did not go through multilineage priming. Our findings provide insight into the divergent mechanisms of the differentiation of the ILC lineage and LTi cell lineage and establish a high-resolution 'blueprint' of their development. PMID:26779601

  5. Ancestral reconstruction of tick lineages.

    PubMed

    Mans, Ben J; de Castro, Minique H; Pienaar, Ronel; de Klerk, Daniel; Gaven, Philasande; Genu, Siyamcela; Latif, Abdalla A

    2016-06-01

    Ancestral reconstruction in its fullest sense aims to describe the complete evolutionary history of a lineage. This depends on accurate phylogenies and an understanding of the key characters of each parental lineage. An attempt is made to delineate our current knowledge with regard to the ancestral reconstruction of the tick (Ixodida) lineage. Tick characters may be assigned to Core of Life, Lineages of Life or Edges of Life phenomena depending on how far back these characters may be assigned in the evolutionary Tree of Life. These include housekeeping genes, sub-cellular systems, heme processing (Core of Life), development, moulting, appendages, nervous and organ systems, homeostasis, respiration (Lineages of Life), specific adaptations to a blood-feeding lifestyle, including the complexities of salivary gland secretions and tick-host interactions (Edges of Life). The phylogenetic relationships of lineages, their origins and importance in ancestral reconstruction are discussed. Uncertainties with respect to systematic relationships, ancestral reconstruction and the challenges faced in comparative transcriptomics (next-generation sequencing approaches) are highlighted. While almost 150 years of information regarding tick biology have been assembled, progress in recent years indicates that we are in the infancy of understanding tick evolution. Even so, broad reconstructions can be made with relation to biological features associated with various lineages. Conservation of characters shared with sister and parent lineages are evident, but appreciable differences are present in the tick lineage indicating modification with descent, as expected for Darwinian evolutionary theory. Many of these differences can be related to the hematophagous lifestyle of ticks. PMID:26868413

  6. A complex MLL rearrangement identified five years after initial MDS diagnosis results in out-of-frame fusions without progression to acute leukemia.

    PubMed

    Meyer, Claus; Kowarz, Eric; Yip, Sze-Fai; Wan, Thomas Shek-Kong; Chan, Tai-Kwong; Dingermann, Theo; Chan, Li-Chong; Marschalek, Rolf

    2011-10-01

    Chromosomal rearrangements of the MLL gene are uncommon in myelodysplastic syndromes (MDSs), and few studies of their molecular structures and oncogenic mechanisms exist. Here, we present a case of de novo MDS with a normal karyotype at initial diagnosis and a mild clinical course. Five years after the initial diagnosis, investigators identified a complex rearrangement of the MLL gene without progression to acute leukemia. The 5' part of the MLL gene is fused out of frame with the LOC100131626 gene, and the 3' part of the MLL gene out of frame with the TCF12 gene. Rapid amplification of complementary DNA 3' ends yielded two main fusion transcripts, which is in concordance with the two described isoforms of the LOC100131626 gene. For both isoform-fusion transcripts, the open reading frame terminates shortly after the breakpoint that is predicted to form two de facto truncated MLL proteins and disrupts the open reading frame of the LOC100131626, TCF12, and UBE4A genes. Neither dimerization nor a transcriptional activation domain, each of which is causally linked to MLL protein-mediated transformation, is present. This and other unusual MLL rearrangements probably represent a subclass of MLL gene abnormalities that have intrinsically no ability or only a weak ability to transform hematopoeitic cells and are identified only in the context of other hematopoetic malignancies. PMID:22137486

  7. Pro Isomerization in MLL1 PHD3-Bromo Cassette Connects H3K4me Readout to CyP33 and HDAC-Mediated Repression

    SciTech Connect

    Wang, Zhanxin; Song, Jikui; Milne, Thomas A.; Wang, Gang G.; Li, Haitao; Allis, C. David; Patel, Dinshaw J.

    2010-09-13

    The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.

  8. Acute myelogenous leukemia cells with the MLL-ELL translocation convert morphologically and functionally into adherent myofibroblasts

    SciTech Connect

    Tashiro, Haruko; Mizutani-Noguchi, Mitsuho; Shirasaki, Ryosuke

    2010-01-01

    Bone marrow-myofibroblasts, a major component of bone marrow-stroma, are reported to originate from hematopoietic stem cells. We show in this paper that non-adherent leukemia blasts can change into myofibroblasts. When myeloblasts from two cases of acute myelogenous leukemia with a fusion product comprising mixed lineage leukemia and RNA polymerase II elongation factor, were cultured long term, their morphology changed to that of myofibroblasts with similar molecular characteristics to the parental myeloblasts. The original leukemia blasts, when cultured on the leukemia blast-derived myofibroblasts, grew extensively. Leukemia blasts can create their own microenvironment for proliferation.

  9. Coexistence of alternative MLL-SEPT9 fusion transcripts in an acute myeloid leukemia with t(11;17)(q23;q25).

    PubMed

    Santos, Joana; Cerveira, Nuno; Correia, Cecília; Lisboa, Susana; Pinheiro, Manuela; Torres, Lurdes; Bizarro, Susana; Vieira, Joana; Viterbo, Luisa; Mariz, José M; Teixeira, Manuel R

    2010-02-01

    We present the characterization at the RNA level of an acute myeloid leukemia with a t(11;17)(q23;q25) and a MLL rearrangement demonstrated by FISH. Molecular analysis led to the identification of two coexistent in-frame MLL-SEPT9 fusion transcripts (variants 1 and 2), presumably resulting from alternative splicing. Real-time quantitative RT-PCR analysis showed that the relative expression of the MLL-SEPT9 fusion variant 2 was 1.88 fold higher than the relative expression of MLL-SEPT9 fusion variant 1. This is the first description of a MLL-SEPT9 fusion resulting in coexistence of two alternative splicing variants, each of which previously found isolated in myeloid leukemias. PMID:20113838

  10. [Refractory primary myeloid sarcoma of the breast with MLL-AF9 rearrangement].

    PubMed

    Uchida, Emi; Watanabe, Ken; Oshikawa, Gaku; Sakashita, Chizuko; Kurosu, Tetsuya; Fukuda, Tetsuya; Arai, Ayako; Murakami, Naomi; Miura, Osamu; Yamamoto, Masahide

    2016-01-01

    A 28-year-old woman presented with a right breast mass and axillary lymphadenopathy. Biopsy of the breast mass revealed myeloid sarcoma (MS) staining positive for CD4, CD13, CD33, and CD68/KP-1. Bone marrow aspiration revealed leukemic cell infiltration (9%). Leukemic cells possessed cytogenetic abnormalities of +8 and t(9;11)(p22;q23) with +22 (lymph node only), and molecular analyses confirmed the MLL-AF9 fusion gene. After induction chemotherapy and 2(nd) consolidation therapy, complete remission was maintained. However, during consolidation radiotherapy for the breast mass, the disease progressed in both the breast and bone marrow. She received re-induction therapy and proceeded to allogeneic stem cell transplantation. However, the disease relapsed in the breast soon after transplantation, and she died from disease progression. Trisomy 8 and the MLL-AF9 fusion gene have been reported in cases with MS in the breast. Trisomy 22 found additionally and exclusively in the extramedullary lesion implies extramedullary progression of MS from the medullary site of origin and may have been associated with the distinctive therapy resistance of these lesions in our case. PMID:26861104

  11. ARTD1 Suppresses Interleukin 6 Expression by Repressing MLL1-Dependent Histone H3 Trimethylation.

    PubMed

    Minotti, Roberta; Andersson, Anneli; Hottiger, Michael O

    2015-09-01

    ADP-ribosyltransferase diphtheria-toxin like 1/poly(ADP-ribose) polymerase 1 (ARTD1/PARP1) is a chromatin-associated protein in the nucleus and plays an important role in different cellular processes such as regulation of gene transcription. ARTD1 has been shown to coregulate the inflammatory response by modulating the activity of the transcription factor nuclear factor κB (NF-κB), the principal regulator of interleukin 6 (IL-6), an important inflammatory cytokine implicated in a variety of diseases such as cancer. However, to what extent and how ARTD1 regulates IL-6 transcription has not been clear. Here, we show that ARTD1 suppresses lipopolysaccharide (LPS)-induced IL-6 expression in macrophages, without affecting the recruitment of the NF-κB subunit RelA to the IL-6 promoter and independent of its enzymatic activity. Interestingly, knockdown of ARTD1 did not alter H3 occupancy but increased LPS-induced trimethylation of histone 3 at lysine 4 (H3K4me3), a hallmark of transcriptionally active genes. We found that ARTD1 mediates its effect through the methyltransferase MLL1, by catalyzing H3K4me3 at the IL-6 promoter and forming a complex with NF-κB. These results demonstrate that ARTD1 modulates IL-6 expression by regulating the function of an NF-κB enhanceosome complex, which involves MLL1 and does not require ADP-ribosylation. PMID:26149390

  12. Context, Context, Context: New Gene Programs Linked to Bad Behavior in MLL-AF9-Initiated Leukemia.

    PubMed

    Watanabe, Tatsuro; Ernst, Patricia

    2016-07-11

    In this issue of Cancer Cell, Stavropoulou et al. report that expression of the MLL-AF9 fusion results in acute myelogenous leukemia (AML) with different behaviors depending on cell context, which leads them to identify a transcriptional signature surprisingly resembling that of the epithelial-to-mesenchymal (EMT) transition, correlating with aggressiveness of disease. PMID:27411579

  13. Physical Interactions and Functional Coordination between the Core Subunits of Set1/Mll Complexes and the Reprogramming Factors

    PubMed Central

    Yang, Zhenhua; Augustin, Jonathan; Hu, Jing; Jiang, Hao

    2015-01-01

    Differentiated cells can be reprogrammed to the pluripotent state by overexpression of defined factors, and this process is profoundly influenced by epigenetic mechanisms including dynamic histone modifications. Changes in H3K4 methylation have been shown to be the predominant activating response in the early stage of cellular reprogramming. Mechanisms underlying such epigenetic priming, however, are not well understood. Here we show that the expression of the reprogramming factors (Yamanaka factors, Oct4, Sox2, Klf4 and Myc), especially Myc, directly promotes the expression of certain core subunits of the Set1/Mll family of H3K4 methyltransferase complexes. A dynamic recruitment of the Set1/Mll complexes largely, though not sufficiently in its own, explains the dynamics of the H3K4 methylation during cellular reprogramming. We then demonstrate that the core subunits of the Set1/Mll complexes physically interact with mainly Sox2 and Myc among the Yamanaka factors. We further show that Sox2 directly binds the Ash2l subunit in the Set1/Mll complexes and this binding is mediated by the HMG domain of Sox2. Functionally, we show that the Set1/Mll complex core subunits are required for efficient cellular reprogramming. We also show that Dpy30, one of the core subunits in the complexes, is required for the efficient target binding of the reprogramming factors. Interestingly, such requirement is not necessarily dependent on locus-specific H3K4 methylation. Our work provides a better understanding of how the reprogramming factors physically interact and functionally coordinate with a key group of epigenetic modulators to mediate transitions of the chromatin state involved in cellular reprogramming. PMID:26691508

  14. Physical Interactions and Functional Coordination between the Core Subunits of Set1/Mll Complexes and the Reprogramming Factors.

    PubMed

    Yang, Zhenhua; Augustin, Jonathan; Hu, Jing; Jiang, Hao

    2015-01-01

    Differentiated cells can be reprogrammed to the pluripotent state by overexpression of defined factors, and this process is profoundly influenced by epigenetic mechanisms including dynamic histone modifications. Changes in H3K4 methylation have been shown to be the predominant activating response in the early stage of cellular reprogramming. Mechanisms underlying such epigenetic priming, however, are not well understood. Here we show that the expression of the reprogramming factors (Yamanaka factors, Oct4, Sox2, Klf4 and Myc), especially Myc, directly promotes the expression of certain core subunits of the Set1/Mll family of H3K4 methyltransferase complexes. A dynamic recruitment of the Set1/Mll complexes largely, though not sufficiently in its own, explains the dynamics of the H3K4 methylation during cellular reprogramming. We then demonstrate that the core subunits of the Set1/Mll complexes physically interact with mainly Sox2 and Myc among the Yamanaka factors. We further show that Sox2 directly binds the Ash2l subunit in the Set1/Mll complexes and this binding is mediated by the HMG domain of Sox2. Functionally, we show that the Set1/Mll complex core subunits are required for efficient cellular reprogramming. We also show that Dpy30, one of the core subunits in the complexes, is required for the efficient target binding of the reprogramming factors. Interestingly, such requirement is not necessarily dependent on locus-specific H3K4 methylation. Our work provides a better understanding of how the reprogramming factors physically interact and functionally coordinate with a key group of epigenetic modulators to mediate transitions of the chromatin state involved in cellular reprogramming. PMID:26691508

  15. Polynesian mitochondrial DNAs reveal three deep maternal lineage clusters.

    PubMed

    Lum, J K; Rickards, O; Ching, C; Cann, R L

    1994-08-01

    The 4000-year-old human population expansion into Remote Oceania has been studied from a variety of genetic perspectives. Here, we report the discovery that Polynesians, traditionally considered to be a single cohesive linguistic and cultural unit, exhibit at least three distinct mitochondrial DNA (mtDNA) groups that probably shared a common maternal ancestor more than 85,000 years ago. The major lineage groups were first identified by PCR amplification of the mitochondrial region V deletion marker, known to be present at high frequency in Polynesian populations. Sequence analysis of mtDNA hypervariable control regions reveals a surprising number of lineages in Polynesia. We also note high sequence divergence between lineage groups deleted and not deleted in region V. Major group I lineages are common in Remote Oceania and include about 95% of the Native Hawaiian, 90% of the Samoan, and 100% of the Tongan donors in our sample. They contain the region V deletion and generally share three control region transition substitutions. This group also contains non-Polynesian individuals, such as Indonesians, Native Americans, Micronesians, Malaysians, Japanese, and Chinese. The group I Polynesians differ by 4.4% in sequence identity from major lineage group II Polynesians, who do not have the region V deletion and who share among themselves four distinct single-base substitutions. Group II individuals are seen at low frequency (< 10%) in Hawaii, Samoa, and the Cook Islands and may represent the predominant maternal lineage group of Papuan Melanesia. Major lineage group III, not found in Hawaii, tentatively links Samoa to Indonesia. Our observation of deep maternal genetic branches in Polynesia today confirms the notion that during the colonization of the Pacific, mainland Asian immigrants mixed with Melanesian peoples already inhabiting Near Oceania and carried a complex assortment of maternal genotypes derived from two distinct geographic sources to isolated island

  16. Archaic lineages in the history of modern humans.

    PubMed Central

    Labuda, D; Zietkiewicz, E; Yotova, V

    2000-01-01

    An important question in the ongoing debate on the origin of Homo sapiens is whether modern human populations issued from a single lineage or whether several, independently evolving lineages contributed to their genetic makeup. We analyzed haplotypes composed of 35 polymorphisms from a segment of the dystrophin gene. We find that the bulk of a worldwide sample of 868 chromosomes represents haplotypes shared by different continental groups. The remaining chromosomes carry haplotypes specific for the continents or for local populations. The haplotypes specific for non-Africans can be derived from the most frequent ones through simple recombination or a mutation. In contrast, chromosomes specific for sub-Saharan Africans represent a distinct group, as shown by principal component analysis, maximum likelihood tree, structural comparison, and summary statistics. We propose that African chromosomes descend from at least two lineages that have been evolving separately for a period of time. One of them underwent range expansion colonizing different continents, including Africa, where it mixed with another, local lineage represented today by a large fraction of African-specific haplotypes. Genetic admixture involving archaic lineages appears therefore to have occurred within Africa rather than outside this continent, explaining greater diversity of sub-Saharan populations observed in a variety of genetic systems. PMID:11014825

  17. Reproduction barrier between two lineages of bed bug (Cimex lectularius) (Heteroptera: Cimicidae).

    PubMed

    Wawrocka, Kamila; Balvín, Ondřej; Bartonička, Tomáš

    2015-08-01

    Populations of bed bugs, Cimex lectularius, have increased in recent years spreading into numerous urban areas across the Western world and making them an increasingly important pest of the twenty-first century. Research into hybridization within and between different lineages of bed bugs can help us to understand processes of micro- and macro-evolution in these ectoparasites and may inform the control of this pest species. Hybridization experiments between two host lineages of bed bug (C. lectularius) from Central Europe (Czech Republic), those associated with humans and those with bats, were conducted under laboratory conditions. Number of eggs and early instars were compared between crosses of mixed host lineages (interspecific mating) with pairs from the same host lineage, those from the same locality and same lineage from different localities (intraspecific mating). While crosses within host lineages resulted in egg production and later instars, crosses between different host lineages were unsuccessful, although of the mated females possessed sperm in their mesospermaleges and/or seminal conceptacles. These crosses did not even result in egg production. Moreover, in the mixed lineage crosses, mortality rates in adults were higher (51 and 50% higher in bat and human lineage, respectively) than in those animals from the same lineage. Survival of adults was in pairs from the same locality slightly higher than in pairs from different localities and differed statistically. These results support the existence of post-mating barriers and show reproductive isolation between two lineages of C. lectularius. Bat and human host adaptations can promote evolving of such barriers and can be product of alloxenic speciation. PMID:25952703

  18. Molecular characterization of the MLL-SEPT6 fusion gene in acute myeloid leukemia: identification of novel fusion transcripts and cloning of genomic breakpoint junctions.

    PubMed

    Cerveira, Nuno; Micci, Francesca; Santos, Joana; Pinheiro, Manuela; Correia, Cecília; Lisboa, Susana; Bizarro, Susana; Norton, Lucília; Glomstein, Anders; Asberg, Ann E; Heim, Sverre; Teixeira, Manuel R

    2008-07-01

    One of the MLL fusion partners in leukemia is the SEPT6 gene, which belongs to the evolutionarily conserved family of genes of septins. In this work we aimed to characterize at both the RNA and DNA levels three acute myeloid leukemias with cytogenetic evidence of a rearrangement between 11q23 and Xq24. Molecular analysis led to the identification of several MLL-SEPT6 fusion transcripts in all cases, including a novel MLL-SEPT6 rearrangement (MLL exon 6 fused with SEPT6 exon 2). Genomic DNA breakpoints were found inside or near Alu or LINE repeats in the MLL breakpoint cluster region, whereas the breakpoint junctions in the SEPT6 intron 1 mapped to the vicinity of GC-rich low-complexity repeats, Alu repeats, and a topoisomerase II consensus cleavage site. These data suggest that a non-homologous end-joining repair mechanism may be involved in the generation of MLL-SEPT6 rearrangements in acute myeloid leukemia. PMID:18492691

  19. [Effect of down-regulating mll-af9 gene expression on proliferation of acute monocytic leukemia cell line THP-1].

    PubMed

    Li, Lei; Zhang, Ai-Hua; Liu, Ling-Bo; Bi, Lan; Wang, Li; Zhao, Ya-Jie; Zou, Ping

    2008-04-01

    This study was aimed to investigate the effect of small interfering RNA (siRNA) on the expression of mll-af9 oncogene and the proliferation of human acute monocytic leukemia cell line THP-1. One group of siRNA was designed targeting mll-af9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured human acute monocytic leukemia cell line THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of mll-af9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). The cell proliferation rate was assayed by MTT. The change of cell cycles and apoptosis rate was detected by flow cytometry. The results showed that the siRNA transfection efficiency was 69.1%+/-1.8%. The level of mll-af9 mRNA expression was significantly inhibited in siRNA-transfected cells as compared with the controls. mll-af9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. The percentage of G0/G1 phase cells significantly increased in siRNA-transfected cells in comparion with the control cells, but the percentage of S phase cells significantly decreased. It is concluded that the mll-af9-targeted siRNA can effectively inhibit the proliferation of human acute monocytic leukemia cell line THP-1. PMID:18426643

  20. Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic

    SciTech Connect

    Avdic, Vanja; Zhang, Pamela; Lanouette, Sylvain; Voronova, Anastassia; Skerjanc, Ilona; Couture, Jean-Francois

    2011-08-24

    The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an N{alpha} acetylated form of histone H3 peptide (N{alpha}H3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of N{alpha}H3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.-Avdic, V., Zhang, P., Lanouette, S., Voronova, A., Skerjanc, I., Couture, J.-F. Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic.

  1. Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development.

    PubMed

    Barrett, Neil A; Malouf, Camille; Kapeni, Chrysa; Bacon, Wendi A; Giotopoulos, George; Jacobsen, Sten Eirik W; Huntly, Brian J; Ottersbach, Katrin

    2016-07-26

    MLL-AF4+ infant B cell acute lymphoblastic leukemia is characterized by an early onset and dismal survival. It initiates before birth, and very little is known about the early stages of the disease's development. Using a conditional Mll-AF4-expressing mouse model in which fusion expression is targeted to the earliest definitive hematopoietic cells generated in the mouse embryo, we demonstrate that Mll-AF4 imparts enhanced B lymphoid potential and increases repopulation and self-renewal capacity during a putative pre-leukemic state. This occurs between embryonic days 12 and 14 and manifests itself most strongly in the lymphoid-primed multipotent progenitor population, thus pointing to a window of opportunity and a potential cell of origin. However, this state alone is insufficient to generate disease, with the mice succumbing to B cell lymphomas only after a long latency. Future analysis of the molecular details of this pre-leukemic state will shed light on additional events required for progression to acute leukemia. PMID:27396339

  2. Lineage analysis by microsatellite loci deep sequencing in mice.

    PubMed

    Luo, Tao; He, Xionglei; Xing, Ke

    2016-05-01

    Lineage analysis is the identification of all the progeny of a single progenitor cell, and has become particularly useful for studying developmental processes and cancer biology. Here, we propose a novel and effective method for lineage analysis that combines sequence capture and next-generation sequencing technology. Genome-wide mononucleotide and dinucleotide microsatellite loci in eight samples from two mice were identified and used to construct phylogenetic trees based on somatic indel mutations at these loci, which were unique enough to distinguish and parse samples from different mice into different groups along the lineage tree. For example, biopsies from the liver and stomach, which originate from the endoderm, were located in the same clade, while samples in kidney, which originate from the mesoderm, were located in another clade. Yet, tissue with a common developmental origin may still contain cells of a mixed ancestry. This genome-wide approach thus provides a non-invasive lineage analysis method based on mutations that accumulate in the genomes of opaque multicellular organism somatic cells. Mol. Reprod. Dev. 83: 387-391, 2016. © 2016 Wiley Periodicals, Inc. PMID:26932355

  3. Phylogenetic lineages in the Botryosphaeriaceae

    PubMed Central

    Crous, Pedro W.; Slippers, Bernard; Wingfield, Michael J.; Rheeder, John; Marasas, Walter F.O.; Philips, Alan J.L.; Alves, Artur; Burgess, Treena; Barber, Paul; Groenewald, Johannes Z.

    2006-01-01

    Botryosphaeria is a species-rich genus with a cosmopolitan distribution, commonly associated with dieback and cankers of woody plants. As many as 18 anamorph genera have been associated with Botryosphaeria, most of which have been reduced to synonymy under Diplodia (conidia mostly ovoid, pigmented, thick-walled), or Fusicoccum (conidia mostly fusoid, hyaline, thin-walled). However, there are numerous conidial anamorphs having morphological characteristics intermediate between Diplodia and Fusicoccum, and there are several records of species outside the Botryosphaeriaceae that have anamorphs apparently typical of Botryosphaeria s.str. Recent studies have also linked Botryosphaeria to species with pigmented, septate ascospores, and Dothiorella anamorphs, or Fusicoccum anamorphs with Dichomera synanamorphs. The aim of this study was to employ DNA sequence data of the 28S rDNA to resolve apparent lineages within the Botryosphaeriaceae. From these data, 12 clades are recognised. Two of these lineages clustered outside the Botryosphaeriaceae, namely Diplodia-like anamorphs occurring on maize, which are best accommodated in Stenocarpella (Diaporthales), as well as an unresolved clade including species of Camarosporium/Microdiplodia. We recognise 10 lineages within the Botryosphaeriaceae, including an unresolved clade (Diplodia/Lasiodiplodia/Tiarosporella), Botryosphaeria s.str. (Fusicoccum anamorphs), Macrophomina, Neoscytalidium gen. nov., Dothidotthia (Dothiorella anamorphs), Neofusicoccum gen. nov. (Botryosphaeria-like teleomorphs, Dichomera-like synanamorphs), Pseudofusicoccum gen. nov., Saccharata (Fusicoccum- and Diplodia-like synanamorphs), “Botryosphaeria” quercuum (Diplodia-like anamorph), and Guignardia (Phyllosticta anamorphs). Separate teleomorph and anamorph names are not provided for newly introduced genera, even where both morphs are known. The taxonomy of some clades and isolates (e.g. B. mamane) remains unresolved due to the absence of ex

  4. MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3)

    PubMed Central

    Sobulo, Olatoyosi M.; Borrow, Julian; Tomek, Ronald; Reshmi, Shalini; Harden, Alanna; Schlegelberger, Brigitte; Housman, David; Doggett, Norman A.; Rowley, Janet D.; Zeleznik-Le, Nancy J.

    1997-01-01

    The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis. PMID:9238046

  5. SON and Its Alternatively Spliced Isoforms Control MLL Complex-Mediated H3K4me3 and Transcription of Leukemia-Associated Genes.

    PubMed

    Kim, Jung-Hyun; Baddoo, Melody C; Park, Eun Young; Stone, Joshua K; Park, Hyeonsoo; Butler, Thomas W; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M; Tan, Ming; Flemington, Erik K; Lim, Ssang-Taek; Ahn, Eun-Young Erin

    2016-03-17

    Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

  6. Atg5-dependent autophagy contributes to the development of acute myeloid leukemia in an MLL-AF9-driven mouse model.

    PubMed

    Liu, Qiang; Chen, Longgui; Atkinson, Jennifer M; Claxton, David F; Wang, Hong-Gang

    2016-01-01

    Acute myeloid leukemia (AML) is a hierarchical hematopoietic malignancy originating from leukemic stem cells (LSCs). Autophagy is a lysosomal degradation pathway that is hypothesized to be important for the maintenance of AML as well as contribute to chemotherapy response. Here we employ a mouse model of AML expressing the fusion oncogene MLL-AF9 and explore the effects of Atg5 deletion, a key autophagy protein, on the malignant transformation and progression of AML. Consistent with a transient decrease in colony-forming potential in vitro, the in vivo deletion of Atg5 in MLL-AF9-transduced bone marrow cells during primary transplantation prolonged the survival of recipient mice, suggesting that autophagy has a role in MLL-AF9-driven leukemia initiation. In contrast, deletion of Atg5 in malignant AML cells during secondary transplantation did not influence the survival or chemotherapeutic response of leukemic mice. Interestingly, autophagy was found to be involved in the survival of differentiated myeloid cells originating from MLL-AF9-driven LSCs. Taken together, our data suggest that Atg5-dependent autophagy may contribute to the development but not chemotherapy sensitivity of murine AML induced by MLL-AF9. PMID:27607576

  7. Theory and Practice of Lineage Tracing.

    PubMed

    Hsu, Ya-Chieh

    2015-11-01

    Lineage tracing is a method that delineates all progeny produced by a single cell or a group of cells. The possibility of performing lineage tracing initiated the field of Developmental Biology and continues to revolutionize Stem Cell Biology. Here, I introduce the principles behind a successful lineage-tracing experiment. In addition, I summarize and compare different methods for conducting lineage tracing and provide examples of how these strategies can be implemented to answer fundamental questions in development and regeneration. The advantages and limitations of each method are also discussed. PMID:26284340

  8. Pygo2 functions as a prognostic factor for glioma due to its up-regulation of H3K4me3 and promotion of MLL1/MLL2 complex recruitment

    PubMed Central

    Zhou, Cefan; Zhang, Yi; Dai, Jun; Zhou, Mengzhou; Liu, Miao; Wang, Yefu; Chen, Xing-Zhen; Tang, Jingfeng

    2016-01-01

    Pygo2 has been discovered as an important Wnt signaling component contributing to the activation of Wnt-target gene transcription. In the present study, we discovered that Pygo2 mRNA and protein levels were up-regulated in the majority of (152/209) human brain glioma tissues and five glioma cell lines, and significantly correlated with the age, the WHO tumor classification and poor patient survival. The histone methyltransferase complex components (WDR5, Ash2, and menin, but not CXCC1 or NCOA6) were down-regulated at the promoter loci of Wnt target genes after Pygo2 knockdown, and this was accompanied by the down-regulation of Wnt/β-catenin pathway activity. Further, we demonstrated that the involvement of Pygo2 in the activation of the Wnt pathway in human glioma progression is through up-regulation of the H3K4me3 (but not H3K4me2) by promoting the recruitment of the histone methyltransferase MLL1/MLL2 complex to Wnt target gene promoters. Thus, our study provided evidence that Pygo2 functions as a novel prognostic marker and represents a potential therapeutic target. PMID:26902498

  9. DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

    PubMed Central

    Stanulla, M; Wang, J; Chervinsky, D S; Thandla, S; Aplan, P D

    1997-01-01

    A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML. PMID:9199342

  10. Regulation of CDX4 gene transcription by HoxA9, HoxA10, the Mll-Ell oncogene and Shp2 during leukemogenesis

    PubMed Central

    Bei, L; Shah, C; Wang, H; Huang, W; Platanias, L C; Eklund, E A

    2014-01-01

    Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in

  11. Set1 and MLL1/2 target distinct sets of functionally different genomic loci in vivo

    PubMed Central

    Duncan, Elizabeth M.; Chitsazan, Alex D.; Seidel, Chris W.; Alvarado, Alejandro Sánchez

    2015-01-01

    SUMMARY Histone H3 lysine 4 trimethylation (H3K4me3) is known to correlate with both active and poised genomic loci, yet many questions remain regarding its functional roles in vivo. We identify functional genomic targets of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells and differentiated tissue of the planarian flatworm Schmidtea mediterranea. We show that, despite their common substrate, these enzymes target distinct genomic loci in vivo, which are distinguishable by the pattern each enzyme leaves on the chromatin template, i.e., the breadth of the H3K4me3 peak. Whereas Set1 targets are largely associated with the maintenance of the stem cell population, MLL1/2 targets are specifically enriched for genes involved in ciliogenesis. These data not only confirm that chromatin regulation is fundamental to planarian stem cell function, but also provide evidence for post-embryonic functional specificity of H3K4me3 methyltransferases in vivo. PMID:26711341

  12. Infant acute lymphoblastic leukemia with MLL gene rearrangements: outcome following intensive chemotherapy and hematopoietic stem cell transplantation.

    PubMed

    Kosaka, Yoshiyuki; Koh, Katsuyoshi; Kinukawa, Naoko; Wakazono, Yoshihiro; Isoyama, Keiichi; Oda, Takanori; Hayashi, Yasuhide; Ohta, Shigeru; Moritake, Hiroshi; Oda, Megumi; Nagatoshi, Yoshihisa; Kigasawa, Hisato; Ishida, Yasushi; Ohara, Akira; Hanada, Ryouji; Sako, Masahiro; Sato, Takeyuki; Mizutani, Shuki; Horibe, Keizo; Ishii, Eiichi

    2004-12-01

    Forty-four infants with acute lymphoblastic leukemia (ALL) characterized by MLL gene rearrangements were treated on a protocol of intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT) between November 1998 and June 2002. The remission induction rate was 91.0%, and the 3-year overall survival and event-free survival (EFS) rates, with 95% confidence intervals, were 58.2% (43.5%-72.9%) and 43.6% (28.5%-58.7%), respectively. Univariate analysis of EFS by presenting features indicated a poorer outcome in patients younger than 6 months of age with high white blood cell counts (>/= 100 x 10(9)/L; EFS rate, 9.4% versus 55.1% for all others, P = .0036) and in those with central nervous system invasion (EFS rate, 10.0% versus 56.9% for all others, P = .0073). The 3-year posttransplantation EFS rate for the 29 patients who underwent HSCT in first remission was 64.4% (46.4%-82.4%). In this subgroup, only the timing of HSCT (first remission versus others) was a significant risk factor by multivariate analysis (P < .0001). These results suggest that early introduction of HSCT, possibly with a less toxic conditioning regimen, may improve the prognosis for infants with MLL(+) ALL. Identification of subgroups or patients who respond well to intensified chemotherapy alone should have a high priority in future investigations. PMID:15297313

  13. Set1 and MLL1/2 Target Distinct Sets of Functionally Different Genomic Loci In Vivo.

    PubMed

    Duncan, Elizabeth M; Chitsazan, Alex D; Seidel, Chris W; Sánchez Alvarado, Alejandro

    2015-12-29

    Histone H3 lysine 4 trimethylation (H3K4me3) is known to correlate with both active and poised genomic loci, yet many questions remain regarding its functional roles in vivo. We identify functional genomic targets of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells and differentiated tissue of the planarian flatworm Schmidtea mediterranea. We show that, despite their common substrate, these enzymes target distinct genomic loci in vivo, which are distinguishable by the pattern each enzyme leaves on the chromatin template, i.e., the breadth of the H3K4me3 peak. Whereas Set1 targets are largely associated with the maintenance of the stem cell population, MLL1/2 targets are specifically enriched for genes involved in ciliogenesis. These data not only confirm that chromatin regulation is fundamental to planarian stem cell function but also provide evidence for post-embryonic functional specificity of H3K4me3 methyltransferases in vivo. PMID:26711341

  14. Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding.

    PubMed

    Hamilton, P B; Lewis, M D; Cruickshank, C; Gaunt, M W; Yeo, M; Llewellyn, M S; Valente, S A; Maia da Silva, F; Stevens, J R; Miles, M A; Teixeira, M M G

    2011-01-01

    Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi, T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes. PMID:21029792

  15. Colponemids represent multiple ancient alveolate lineages.

    PubMed

    Janouškovec, Jan; Tikhonenkov, Denis V; Mikhailov, Kirill V; Simdyanov, Timur G; Aleoshin, Vladimir V; Mylnikov, Alexander P; Keeling, Patrick J

    2013-12-16

    The alveolates comprise three well-studied protist lineages of significant environmental, medical, and economical importance: apicomplexans (e.g., Plasmodium), dinoflagellates (e.g., Symbiodinium), and ciliates (e.g., Tetrahymena). These major lineages have evolved distinct and unusual characteristics, the origins of which have proved to be difficult evolutionary puzzles. Mitochondrial genomes are a prime example: all three groups depart from canonical form and content, but in different ways. Reconstructing such ancient transitions is difficult without deep-branching lineages that retain ancestral characteristics. Here we describe two such lineages and how they illuminate the ancestral state of alveolate mitochondrial genomes. We established five clonal cultures of colponemids, predatory alveolates without cultured representatives and molecular data. Colponemids represent at least two independent lineages at the phylum level in multilocus phylogenetic analysis; one sister to apicomplexans and dinoflagellates, and the other at a deeper position. A genome survey from one strain showed that ancestral state of the mitochondrial genomes in the three major alveolate lineages consisted of an unusual linear chromosome with telomeres and a substantially larger gene set than known alveolates. Colponemid sequences also identified several environmental lineages as colponemids, altogether suggesting an untapped potential for understanding the origin and evolution of apicomplexans, dinoflagellates, and ciliates. PMID:24316202

  16. Importance of a Specific Amino Acid Pairing for Murine MLL Leukemias Driven by MLLT1/3 or AFF1/4

    PubMed Central

    Lokken, Alyson A.; Achille, Nicholas J.; Chang, Ming-jin; Lin, Jeffrey J.; Kuntimaddi, Aravinda; Leach, Benjamin I.; Malik, Bhavna; Nesbit, Jacqueline B.; Zhang, Shubin; Bushweller, John H.; Zeleznik-Le, Nancy J.; Hemenway, Charles S.

    2014-01-01

    Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins. PMID:25282333

  17. Spontaneous remission of acute myeloid leukemia relapse after hematopoietic cell transplantation in a high-risk patient with 11q23/MLL abnormality.

    PubMed

    Hudecek, Michael; Bartsch, Kristina; Jäkel, Nadja; Heyn, Simone; Pfannes, Roald; Al-Ali, Haifa Kathrin; Cross, Michael; Pönisch, Wolfram; Gerecke, Ulrich; Edelmann, Jeanett; Ittel, Thomas; Niederwieser, Dietger

    2008-01-01

    A 35-year-old female patient was diagnosed with acute myeloid leukemia with multiple genetic aberrations [48 XX, del(3)(q21), +6, t(11;15)(q23;q15), +21] including an 11q23/MLL abnormality. The patient achieved a complete remission after one induction chemotherapy cycle. After three courses of consolidation, a matched unrelated hematopoietic cell transplantation (HCT) was performed. Following an upper respiratory tract infection 7 years after transplant, her blood counts declined to leukocytes of 1 x 10(9)/l, platelets of 51 x 10(9)/l and hemoglobin of 7.5 g/dl. A bone marrow aspirate revealed 55% leukemic blasts carrying the unfavorable genetic aberrations seen at initial diagnosis (11q23/MLL). In the absence of any disease-specific treatment, the leukemic blasts cleared from the bone marrow within 6 days after diagnosis of relapse and peripheral blood counts returned to normal. Molecular analysis of the 11q23/MLL rearrangement was used to evaluate minimal residual disease, which became undetectable in repetitive FISH analyses. This is the first report of spontaneous remission in a patient with initially a multiaberrant leukemic cell clone and a proven 11q23/MLL abnormality at relapse after HCT. PMID:18367831

  18. All-trans retinoic acid combined with 5-Aza-2 Prime -deoxycitidine induces C/EBP{alpha} expression and growth inhibition in MLL-AF9-positive leukemic cells

    SciTech Connect

    Fujiki, Atsushi; Imamura, Toshihiko; Sakamoto, Kenichi; Kawashima, Sachiko; Yoshida, Hideki; Hirashima, Yoshifumi; Miyachi, Mitsuru; Yagyu, Shigeki; Nakatani, Takuya; Sugita, Kanji; Hosoi, Hajime

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer We tested whether ATRA and 5-Aza affect AML cell differentiation and growth. Black-Right-Pointing-Pointer Cell differentiation and growth arrest were induced in MLL-AF9-expressing cells. Black-Right-Pointing-Pointer Increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1 were also observed. Black-Right-Pointing-Pointer MLL-AF4/AF5q31-expressing cells are less sensitive to ATRA and 5-Aza. Black-Right-Pointing-Pointer Different MLL fusion has distinct epigenetic properties related to RA pathway. -- Abstract: The present study tested whether all-trans retinoic acid (ATRA) and 5-Aza-2 Prime -deoxycitidine (5-Aza) affect AML cell differentiation and growth in vitro by acting on the CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) and c-Myc axis. After exposure to a combination of these agents, cell differentiation and growth arrest were significantly higher in human and murine MLL-AF9-expressing cells than in MLL-AF4/AF5q31-expressing cells, which were partly associated with increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1, and decreased expression of c-Myc. These findings indicate that MLL-AF9-expressing cells are more sensitive to ATRA and 5-Aza, indicating that different MLL fusion proteins possess different epigenetic properties associated with retinoic acid pathway inactivation.

  19. Degree of Recruitment of DOT1L to MLL-AF9 Defines Level of H3K79 Di- and Tri-methylation on Target Genes and Transformation Potential

    PubMed Central

    Kuntimaddi, Aravinda; Achille, Nicholas J.; Thorpe, Jeremy; Lokken, Alyson A.; Singh, Ritambhara; Hemenway, Charles S.; Adli, Mazhar; Zeleznik-Le, Nancy J.; Bushweller, John H.

    2015-01-01

    SUMMARY The MLL gene is a common target of chromosomal translocations found in human leukemia. MLL-fusion leukemia has a consistently poor outcome. One of the most common translocation partners is AF9 (MLLT3). MLL-AF9 recruits DOT1L, a histone 3 lysine 79 methyltransferase (H3K79me1/me2/me3), leading to aberrant gene transcription. We show that DOT1L has three AF9 binding sites, and present the NMR solution structure of a DOT1L-AF9 complex. We generate structure-guided point mutations and find they have graded effects on recruitment of DOT1L to MLL-AF9. ChIP-Seq analyses of H3K79me2 and H3K79me3 show that graded reduction of the DOT1L interaction with MLL-AF9 results in differential loss of H3K79me2 and me3 at MLL-AF9 target genes. Furthermore, the degree of DOT1L recruitment is linked to the level of MLL-AF9 hematopoietic transformation. PMID:25921540

  20. Cloning of ELL, a gene that fuses to MLL in a t(11; 19)(q23; p13. 1) in acute myeloid leukemia

    SciTech Connect

    Thirman, M.J.; Levitan, D.A.; Kobayashi, H.; Simon, M.C.; Rowley, J.D. )

    1994-12-06

    To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient's leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On [open quotes]zoo blots,[close quotes] this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis. 30 refs., 7 figs.

  1. Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia.

    PubMed Central

    Thirman, M J; Levitan, D A; Kobayashi, H; Simon, M C; Rowley, J D

    1994-01-01

    To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient's leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On "zoo blots," this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis. Images PMID:7991593

  2. Amplification of c-MYC and MLL Genes as a Marker of Clonal Cell Progression in Patients with Myeloid Malignancy and Trisomy of Chromosomes 8 or 11.

    PubMed

    Angelova, S; Jordanova, M; Spassov, B; Shivarov, V; Simeonova, M; Christov, I; Angelova, P; Alexandrova, K; Stoimenov, A; Nikolova, V; Dimova, I; Ganeva, P; Tzvetkov, N; Hadjiev, E; Toshkov, S

    2011-12-01

    Gene amplification (amp) is one of the basic mechanisms connected with overexpression of oncogenes. The c-MYC (located in 8q24) and MLL (located in 11q23) are the most often over represented genes that lead to a rapid proliferation of the affected cell clone in patients with myeloid neoplasms. Assessment of the level of amp c-MYC or amp MLL in the cases with trisomy 8 (+8) or trisomy 11 (+11) and myeloid malignances is necessary for a more precise estimation of the disease progression. A total of 26 patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) were included in the study: 18 with +8, six with +11 and two with complex karyotypes suspected of the partial trisomy. Routine cytogenetic analysis and fluorescent in situ hybridization (FISH) were applied to indicate the chromosome alterations and genes amp in the bone marrow cells. Amp c-MYC was observed in 12 from 18 (66.7%) patients with +8. All the patients with +11 demonstrated a different level of amp MLL. In most of the cases with MDS (9/10), the coincidence of the +8 or +11 with amp c-MYC or amp MLL, respectively, leads to transformation to AML and/or short overall survival. Our data suggest that amp c-MYC and amp MLL develop in conformity with +8 and +11, especially in cases with progressive deviations in the karyotype as an aggressive expansion of an aberrant cell clone and appearance of additional chromosome anomalies. PMID:24052708

  3. Development and Use of Assay Conditions Suited to Screening for and Profiling of SET-Domain-Targeted Inhibitors of the MLL/SET1 Family of Lysine Methyltransferases

    PubMed Central

    Ferry, Joseph J.; Smith, Robert F.; Denney, Natalie; Walsh, Colin P.; McCauley, Lauren; Qian, Jie; Ma, Haiching; Horiuchi, Kurumi Y.

    2015-01-01

    Abstract Methylation of histone H3 lysine-4 (H3K4) is an important, regulatory, epigenetic post-translational modification associated with actively transcribed genes. In humans, the principal mediators of this modification are part of the MLL/SET1 family of methyltransferases, which comprises six members, MLLs1–4 and SET1A/SET1B. Aberrations in the structure, expression, and regulation of these enzymes are implicated in various disease states, making them important potential targets for drug discovery, particularly for oncology indications. The MLL/SET1 family members are most enzymatically active when part of a “core complex,” the catalytic SET-domain-containing subunits bound to a subcomplex consisting of the proteins WDR5, RbBP5, Ash2L and a homodimer of DPY-30 (WRAD2). The necessity of MLL/SET1 members to bind WRAD2 for full activity is the basis of a particular drug development strategy, which seeks to disrupt the interaction between the MLL/SET1 subunits and WDR5. This strategy is not without its theoretical and practical drawbacks, some of which relate to the ease with which complexes of Escherichia coli-expressed MLL/SET1 and WRAD2 fall apart. As an alternative strategy, we explore ways to stabilize the complex, focusing on the use of an excess of WRAD2 to drive the binding equilibria toward complex formation while maintaining low concentrations of the catalytic subunits. The purpose of this approach is to seek inhibitors that bind the SET domain, an approach proven successful with the related, but inherently more stable, enhancer of zeste homolog 2 (EZH2) complex. PMID:26065558

  4. Lineage Switching in Acute Leukemias: A Consequence of Stem Cell Plasticity?

    PubMed Central

    Dorantes-Acosta, Elisa; Pelayo, Rosana

    2012-01-01

    Acute leukemias are the most common cancer in childhood and characterized by the uncontrolled production of hematopoietic precursor cells of the lymphoid or myeloid series within the bone marrow. Even when a relatively high efficiency of therapeutic agents has increased the overall survival rates in the last years, factors such as cell lineage switching and the rise of mixed lineages at relapses often change the prognosis of the illness. During lineage switching, conversions from lymphoblastic leukemia to myeloid leukemia, or vice versa, are recorded. The central mechanisms involved in these phenomena remain undefined, but recent studies suggest that lineage commitment of plastic hematopoietic progenitors may be multidirectional and reversible upon specific signals provided by both intrinsic and environmental cues. In this paper, we focus on the current knowledge about cell heterogeneity and the lineage switch resulting from leukemic cells plasticity. A number of hypothetical mechanisms that may inspire changes in cell fate decisions are highlighted. Understanding the plasticity of leukemia initiating cells might be fundamental to unravel the pathogenesis of lineage switch in acute leukemias and will illuminate the importance of a flexible hematopoietic development. PMID:22852088

  5. Lineage Selection and the Maintenance of Sex

    PubMed Central

    de Vienne, Damien M.; Giraud, Tatiana; Gouyon, Pierre-Henri

    2013-01-01

    Sex predominates in eukaryotes, despite its short-term disadvantage when compared to asexuality. Myriad models have suggested that short-term advantages of sex may be sufficient to counterbalance its twofold costs. However, despite decades of experimental work seeking such evidence, no evolutionary mechanism has yet achieved broad recognition as explanation for the maintenance of sex. We explore here, through lineage-selection models, the conditions favouring the maintenance of sex. In the first model, we allowed the rate of transition to asexuality to evolve, to determine whether lineage selection favoured species with the strongest constraints preventing the loss of sex. In the second model, we simulated more explicitly the mechanisms underlying the higher extinction rates of asexual lineages than of their sexual counterparts. We linked extinction rates to the ecological and/or genetic features of lineages, thereby providing a formalisation of the only figure included in Darwin's “The origin of species”. Our results reinforce the view that the long-term advantages of sex and lineage selection may provide the most satisfactory explanations for the maintenance of sex in eukaryotes, which is still poorly recognized, and provide figures and a simulation website for training and educational purposes. Short-term benefits may play a role, but it is also essential to take into account the selection of lineages for a thorough understanding of the maintenance of sex. PMID:23825582

  6. Philadelphia chromosome-negative acute myeloid leukemia with 11q23/MLL translocation in a patient with chronic myelogenous leukemia

    PubMed Central

    Jaitly, Vanya; Wang, Wei

    2015-01-01

    Although defined by the presence of t(9;22), chronic myelogenous leukemia (CML) can have other concurrent additional cytogenetic changes, especially during disease progression. Additional chromosomal changes (ACAs) in CML often occur in Philadelphia chromosome (Ph)-positive cells and are associated with disease acceleration and treatment resistance. Occasionally chromosomal changes occur in Ph-negative cells and this phenomenon is often transient and does not correlate with disease progression. Very rarely myelodysplastic syndrome or acute leukemia can develop in Ph-negative cells. In this study, we report an unusual case of acute myeloid leukemia (AML) with 11q23/MLL translocation emerging from Ph-negative cells in a patient with CML. PMID:27358881

  7. The epigenetic factor Kmt2a/Mll1 regulates neural progenitor proliferation and neuronal and glial differentiation.

    PubMed

    Huang, Yin-Cheng; Shih, Hung-Yu; Lin, Sheng-Jia; Chiu, Ching-Chi; Ma, Tsu-Lin; Yeh, Tu-Hsueh; Cheng, Yi-Chuan

    2015-05-01

    Multiple epigenetic factors play a critical role in cell proliferation and differentiation. However, their function in embryogenesis, especially in neural development, is currently unclear. The Trithorax group (TrxG) homolog KMT2A (MLL1) is an important epigenetic regulator during development and has an especially well-defined role in hematopoiesis. Translocation and aberrant expression of KMT2A is often observed in many tumors, indicating its proto-oncogenic character. Here, we show that Kmt2a was essential for neural development in zebrafish embryos. Disrupting the expression of Kmt2a using morpholino antisense oligonucleotides and a dominant-negative variant resulted in neurogenic phenotypes, including downregulated proliferation of neural progenitors, premature differentiation of neurons, and impaired gliogenesis. This study therefore revealed a novel function of Kmt2a in cell proliferation and differentiation, providing further insight into the function of TrxG proteins in neural development and brain tumors. PMID:25284327

  8. Postembryonic lineages of the Drosophila brain: I. Development of the lineage-associated fiber tracts

    PubMed Central

    Lovick, Jennifer K.; Ngo, Kathy T.; Omoto, Jaison J.; Wong, Darren C.; Nguyen, Joseph D.; Hartenstein, Volker

    2013-01-01

    Neurons of the Drosophila central brain fall into approximately 100 paired groups, termed lineages. Each lineage is derived from a single asymmetrically-dividing neuroblast. Embryonic neuroblasts produce 1,500 primary neurons (per hemisphere) that make up the larval CNS followed by a second mitotic period in the larva that generates approximately 10,000 secondary, adult-specific neurons. Clonal analyses based on previous works using lineage-specific Gal4 drivers have established that such lineages form highly invariant morphological units. All neurons of a lineage project as one or a few axon tracts (secondary axon tracts, SATs) with characteristic trajectories, thereby representing unique hallmarks. In the neuropil, SATs assemble into larger fiber bundles (fascicles) which interconnect different neuropil compartments. We have analyzed the SATs and fascicles formed by lineages during larval, pupal, and adult stages using antibodies against membrane molecules (Neurotactin/Neuroglian) and synaptic proteins (Bruchpilot/N-Cadherin). The use of these markers allows one to identify fiber bundles of the adult brain and associate them with SATs and fascicles of the larval brain. This work lays the foundation for assigning the lineage identity of GFP-labeled MARCM clones on the basis of their close association with specific SATs and neuropil fascicles, as described in the accompanying paper (Wong et al., 2013. Postembryonic lineages of the Drosophila brain: II. Identification of lineage projection patterns based on MARCM clones. Submitted.). PMID:23880429

  9. Postembryonic lineages of the Drosophila brain: I. Development of the lineage-associated fiber tracts.

    PubMed

    Lovick, Jennifer K; Ngo, Kathy T; Omoto, Jaison J; Wong, Darren C; Nguyen, Joseph D; Hartenstein, Volker

    2013-12-15

    Neurons of the Drosophila central brain fall into approximately 100 paired groups, termed lineages. Each lineage is derived from a single asymmetrically-dividing neuroblast. Embryonic neuroblasts produce 1,500 primary neurons (per hemisphere) that make up the larval CNS followed by a second mitotic period in the larva that generates approximately 10,000 secondary, adult-specific neurons. Clonal analyses based on previous works using lineage-specific Gal4 drivers have established that such lineages form highly invariant morphological units. All neurons of a lineage project as one or a few axon tracts (secondary axon tracts, SATs) with characteristic trajectories, thereby representing unique hallmarks. In the neuropil, SATs assemble into larger fiber bundles (fascicles) which interconnect different neuropil compartments. We have analyzed the SATs and fascicles formed by lineages during larval, pupal, and adult stages using antibodies against membrane molecules (Neurotactin/Neuroglian) and synaptic proteins (Bruchpilot/N-Cadherin). The use of these markers allows one to identify fiber bundles of the adult brain and associate them with SATs and fascicles of the larval brain. This work lays the foundation for assigning the lineage identity of GFP-labeled MARCM clones on the basis of their close association with specific SATs and neuropil fascicles, as described in the accompanying paper (Wong et al., 2013. Postembryonic lineages of the Drosophila brain: II. Identification of lineage projection patterns based on MARCM clones. Submitted.). PMID:23880429

  10. MLL-AF9 Expression in Hematopoietic Stem Cells Drives a Highly Invasive AML Expressing EMT-Related Genes Linked to Poor Outcome.

    PubMed

    Stavropoulou, Vaia; Kaspar, Susanne; Brault, Laurent; Sanders, Mathijs A; Juge, Sabine; Morettini, Stefano; Tzankov, Alexandar; Iacovino, Michelina; Lau, I-Jun; Milne, Thomas A; Royo, Hélène; Kyba, Michael; Valk, Peter J M; Peters, Antoine H F M; Schwaller, Juerg

    2016-07-11

    To address the impact of cellular origin on acute myeloid leukemia (AML), we generated an inducible transgenic mouse model for MLL-AF9-driven leukemia. MLL-AF9 expression in long-term hematopoietic stem cells (LT-HSC) in vitro resulted in dispersed clonogenic growth and expression of genes involved in migration and invasion. In vivo, 20% LT-HSC-derived AML were particularly aggressive with extensive tissue infiltration, chemoresistance, and expressed genes related to epithelial-mesenchymal transition (EMT) in solid cancers. Knockdown of the EMT regulator ZEB1 significantly reduced leukemic blast invasion. By classifying mouse and human leukemias according to Evi1/EVI1 and Erg/ERG expression, reflecting aggressiveness and cell of origin, and performing comparative transcriptomics, we identified several EMT-related genes that were significantly associated with poor overall survival of AML patients. PMID:27344946

  11. Postembryonic lineages of the Drosophila brain: II. Identification of lineage projection patterns based on MARCM clones

    PubMed Central

    Wong, Darren C.; Lovick, Jennifer K.; Ngo, Kathy T.; Borisuthirattana, Wichanee; Omoto, Jaison J.; Hartenstein, Volker

    2014-01-01

    The Drosophila central brain is largely composed of lineages, units of sibling neurons derived from a single progenitor cell or neuroblast. During the early embryonic period neuroblast generate the primary neurons that constitute the larval brain. Neuroblasts reactivate in the larva, adding to their lineages a large number of secondary neurons which, according to previous studies in which selected lineages were labeled by stably expressed markers, differentiate during metamorphosis, sending terminal axonal and dendritic branches into defined volumes of the brain neuropil. We call the overall projection pattern of neurons forming a given lineage the “projection envelope” of that lineage. By inducing MARCM clones at the early larval stage, we labeled the secondary progeny of each neuroblast. For the supraesophageal ganglion excluding mushroom body (the part of the brain investigated in the present work) we obtained 81 different types of clones, Based on the trajectory of their secondary axon tracts (described in the accompanying paper), we assigned these clones to specific lineages defined in the larva. Since a labeled clone reveals all aspects (cell bodies, axon tracts, terminal arborization) of a lineage, we were able to describe projection envelopes for all secondary lineages of the supraesophageal ganglion. This work provides a framework by which the secondary neurons (forming the vast majority of adult brain neurons) can be assigned to genetically and developmentally defined groups. It also represents a step towards the goal to establish, for each lineage, the link between its mature anatomical and functional phenotype, and the genetic make-up of the neuroblast it descends from. PMID:23872236

  12. Postembryonic lineages of the Drosophila brain: II. Identification of lineage projection patterns based on MARCM clones.

    PubMed

    Wong, Darren C; Lovick, Jennifer K; Ngo, Kathy T; Borisuthirattana, Wichanee; Omoto, Jaison J; Hartenstein, Volker

    2013-12-15

    The Drosophila central brain is largely composed of lineages, units of sibling neurons derived from a single progenitor cell or neuroblast. During the early embryonic period, neuroblasts generate the primary neurons that constitute the larval brain. Neuroblasts reactivate in the larva, adding to their lineages a large number of secondary neurons which, according to previous studies in which selected lineages were labeled by stably expressed markers, differentiate during metamorphosis, sending terminal axonal and dendritic branches into defined volumes of the brain neuropil. We call the overall projection pattern of neurons forming a given lineage the "projection envelope" of that lineage. By inducing MARCM clones at the early larval stage, we labeled the secondary progeny of each neuroblast. For the supraesophageal ganglion excluding mushroom body (the part of the brain investigated in the present work) we obtained 81 different types of clones. Based on the trajectory of their secondary axon tracts (described in the accompanying paper, Lovick et al., 2013), we assigned these clones to specific lineages defined in the larva. Since a labeled clone reveals all aspects (cell bodies, axon tracts, terminal arborization) of a lineage, we were able to describe projection envelopes for all secondary lineages of the supraesophageal ganglion. This work provides a framework by which the secondary neurons (forming the vast majority of adult brain neurons) can be assigned to genetically and developmentally defined groups. It also represents a step towards the goal to establish, for each lineage, the link between its mature anatomical and functional phenotype, and the genetic make-up of the neuroblast it descends from. PMID:23872236

  13. C/EBPalpha and C/EBPvarepsilon induce the monocytic differentiation of myelomonocytic cells with the MLL-chimeric fusion gene.

    PubMed

    Matsushita, H; Nakajima, H; Nakamura, Y; Tsukamoto, H; Tanaka, Y; Jin, G; Yabe, M; Asai, S; Ono, R; Nosaka, T; Sugita, K; Morimoto, A; Hayashi, Y; Hotta, T; Ando, K; Miyachi, H

    2008-12-01

    CCAAT/enhancer binding proteins (C/EBPs) have an important function in granulocytic differentiation, and are also involved in the leukemogenesis of acute myeloid leukemia (AML). Their involvement in myelomonocytic leukemia, however, is still unclear. Therefore, the expression and function of C/EBPs in myelomonocytic cells with MLL-fusion genes were investigated. Retinoic acid (RA) induced monocytic differentiation in the myelomonocytic cell lines with MLL-fusion genes, THP-1, MOLM-14 and HF-6 cells, accompanied by monocytic differentiation with the upregulation of C/EBPalpha and C/EBPepsilon. Monocytic differentiation by RA treatment was confirmed in primary AML cells using a clonogenic assay. When the activity of C/EBPalpha or C/EBPepsilon was introduced into HF-6 cells, their cellular growth was arrested through differentiation into monocytes with the concomitant marked downregulation of Myc. Cebpe mRNA was upregulated by the induction of C/EBPalpha-ER, but not vice versa, thus suggesting that C/EBPepsilon may have an important function in the differentiation process. Introduction of Myc isoforms into HF-6 cells partially antagonized the C/EBPs effects. These findings suggest that the ectopic expression of C/EBPepsilon, as well as C/EBPalpha, can induce the monocytic differentiation of myelomonocytic leukemic cells with MLL-fusion gene through the downregulation of Myc, thus providing insight into the development of novel therapeutic approaches. PMID:18776924

  14. MLL-AF9– and HOXA9-mediated acute myeloid leukemia stem cell self-renewal requires JMJD1C

    PubMed Central

    Zhu, Nan; Chen, Mo; Eng, Rowena; DeJong, Joshua; Sinha, Amit U.; Rahnamay, Noushin F.; Koche, Richard; Al-Shahrour, Fatima; Minehart, Janna C.; Chen, Chun-Wei; Deshpande, Aniruddha J.; Xu, Haiming; Chu, S. Haihua; Ebert, Benjamin L.; Roeder, Robert G.; Armstrong, Scott A.

    2016-01-01

    Self-renewal is a hallmark of both hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs); therefore, the identification of mechanisms that are required for LSC, but not HSC, function could provide therapeutic opportunities that are more effective and less toxic than current treatments. Here, we employed an in vivo shRNA screen and identified jumonji domain–containing protein JMJD1C as an important driver of MLL-AF9 leukemia. Using a conditional mouse model, we showed that loss of JMJD1C substantially decreased LSC frequency and caused differentiation of MLL-AF9– and homeobox A9–driven (HOXA9-driven) leukemias. We determined that JMJD1C directly interacts with HOXA9 and modulates a HOXA9-controlled gene-expression program. In contrast, loss of JMJD1C led to only minor defects in blood homeostasis and modest effects on HSC self-renewal. Together, these data establish JMJD1C as an important mediator of MLL-AF9– and HOXA9-driven LSC function that is largely dispensable for HSC function. PMID:26878175

  15. An Lnc RNA (GAS5)/SnoRNA-derived piRNA induces activation of TRAIL gene by site-specifically recruiting MLL/COMPASS-like complexes

    PubMed Central

    He, Xin; Chen, Xinxin; Zhang, Xue; Duan, Xiaobing; Pan, Ting; Hu, Qifei; Zhang, Yijun; Zhong, Fudi; Liu, Jun; Zhang, Hong; Luo, Juan; Wu, Kang; Peng, Gao; Luo, Haihua; Zhang, Lehong; Li, Xiaoxi; Zhang, Hui

    2015-01-01

    PIWI-interacting RNA (piRNA) silences the transposons in germlines or induces epigenetic modifications in the invertebrates. However, its function in the mammalian somatic cells remains unknown. Here we demonstrate that a piRNA derived from Growth Arrest Specific 5, a tumor-suppressive long non-coding RNA, potently upregulates the transcription of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a proapoptotic protein, by inducing H3K4 methylation/H3K27 demethylation. Interestingly, the PIWIL1/4 proteins, which bind with this piRNA, directly interact with WDR5, resulting in a site-specific recruitment of the hCOMPASS-like complexes containing at least MLL3 and UTX (KDM6A). We have indicated a novel pathway for piRNAs to specially activate gene expression. Given that MLL3 or UTX are frequently mutated in various tumors, the piRNA/MLL3/UTX complex mediates the induction of TRAIL, and consequently leads to the inhibition of tumor growth. PMID:25779046

  16. Diversity rankings among bacterial lineages in soil.

    PubMed

    Youssef, Noha H; Elshahed, Mostafa S

    2009-03-01

    We used rarefaction curve analysis and diversity ordering-based approaches to rank the 11 most frequently encountered bacterial lineages in soil according to diversity in 5 previously reported 16S rRNA gene clone libraries derived from agricultural, undisturbed tall grass prairie and forest soils (n=26,140, 28 328, 31 818, 13 001 and 53 533). The Planctomycetes, Firmicutes and the delta-Proteobacteria were consistently ranked among the most diverse lineages in all data sets, whereas the Verrucomicrobia, Gemmatimonadetes and beta-Proteobacteria were consistently ranked among the least diverse. On the other hand, the rankings of alpha-Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes and Chloroflexi varied widely in different soil clone libraries. In general, lineages exhibiting largest differences in diversity rankings also exhibited the largest difference in relative abundance in the data sets examined. Within these lineages, a positive correlation between relative abundance and diversity was observed within the Acidobacteria, Actinobacteria and Chloroflexi, and a negative diversity-abundance correlation was observed within the Bacteroidetes. The ecological and evolutionary implications of these results are discussed. PMID:18987677

  17. Towards One Generic Name for Monophyletic Lineages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With the integration of asexually reproducing fungi into meaningful phylogenies, the need to use the same generic name for a monophyletic lineage has become urgent. At present Article 59 of the International Code of Botanical Nomenclature (ICBN) requires the use of a sexual state name for sexually r...

  18. SWI/SNF Subunits SMARCA4, SMARCD2 and DPF2 Collaborate in MLL-Rearranged Leukaemia Maintenance

    PubMed Central

    Sankar, Aditya; Miyagi, Satoru; Rundsten, Carsten Friis; Johansen, Jens Vilstrup; Helin, Kristian

    2015-01-01

    Alterations in chromatin structure caused by deregulated epigenetic mechanisms collaborate with underlying genetic lesions to promote cancer. SMARCA4/BRG1, a core component of the SWI/SNF ATP-dependent chromatin-remodelling complex, has been implicated by its mutational spectrum as exerting a tumour-suppressor function in many solid tumours; recently however, it has been reported to sustain leukaemogenic transformation in MLL-rearranged leukaemia in mice. Here we further explore the role of SMARCA4 and the two SWI/SNF subunits SMARCD2/BAF60B and DPF2/BAF45D in leukaemia. We observed the selective requirement for these proteins for leukaemic cell expansion and self-renewal in-vitro as well as in leukaemia. Gene expression profiling in human cells of each of these three factors suggests that they have overlapping functions in leukaemia. The gene expression changes induced by loss of the three proteins demonstrate that they are required for the expression of haematopoietic stem cell associated genes but in contrast to previous results obtained in mouse cells, the three proteins are not required for the expression of c-MYC regulated genes. PMID:26571505

  19. Evolution of a reassortant North American gull influenza virus lineage: drift, shift and stability

    PubMed Central

    2013-01-01

    Background The role of gulls in the ecology of avian influenza (AI) is different than that of waterfowl. Different constellations of subtypes circulate within the two groups of birds and AI viruses isolated from North American gulls frequently possess reassortant genomes with genetic elements from both North America and Eurasian lineages. A 2008 isolate from a Newfoundland Great Black-backed Gull contained a mix of North American waterfowl, North American gull and Eurasian lineage genes. Methods We isolated, sequenced and phylogenetically compared avian influenza viruses from 2009 Canadian wild birds. Results We analyzed six 2009 virus isolates from Canada and found the same phylogenetic lineage had persisted over a larger geographic area, with an expanded host range that included dabbling and diving ducks as well as gulls. All of the 2009 virus isolates contained an internal protein coding set of genes of the same Eurasian lineage genes except PB1 that was from a North American lineage, and these genes continued to evolve by genetic drift. We show evidence that the 2008 Great Black-backed Gull virus was derived from this lineage with a reassortment of a North American PA gene into the more stable core set of internal protein coding genes that has circulated in avian populations for at least 2 years. From this core, the surface glycoprotein genes have switched several times creating H13N6, H13N2, and H16N3 subtypes. These gene segments were from North American lineages except for the H16 and N3 vRNAs. Conclusions This process appears similar to genetic shifts seen with swine influenza where a stable “triple reassortant internal gene” core has circulated in swine populations with genetic shifts occurring with hemaggluttinin and neuraminidase proteins getting periodically switched. Thus gulls may serve as genetic mixing vessels for different lineages of avian influenza, similar to the role of swine with regards to human influenza. These findings illustrate the

  20. Evolution of a reassortant North American gull influenza virus lineage: drift, shift and stability

    USGS Publications Warehouse

    Hall, Jeffrey S.; TeSlaa, Joshua L.; Nashold, Sean W.; Halpin, Rebecca A.; Stockwell, Timothy; Wentworth, David E.; Dugan, Vivien; Ip, Hon S.

    2013-01-01

    Background: The role of gulls in the ecology of avian influenza (AI) is different than that of waterfowl. Different constellations of subtypes circulate within the two groups of birds and AI viruses isolated from North American gulls frequently possess reassortant genomes with genetic elements from both North America and Eurasian lineages. A 2008 isolate from a Newfoundland Great Black-backed Gull contained a mix of North American waterfowl, North American gull and Eurasian lineage genes. Methods: We isolated, sequenced and phylogenetically compared avian influenza viruses from 2009 Canadian wild birds. Results: We analyzed six 2009 virus isolates from Canada and found the same phylogenetic lineage had persisted over a larger geographic area, with an expanded host range that included dabbling and diving ducks as well as gulls. All of the 2009 virus isolates contained an internal protein coding set of genes of the same Eurasian lineage genes except PB1 that was from a North American lineage, and these genes continued to evolve by genetic drift. We show evidence that the 2008 Great Black-backed Gull virus was derived from this lineage with a reassortment of a North American PA gene into the more stable core set of internal protein coding genes that has circulated in avian populations for at least 2 years. From this core, the surface glycoprotein genes have switched several times creating H13N6, H13N2, and H16N3 subtypes. These gene segments were from North American lineages except for the H16 and N3 vRNAs. Conclusions: This process appears similar to genetic shifts seen with swine influenza where a stable "triple reassortant internal gene" core has circulated in swine populations with genetic shifts occurring with hemaggluttinin and neuraminidase proteins getting periodically switched. Thus gulls may serve as genetic mixing vessels for different lineages of avian influenza, similar to the role of swine with regards to human influenza. These findings illustrate the

  1. Ancestral relationships of the major eukaryotic lineages.

    PubMed

    Sogin, M L; Morrison, H G; Hinkle, G; Silberman, J D

    1996-03-01

    Molecular systematics has revolutionized our understanding of microbial evolution. Phylogenetic frameworks relating all organisms in this biosphere can be inferred from comparisons of slowly evolving molecules such as the small and large subunit ribosomal RNAs. Unlike today's text book standard, the "Five Kingdoms" (plants, animals, fungi, protists and bacteria), molecular studies define three primary lines of descent (Eukaryotes, Eubacteria, and Archaebacteria). Within the Eukaryotes, the "higher" kingdoms (Fungi, Plantae, and Animalia) are joined by at least two novel complex evolutionary assemblages, the "Alveolates" (ciliates, dinoflagellates and apicomplexans) and the "Stramenopiles" (diatoms, oomycetes, labyrinthulids, brown algae and chrysophytes). The separation of these eukaryotic groups (described as the eukaryotic "crown") occurred approximately 10(9) years ago and was preceded by a succession of earlier diverging protist lineages, some as ancient as the separation of the prokaryotic domains. The molecular phylogenies suggest that multiple endosymbiotic events introduced plastids into discrete eukaryotic lineages. PMID:9019131

  2. Launching the T-Lineage Developmental Programme

    PubMed Central

    Rothenberg, Ellen V.; Moore, Jonathan E.; Yui, Mary A.

    2011-01-01

    Preface Multipotent blood progenitor cells enter the thymus and begin a protracted differentiation process in which they gradually acquire T-cell characteristics while shedding their legacy of developmental plasticity. Notch signalling and basic helix-loop-helix E-protein transcription factors collaborate repeatedly to trigger and sustain this process throughout the period leading up to T-cell lineage commitment. Nevertheless, the process is discontinuous with separately regulated steps that demand roles for additional collaborating factors. This review discusses new evidence on the coordination of specification and commitment in the early T-cell pathway; effects of microenvironmental signals; the inheritance of stem-cell regulatory factors; and the ensemble of transcription factors that modulate the effects of Notch and E proteins, to distinguish individual stages and to polarize T-lineage fate determination. PMID:18097446

  3. Lineages of varicella-zoster virus.

    PubMed

    McGeoch, Duncan J

    2009-04-01

    Relationships among varicella-zoster virus (VZV; Human herpesvirus 3) genome sequences were examined to evaluate descent of strains, structures of lineages and incidence of recombination events. Eighteen complete, published genome sequences were aligned and 494 single nucleotide polymorphisms (SNPs) extracted, each as two alleles. At 281 SNPs, a single sequence differed from all the others. Distributions of the remaining 213 SNPs indicated that the sequences fell into five groups, which coincided with previously recognized phylogenetic groupings, termed E1, E2, J, M1 and M2. The 213-SNP set was divisible into 104 SNPs that were specific to a single group, and 109 cross-group SNPs that defined relationships among groups. This last set was evaluated by criteria of continuities in relationships between groups and breaks in such patterns, to identify crossover points and ascribe them to lineages. For the 99 cross-group SNPs in the genome's long unique region, it was seen that the E2 and M2 groups were almost completely distinct in their SNP alleles, and the E1 group was derived from a recombinant of E2 and M2. A valid phylogenetic tree could thus be constructed for the four E2 and two M2 strains. There was no substantive evidence for recombination within the E2 group or the E1 group (ten strains). The J and M1 groups each contained only one strain, and both were interpreted as having substantial distinct histories plus possible recombinant elements from the E2 and M2 lineages. The view of VZV recombination and phylogeny reached represents a major clarification of deep relationships among VZV lineages. PMID:19264671

  4. Genome sequesnce of lineage III Listeria monocytogenes strain HCC23

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More than 98% of reported human listeriosis cases are caused by Listeria monocytogenes serotypes within lineages I and II. Serotypes within lineage III (4a and 4c) are commonly isolated from environmental and food specimens. We report the first complete genome sequence of a lineage III isolate, HCC2...

  5. Phylogenomics of the Zygomycete lineages: Exploring phylogeny and genome evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Zygomycete lineages mark the major transition from zoosporic life histories of the common ancestors of Fungi and the earliest diverging chytrid lineages (Chytridiomycota and Blastocladiomycota). Genome comparisons from these lineages may reveal gene content changes that reflect the transition to...

  6. Genetic Mosaics and the Germ Line Lineage

    PubMed Central

    Samuels, Mark E.; Friedman, Jan M.

    2015-01-01

    Genetic mosaics provide information about cellular lineages that is otherwise difficult to obtain, especially in humans. De novo mutations act as cell markers, allowing the tracing of developmental trajectories of all descendants of the cell in which the new mutation arises. De novo mutations may arise at any time during development but are relatively rare. They have usually been observed through medical ascertainment, when the mutation causes unusual clinical signs or symptoms. Mutational events can include aneuploidies, large chromosomal rearrangements, copy number variants, or point mutations. In this review we focus primarily on the analysis of point mutations and their utility in addressing questions of germ line versus somatic lineages. Genetic mosaics demonstrate that the germ line and soma diverge early in development, since there are many examples of combined somatic and germ line mosaicism for de novo mutations. The occurrence of simultaneous mosaicism in both the germ line and soma also shows that the germ line is not strictly clonal but arises from at least two, and possibly multiple, cells in the embryo with different ancestries. Whole genome or exome DNA sequencing technologies promise to expand the range of studies of genetic mosaics, as de novo mutations can now be identified through sequencing alone in the absence of a medical ascertainment. These technologies have been used to study mutation patterns in nuclear families and in monozygotic twins, and in animal model developmental studies, but not yet for extensive cell lineage studies in humans. PMID:25898403

  7. Environmental biology of the marine Roseobacter lineage.

    PubMed

    Wagner-Döbler, Irene; Biebl, Hanno

    2006-01-01

    The Roseobacter lineage is a phylogenetically coherent, physiologically heterogeneous group of alpha-Proteobacteria comprising up to 25% of marine microbial communities, especially in coastal and polar oceans, and it is the only lineage in which cultivated bacteria are closely related to environmental clones. Currently 41 subclusters are described, covering all major marine ecological niches (seawater, algal blooms, microbial mats, sediments, sea ice, marine invertebrates). Members of the Roseobacter lineage play an important role for the global carbon and sulfur cycle and the climate, since they have the trait of aerobic anoxygenic photosynthesis, oxidize the greenhouse gas carbon monoxide, and produce the climate-relevant gas dimethylsulfide through the degradation of algal osmolytes. Production of bioactive metabolites and quorum-sensing-regulated control of gene expression mediate their success in complex communities. Studies of representative isolates in culture, whole-genome sequencing, e.g., of Silicibacter pomeroyi, and the analysis of marine metagenome libraries have started to reveal the environmental biology of this important marine group. PMID:16719716

  8. Lymphatic endothelial lineage assemblage during corneal lymphangiogenesis.

    PubMed

    Connor, Alicia L; Kelley, Philip M; Tempero, Richard M

    2016-03-01

    Postnatal inflammatory lymphangiogenesis presumably requires precise regulatory processes to properly assemble proliferating lymphatic endothelial cells (LECs). The specific mechanisms that regulate the assembly of LECs during new lymphatic vessel synthesis are unclear. Dynamic endothelial shuffling and rearrangement has been proposed as a mechanism of blood vessel growth. We developed genetic lineage-tracing strategies using an inductive transgenic technology to track the fate of entire tandem dimer tomato-positive (tdT) lymphatic vessels or small, in some cases clonal, populations of LECs. We coupled this platform with a suture-induced mouse model of corneal lymphangiogenesis and used different analytic microscopy techniques including serial live imaging to study the spatial properties of proliferating tdT(+) LEC progenies. LEC precursors and their progeny expanded from the corneal limbal lymphatic vessel and were assembled contiguously to comprise a subunit within a new lymphatic vessel. VE-cadherin blockade induced morphologic abnormalities in newly synthesized lymphatic vessels, but did not disrupt the tdT(+) lymphatic endothelial lineage assembly. Analysis of this static and dynamic data based largely on direct in vivo observations supports a model of lymphatic endothelial lineage assemblage during corneal inflammatory lymphangiogenesis. PMID:26658452

  9. Matrix elasticity directs stem cell lineage specification

    NASA Astrophysics Data System (ADS)

    Discher, Dennis

    2010-03-01

    Adhesion of stem cells - like most cells - is not just a membrane phenomenon. Most tissue cells need to adhere to a ``solid'' for viability, and over the last decade it has become increasingly clear that the physical ``elasticity'' of that solid is literally ``felt'' by cells. Here we show that Mesenchymal Stem Cells (MSCs) specify lineage and commit to phenotypes with extreme sensitivity to the elasticity typical of tissues [1]. In serum only media, soft matrices that mimic brain appear neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. Inhibition of nonmuscle myosin II activity blocks all elasticity directed lineage specification, which indicates that the cytoskeleton pulls on matrix through adhesive attachments. Results have significant implications for `therapeutic' stem cells and have motivated development of a proteomic-scale method to identify mechano-responsive protein structures [2] as well as deeper physical studies of matrix physics [3] and growth factor pathways [4]. [4pt] [1] A. Engler, et al. Matrix elasticity directs stem cell lineage specification. Cell (2006).[0pt] [2] C.P. Johnson, et al. Forced unfolding of proteins within cells. Science (2007).[0pt] [3] A.E.X. Brown, et al. Multiscale mechanics of fibrin polymer: Gel stretching with protein unfolding and loss of water. Science (2009).[0pt] [4] D.E. Discher, et al. Growth factors, matrices, and forces combine and control stem cells. Science (2009).

  10. Lymphatic endothelial lineage assemblage during corneal lymphangiogenesis

    PubMed Central

    Connor, Alicia L.; Kelley, Philip M.; Tempero, Richard M.

    2015-01-01

    Post natal inflammatory lymphangiogenesis presumably requires precise regulatory processes to properly assemble proliferating lymphatic endothelial cells (LECs). The specific mechanisms that regulate the assembly of LECs during new lymphatic vessel synthesis are unclear. Dynamic endothelial shuffling and rearrangement has been proposed as a mechanism of blood vessel growth. We developed genetic lineage tracing strategies using an inductive transgenic technology to track the fate of entire tandem dimer tomato positive (tdT) lymphatic vessels or small, in some cases clonal, populations of LECs. We coupled this platform with a suture induced mouse model of corneal lymphangiogenesis and used different analytic microscopy techniques including serial live imaging to study the spatial properties of proliferating tdT+ LEC progenies. LEC precursors and their progeny expanded from the corneal limbal lymphatic vessel and were assembled contiguously to comprise a subunit within a new lymphatic vessel. VE-cadherin blockade induced morphologic abnormalities in newly synthesized lymphatic vessels, but did not disrupt the tdT+ lymphatic endothelial lineage assembly. Analysis of this static and dynamic data based largely on direct in vivo observations supports a model of lymphatic endothelial lineage assemblage during corneal inflammatory lymphangiogenesis. PMID:26658452

  11. Bazooka mediates secondary axon morphology in Drosophila brain lineages

    PubMed Central

    2011-01-01

    In the Drosophila brain, neural lineages project bundled axon tracts into a central neuropile. Each lineage exhibits a stereotypical branching pattern and trajectory, which distinguish it from other lineages. In this study, we used a multilineage approach to explore the neural function of the Par-complex member Par3/Bazooka in vivo. Drosophila bazooka is expressed in post-mitotic neurons of the larval brain and localizes within neurons in a lineage-dependent manner. The fact that multiple GAL4 drivers have been mapped to several lineages of the Drosophila brain enables investigation of the role of Bazooka from larval to adult stages Bazooka loss-of-function (LOF) clones had abnormal morphologies, including aberrant pathway choice of ventral projection neurons in the BAla1 lineage, ectopic branching in the DALv2 and BAmv1 lineages, and excess BLD5 lineage axon projections in the optic medulla. Exogenous expression of Bazooka protein in BAla1 neurons rescued defective guidance, supporting an intrinsic requirement for Bazooka in the post-mitotic neuron. Elimination of the Par-complex member Par6 recapitulated Bazooka phenotypes in some but not all lineages, suggesting that the Par complex functions in a lineage-dependent manner, and that Bazooka may act independently in some lineages. Importantly, this study highlights the potential of using a multilineage approach when studying gene function during neural development in Drosophila. PMID:21524279

  12. Bazooka mediates secondary axon morphology in Drosophila brain lineages.

    PubMed

    Spindler, Shana R; Hartenstein, Volker

    2011-01-01

    In the Drosophila brain, neural lineages project bundled axon tracts into a central neuropile. Each lineage exhibits a stereotypical branching pattern and trajectory, which distinguish it from other lineages. In this study, we used a multilineage approach to explore the neural function of the Par-complex member Par3/Bazooka in vivo. Drosophila bazooka is expressed in post-mitotic neurons of the larval brain and localizes within neurons in a lineage-dependent manner. The fact that multiple GAL4 drivers have been mapped to several lineages of the Drosophila brain enables investigation of the role of Bazooka from larval to adult stages Bazooka loss-of-function (LOF) clones had abnormal morphologies, including aberrant pathway choice of ventral projection neurons in the BAla1 lineage, ectopic branching in the DALv2 and BAmv1 lineages, and excess BLD5 lineage axon projections in the optic medulla. Exogenous expression of Bazooka protein in BAla1 neurons rescued defective guidance, supporting an intrinsic requirement for Bazooka in the post-mitotic neuron. Elimination of the Par-complex member Par6 recapitulated Bazooka phenotypes in some but not all lineages, suggesting that the Par complex functions in a lineage-dependent manner, and that Bazooka may act independently in some lineages. Importantly, this study highlights the potential of using a multilineage approach when studying gene function during neural development in Drosophila. PMID:21524279

  13. A new family of cyclophilins with an RNA recognition motif that interact with members of the trx/MLL protein family in Drosophila and human cells.

    PubMed

    Anderson, Melanie; Fair, Keri; Amero, Sally; Nelson, Stephanie; Harte, Peter J; Diaz, Manuel O

    2002-04-01

    A new family of cyclophilins with an RNA recognition motif (RRM) has members in vertebrates, roundworms and flatworms. We have identified a Drosophilacyclophilin, Dcyp33, with a high degree of amino acid sequence identity and similarity with other members of the family. Dcyp33 interacts through its RRM domain with the third PHD finger of trithorax. This interaction is conserved in the human homologues of these proteins, Cyp33 and MLL. Over expression of Dcyp33 in DrosophilaSL1 cells results in down-regulation of AbdominalB Hoxgene expression, mirroring the effect of human Cyp33 on the expression of human HOXgenes. PMID:11976948

  14. Two myogenic lineages within the developing somite.

    PubMed

    Ordahl, C P; Le Douarin, N M

    1992-02-01

    It is well known that the muscles of the vertebrate body are derived from the somite. Precursor cells within the somite proper form the back or axial muscles while other precursor cells migrate away from the somite to populate the muscle of the limbs and ventral body wall. Although both types of muscle are generally thought of as arising from a common progenitor population, the myotome, recent evidence points to developmental differences in these two groups of muscles which may reflect different developmental lineages. To test the lineage hypothesis, we used microsurgery and the chick-quail nucleolar marker system to follow the developmental fate of the lateral and medial halves of somites at the wing level. The results showed that the structures of the mature somite (myotome and sclerotome) are derived virtually exclusively from cells residing in the medial half of the newly formed somite. On the other hand, virtually all of the cells residing in the lateral half of the newly formed somite are destined to leave the somite proper and populate the limb muscle and, probably, other somite-derived mesenchymal structures in the limb and ventral body wall. Switch-graft experiments show that the two halves of newly formed somites are largely interchangeable demonstrating that their ultimate developmental fate is position-dependent and that it becomes fixed as a result of extrinsic influences which act during later stages of somitogenesis. We conclude that at least two distinct myogenic lineages exist in the somite; one giving rise to the muscles of the back and the other giving rise to the limb musculature. PMID:1591996

  15. Direct lineage reprogramming to neural cells

    PubMed Central

    Kim, Janghwan; Ambasudhan, Rajesh; Ding, Sheng

    2016-01-01

    Recently we have witnessed an array of studies on direct reprogramming that describe induced inter conversion of mature cell types from higher organisms including human. While these studies reveal an unexpected level of plasticity of differentiated somatic cells, they also provide unprecedented opportunities to develop regenerative therapies for many debilitating disorders and model these ‘diseases-in-a-dish’ for studying their pathophysiology. Here we review the current state of the art in direct lineage reprogramming to neural cells, and discuss the challenges that need to be addressed toward achieving the full potential of this exciting new technology. PMID:22652035

  16. Cardiac Cell Lineages that Form the Heart

    PubMed Central

    Meilhac, Sigolène M.; Lescroart, Fabienne; Blanpain, Cédric; Buckingham, Margaret E.

    2014-01-01

    Myocardial cells ensure the contractility of the heart, which also depends on other mesodermal cell types for its function. Embryological experiments had identified the sources of cardiac precursor cells. With the advent of genetic engineering, novel tools have been used to reconstruct the lineage tree of cardiac cells that contribute to different parts of the heart, map the development of cardiac regions, and characterize their genetic signature. Such knowledge is of fundamental importance for our understanding of cardiogenesis and also for the diagnosis and treatment of heart malformations. PMID:25183852

  17. Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics.

    PubMed

    Orsi, Renato H; den Bakker, Henk C; Wiedmann, Martin

    2011-02-01

    Listeria monocytogenes consists of at least 4 evolutionary lineages (I, II, III, and IV) with different but overlapping ecological niches. Most L. monocytogenes isolates seem to belong to lineages I and II, which harbor the serotypes more commonly associated with human clinical cases, including serotype 1/2a (lineage II) and serotypes 1/2b and 4b (lineage I). Lineage II strains are common in foods, seem to be widespread in the natural and farm environments, and are also commonly isolated from animal listeriosis cases and sporadic human clinical cases. Most human listeriosis outbreaks are associated with lineage I isolates though. In addition, a number of studies indicate that, in many countries, lineage I strains are overrepresented among human isolates, as compared to lineage II strains. Lineage III and IV strains on the other hand are rare and predominantly isolated from animal sources. The apparent differences in the distribution of strains representing the L. monocytogenes lineages has lead to a number of studies aimed at identifying phenotypic differences among the different lineages. Interestingly, lineage II isolates seem to carry more plasmids than lineage I isolates and these plasmids often confer resistance to toxic metals and possibly other compounds that may be found in the environment. Moreover, lineage II isolates seem to be more resistant to bacteriocins than lineage I isolates, which probably confers an advantage in environments where bacteriocin-producing organisms are abundant. A large number of lineage II isolates and strains have been shown to be virulence-attenuated due to premature stop codon mutations in inlA and mutations in prfA. A subset of lineage I isolates carry a listeriolysin S hemolysin, which is not present in isolates belonging to lineages II, III, or IV. While lineage II isolates also show higher recombination rates than lineage I isolates, possibly facilitating adaptation of lineage II strains to diverse environments, lineage I

  18. Taspase 1: A protease with many biological surprises

    PubMed Central

    Niizuma, Hidetaka; Cheng, Emily H; Hsieh, James J

    2015-01-01

    Taspase 1 (TASP1) cleaves the mixed-lineage leukemia (MLL) and transcription factor (TF) IIA families of nuclear proteins to orchestrate various biological processes. TASP1 is not a classical oncogene, but assists in cell proliferation and permits oncogenic initiation through cleavage of MLL and TFIIA. TASP1 is thus better classified as a “non-oncogene addiction” protease, and targeting TASP1 offers a novel and attractive anticancer therapeutic strategy. PMID:27308523

  19. Conditional Lineage Ablation to Model Human Diseases

    NASA Astrophysics Data System (ADS)

    Lee, Paul; Morley, Gregory; Huang, Qian; Fischer, Avi; Seiler, Stephanie; Horner, James W.; Factor, Stephen; Vaidya, Dhananjay; Jalife, Jose; Fishman, Glenn I.

    1998-09-01

    Cell loss contributes to the pathogenesis of many inherited and acquired human diseases. We have developed a system to conditionally ablate cells of any lineage and developmental stage in the mouse by regulated expression of the diphtheria toxin A (DTA) gene by using tetracycline-responsive promoters. As an example of this approach, we targeted expression of DTA to the hearts of adult mice to model structural abnormalities commonly observed in human cardiomyopathies. Induction of DTA expression resulted in cell loss, fibrosis, and chamber dilatation. As in many human cardiomyopathies, transgenic mice developed spontaneous arrhythmias in vivo, and programmed electrical stimulation of isolated-perfused transgenic hearts demonstrated a strikingly high incidence of spontaneous and inducible ventricular tachycardia. Affected mice showed marked perturbations of cardiac gap junction channel expression and localization, including a subset with disorganized epicardial activation patterns as revealed by optical action potential mapping. These studies provide important insights into mechanisms of arrhythmogenesis and suggest that conditional lineage ablation may have wide applicability for studies of disease pathogenesis.

  20. Lineage and clonal development of gastric glands.

    PubMed

    Nomura, S; Esumi, H; Job, C; Tan, S S

    1998-12-01

    Individual gastric glands of the stomach are composed of cells of different phenotypes. These are derived from multipotent progenitor stem cells located at the isthmus region of the gland. Previous cell lineage analyses suggest that gastric glands, as in the colon and small intestine, are invariably monoclonal by adult stages. However, little is known about the ontogenetic progression of glandular clonality in the stomach. To examine this issue, we employed an in situ cell lineage marker in female mice heterozygous for an X-linked transgene. We found that stomach glands commence development as polyclonal units, but by adulthood (6 weeks), the majority progressed to monoclonal units. Our analysis suggests that at least three progenitor cells are required to initiate the development of individual gastric glands if they are analyzed just after birth. Hence, unlike the colon and small intestine, stomachs showed a significant fraction (10-25%) of polyclonal glands at adult stages. We suggest that these glands persist from polyclonal glands present in the embryonic stomach and hypothesize that they represent a subpopulation of glands with larger numbers of self-renewing stem cells. PMID:9851847

  1. The melanocyte lineage in development and disease

    PubMed Central

    Mort, Richard L.; Jackson, Ian J.; Patton, E. Elizabeth

    2015-01-01

    Melanocyte development provides an excellent model for studying more complex developmental processes. Melanocytes have an apparently simple aetiology, differentiating from the neural crest and migrating through the developing embryo to specific locations within the skin and hair follicles, and to other sites in the body. The study of pigmentation mutations in the mouse provided the initial key to identifying the genes and proteins involved in melanocyte development. In addition, work on chicken has provided important embryological and molecular insights, whereas studies in zebrafish have allowed live imaging as well as genetic and transgenic approaches. This cross-species approach is powerful and, as we review here, has resulted in a detailed understanding of melanocyte development and differentiation, melanocyte stem cells and the role of the melanocyte lineage in diseases such as melanoma. PMID:25670789

  2. Feedback, Lineages and Self-Organizing Morphogenesis

    PubMed Central

    Calof, Anne L.; Lowengrub, John S.; Lander, Arthur D.

    2016-01-01

    Feedback regulation of cell lineage progression plays an important role in tissue size homeostasis, but whether such feedback also plays an important role in tissue morphogenesis has yet to be explored. Here we use mathematical modeling to show that a particular feedback architecture in which both positive and negative diffusible signals act on stem and/or progenitor cells leads to the appearance of bistable or bi-modal growth behaviors, ultrasensitivity to external growth cues, local growth-driven budding, self-sustaining elongation, and the triggering of self-organization in the form of lamellar fingers. Such behaviors arise not through regulation of cell cycle speeds, but through the control of stem or progenitor self-renewal. Even though the spatial patterns that arise in this setting are the result of interactions between diffusible factors with antagonistic effects, morphogenesis is not the consequence of Turing-type instabilities. PMID:26989903

  3. Cell lineage in mammalian craniofacial mesenchyme.

    PubMed

    Yoshida, Toshiyuki; Vivatbutsiri, Philaiporn; Morriss-Kay, Gillian; Saga, Yumiko; Iseki, Sachiko

    2008-01-01

    We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells. PMID:18617001

  4. New native South American Y chromosome lineages.

    PubMed

    Jota, Marilza S; Lacerda, Daniela R; Sandoval, José R; Vieira, Pedro Paulo R; Ohasi, Dominique; Santos-Júnior, José E; Acosta, Oscar; Cuellar, Cinthia; Revollo, Susana; Paz-Y-Miño, Cesar; Fujita, Ricardo; Vallejo, Gustavo A; Schurr, Theodore G; Tarazona-Santos, Eduardo M; Pena, Sergio Dj; Ayub, Qasim; Tyler-Smith, Chris; Santos, Fabrício R

    2016-07-01

    Many single-nucleotide polymorphisms (SNPs) in the non-recombining region of the human Y chromosome have been described in the last decade. High-coverage sequencing has helped to characterize new SNPs, which has in turn increased the level of detail in paternal phylogenies. However, these paternal lineages still provide insufficient information on population history and demography, especially for Native Americans. The present study aimed to identify informative paternal sublineages derived from the main founder lineage of the Americas-haplogroup Q-L54-in a sample of 1841 native South Americans. For this purpose, we used a Y-chromosomal genotyping multiplex platform and conventional genotyping methods to validate 34 new SNPs that were identified in the present study by sequencing, together with many Y-SNPs previously described in the literature. We updated the haplogroup Q phylogeny and identified two new Q-M3 and three new Q-L54*(xM3) sublineages defined by five informative SNPs, designated SA04, SA05, SA02, SA03 and SA29. Within the Q-M3, sublineage Q-SA04 was mostly found in individuals from ethnic groups belonging to the Tukanoan linguistic family in the northwest Amazon, whereas sublineage Q-SA05 was found in Peruvian and Bolivian Amazon ethnic groups. Within Q-L54*, the derived sublineages Q-SA03 and Q-SA02 were exclusively found among Coyaima individuals (Cariban linguistic family) from Colombia, while Q-SA29 was found only in Maxacali individuals (Jean linguistic family) from southeast Brazil. Furthermore, we validated the usefulness of several published SNPs among indigenous South Americans. This new Y chromosome haplogroup Q phylogeny offers an informative paternal genealogy to investigate the pre-Columbian history of South America.Journal of Human Genetics advance online publication, 31 March 2016; doi:10.1038/jhg.2016.26. PMID:27030145

  5. Lineage determinants in early endocrine development

    PubMed Central

    Rieck, Sebastian; Bankaitis, Eric D.; Wright, Christopher V.E.

    2013-01-01

    Pancreatic endocrine cells are produced from a dynamic epithelium in a process that, as in any developing organ, is driven by interacting programs of spatiotemporally regulated intercellular signals and autonomous gene regulatory networks. These algorithms work to push progenitors and their transitional intermediates through a series of railroad-station-like switching decisions to regulate flux along specific differentiation tracks. Extensive research on pancreas organogenesis over the last 20 years, greatly spurred by the potential to restore functional β-cell mass in diabetic patients by transplantation therapy, is advancing our knowledge of how endocrine lineage bias is established and allocation is promoted. The field is working towards the goal of generating a detailed blueprint of how heterogeneous cell populations interact and respond to each other, and other influences such as the extracellular matrix, to move into progressively refined and mature cell states. Here, we highlight how signaling codes and transcriptional networks might determine endocrine lineage within a complex and dynamic architecture, based largely on studies in the mouse. The process begins with the designation of multipotent progenitor cells (MPC) to pancreatic buds that subsequently move through a newly proposed period involving epithelial plexus formation-remodeling, and ends with formation of clustered endocrine islets connected to the vascular and peripheral nervous systems. Developing this knowledge base, and increasing the emphasis on direct comparisons between mouse and human, will yield a more complete and focused picture of pancreas development, and thereby inform β-cell-directed differentiation from human embryonic stem or induced pluripotent stem cells (hESC, iPSC). Additionally, a deeper understanding may provide surprising therapeutic angles by defining conditions that allow the controllable reprogramming of endodermal or pancreatic cell populations. PMID:22728667

  6. Identification and characterization of mouse otic sensory lineage genes

    PubMed Central

    Hartman, Byron H.; Durruthy-Durruthy, Robert; Laske, Roman D.; Losorelli, Steven; Heller, Stefan

    2015-01-01

    Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells. PMID:25852475

  7. Viral load, E2 gene disruption status, and lineage of human papillomavirus type 16 infection in cervical neoplasia.

    PubMed

    Cheung, Jo L K; Lo, Keith W K; Cheung, Tak-Hong; Tang, Julian W; Chan, Paul K S

    2006-12-15

    The clinical utility of human papillomavirus (HPV) load and integration status remains unclear. We applied refined methods to delineate the viral load, integration status, and lineage of 104 women with HPV-16 monotype infection, including 19 with normal cervices, 9 with histologically proven cervical intraepithelial neoplasia (CIN) 1, 24 with CIN 2, 27 with CIN 3, and 25 with squamous cell carcinoma (SCC). Higher crude viral load, as determined by real-time polymerase chain reaction (PCR) targeting the E7 gene, was observed for SCC but became insignificant after normalization for cell content. Integration was located and quantified by real-time PCRs targeting, respectively, the carboxyl, amino, and hinge domains of the E2 gene. Pure episomal, integrated, and mixed forms were observed in all disease groups. Most E2 gene disruptions involved the amino-terminal, but sparing the hinge region that has been frequently used as a surrogate marker of integration. Large-fragment disruption involving all 3 E2 regions was observed only in the CIN 3 and SCC groups. Altogether, 33.3% of the CIN 3 group and 28.0% of the SCC group harbored pure episomal genomes. The Asian lineage was associated with a higher risk for CIN 3/SCC than the European lineage, and 6 of the 7 large-fragment E2 disruptions were from Asian lineage. The link between viral lineage, integration pattern, and oncogenesis deserves further study. PMID:17109343

  8. Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors

    PubMed Central

    Chaabouni, Azza; Chazaud, Bénédicte; Morlé, François

    2016-01-01

    This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells. PMID:27089435

  9. Prognostic and therapeutic role of targetable lesions in B-lineage acute lymphoblastic leukemia without recurrent fusion genes

    PubMed Central

    Fedullo, Anna Lucia; Peragine, Nadia; Gianfelici, Valentina; Piciocchi, Alfonso; Brugnoletti, Fulvia; Di Giacomo, Filomena; Pauselli, Simona; Holmes, Antony B.; Puzzolo, Maria Cristina; Ceglie, Giulia; Apicella, Valerio; Mancini, Marco; te Kronnie, Geertruy; Testi, Anna Maria; Vitale, Antonella; Vignetti, Marco; Guarini, Anna; Rabadan, Raul; Foà, Robin

    2016-01-01

    To shed light into the molecular bases of B-lineage acute lymphoblastic leukemia lacking known fusion transcripts, i.e. BCR-ABL1, ETV6-RUNX1, E2A-PBX1, and MLL rearrangements (B-NEG ALL) and the differences between children, adolescents/young adults (AYA) and adults, we analyzed 168 B-NEG ALLs by genome-wide technologies. This approach showed that B-NEG cases carry 10.5 mutations and 9.1 copy-number aberrations/sample. The most frequently mutated druggable pathways were those pertaining to RAS/RTK (26.8%) and JAK/STAT (12.5%) signaling. In particular, FLT3 and JAK/STAT mutations were detected mainly in AYA and adults, while KRAS and NRAS mutations were more frequent in children. RAS/RTK mutations negatively affected the outcome of AYA and adults, but not that of children. Furthermore, adult B-NEG ALL carrying JAK/STAT mutations had a shorter survival. In vitro experiments showed that FLT3 inhibitors reduced significantly the proliferation of FLT3-mutated primary B-NEG ALL cells. Likewise, PI3K/mTOR inhibitors reduced the proliferation of primary cells harboring RAS and IL7R mutations. These results refine the genetic landscape of B-NEG ALL and suggest that the different distribution of lesions and their prognostic impact might sustain the diverse outcome between children, adults and partly AYA - whose genomic scenario is similar to adults - and open the way to targeted therapeutic strategies. PMID:26883104

  10. Prognostic and therapeutic role of targetable lesions in B-lineage acute lymphoblastic leukemia without recurrent fusion genes.

    PubMed

    Messina, Monica; Chiaretti, Sabina; Wang, Jiguang; Fedullo, Anna Lucia; Peragine, Nadia; Gianfelici, Valentina; Piciocchi, Alfonso; Brugnoletti, Fulvia; Di Giacomo, Filomena; Pauselli, Simona; Holmes, Antony B; Puzzolo, Maria Cristina; Ceglie, Giulia; Apicella, Valerio; Mancini, Marco; Te Kronnie, Geertruy; Testi, Anna Maria; Vitale, Antonella; Vignetti, Marco; Guarini, Anna; Rabadan, Raul; Foà, Robin

    2016-03-22

    To shed light into the molecular bases of B-lineage acute lymphoblastic leukemia lacking known fusion transcripts, i.e. BCR-ABL1, ETV6-RUNX1, E2A-PBX1, and MLL rearrangements (B-NEG ALL) and the differences between children, adolescents/young adults (AYA) and adults, we analyzed 168 B-NEG ALLs by genome-wide technologies. This approach showed that B-NEG cases carry 10.5 mutations and 9.1 copy-number aberrations/sample. The most frequently mutated druggable pathways were those pertaining to RAS/RTK (26.8%) and JAK/STAT (12.5%) signaling. In particular, FLT3 and JAK/STAT mutations were detected mainly in AYA and adults, while KRAS and NRAS mutations were more frequent in children. RAS/RTK mutations negatively affected the outcome of AYA and adults, but not that of children. Furthermore, adult B-NEG ALL carrying JAK/STAT mutations had a shorter survival. In vitro experiments showed that FLT3 inhibitors reduced significantly the proliferation of FLT3-mutated primary B-NEG ALL cells. Likewise, PI3K/mTOR inhibitors reduced the proliferation of primary cells harboring RAS and IL7R mutations. These results refine the genetic landscape of B-NEG ALL and suggest that the different distribution of lesions and their prognostic impact might sustain the diverse outcome between children, adults and partly AYA - whose genomic scenario is similar to adults - and open the way to targeted therapeutic strategies. PMID:26883104