Andrew G. Muntean

Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

The Pathogenesis of Mixed-Lineage Leukemia

Aggressive leukemias arise in both children and adults as a result of rearrangements to the mixed-lineage leukemia gene (MLL) located on chromosome 11q23. MLL encodes a large histone methyltransferase that directly binds DNA and positively regulates gene transcription, including homeobox (HOX) genes. MLL is involved in chromosomal translocations, partial tandem duplications, and amplifications, all of which result in hematopoietic malignancies due to sustained HOX expression and stalled differentiation. MLL lesions are associated with both acute myeloid leukemia and acute lymphoid leukemia and are usually associated with a relatively poor prognosis despite improved treatment options such as allogeneic hematopoietic stem cell transplantation, which underscores the need for new treatment regimens. Recent advances have begun to reveal the molecular mechanisms that drive MLL-associated leukemias, which, in turn, have provided opportunities for therapeutic development. Here, we discuss the etiology of MLL leukemias and potential directions for future therapy.

The PAF complex synergizes with MLL fusion proteins at HOX loci to promote leukemogenesis

MLL is involved in chromosomal rearrangements that generate fusion proteins with deregulated transcriptional activity. The mechanisms of MLL fusion protein-mediated transcriptional activation are poorly understood. Here we show MLL interacts directly with the polymerase associated factor complex (PAFc) through sequences flanking the CxxC domain. PAFc interacts with RNA polymerase II and stimulates posttranslational histone modifications. PAFc augments MLL and MLL-AF9 mediated transcriptional activation of Hoxa9. Conversely, knockdown of PAFc disrupts MLL fusion protein-mediated transcriptional activation and MLL recruitment to target loci. PAFc gene expression is downregulated during hematopoiesis and likely serves to regulate MLL function. Deletions of MLL that abolish interactions with PAFc also eliminate MLL-AF9 mediated immortalization indicating an essential function for this interaction in leukemogenesis.

CBX8, a polycomb group protein, is essential for MLL-AF9-induced leukemogenesis.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene lead to the development of acute leukemias. Constitutive HOX gene activation by MLL fusion proteins is required for MLL-mediated leukemogenesis; however, the underlying mechanisms remain elusive. Here, we show that chromobox homolog 8 (CBX8), a Polycomb Group protein that interacts with MLL-AF9 and TIP60, is required for MLL-AF9-induced transcriptional activation and leukemogenesis. Conversely, both CBX8 ablation and specific disruption of the CBX8 interaction by point mutations in MLL-AF9 abrogate HOX gene upregulation and abolish MLL-AF9 leukemic transformation. Surprisingly, Cbx8-deficient mice are viable and display no apparent hematopoietic defects. Together, our findings demonstrate that CBX8 plays an essential role in MLL-AF9 transcriptional regulation and leukemogenesis.

STAT1 promotes megakaryopoiesis downstream of GATA-1 in mice

Thrombocytosis is associated with inflammation, and certain inflammatory cytokines, including IFN-gamma, stimulate megakaryocyte and platelet production. However, the roles of IFN-gamma and its downstream effector STAT1 in megakaryocyte development are poorly understood. We previously reported that STAT1 expression was significantly downregulated in Gata1-knockdown murine megakaryocytes, which also have impaired terminal maturation. Here, we show that ectopic expression of STAT1, or its target effector IRF-1, rescued multiple defects in Gata1-deficient megakaryopoiesis in mice, inducing polyploidization and expression of a subset of platelet-expressing genes. Enforced expression of STAT1, IRF-1, or GATA-1 enhanced phosphorylation of STAT1, STAT3, and STAT5 in cultured Gata1-deficient murine megakaryocytes, with concomitant megakaryocyte maturation. In contrast, enhanced thrombopoietin signaling, conferred by enforced expression of constitutively active JAK2 or c-MPL, induced phosphorylation of STAT3 and STAT5, but not STAT1, and failed to rescue megakaryocyte maturation. Finally, megakaryocytes from Stat1(-/-) mice were defective in polyploidization. Together, these findings reveal a unique role for STAT1 in megakaryopoiesis and provide new insights into how GATA-1 regulates this process. Our studies elucidate potential mechanisms by which various inflammatory disorders can cause elevated platelet counts.

The PHD fingers of MLL block MLL fusion protein-mediated transformation.

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.

ECSASB2 mediates MLL degradation during hematopoietic differentiation

Mixed lineage leukemia (MLL) is a key epigenetic regulator of normal hematopoietic development and chromosomal translocations involving MLL are one of the most common genetic alterations in human leukemia. Here we show that ASB2, a component of the ECS(ASB) E3 ubiquitin ligase complex, mediates MLL degradation through interaction with the PHD/Bromodomain region of MLL. Forced expression of ASB2 degrades MLL and reduces MLL transactivation activity. In contrast, the MLL-AF9 fusion protein does not interact with ASB2 and is resistant to ASB2 mediated degradation. Increased expression of ASB2 during hematopoietic differentiation is associated with decreased levels of MLL protein and down-regulation of MLL target genes. Knockdown of ASB2 leads to increased expression of HOXA9 and delayed cell differentiation. Our data support a model whereby ASB2 contributes to hematopoietic differentiation, in part, through MLL degradation and HOX gene down-regulation. Moreover, deletion of the PHD/Bromo region renders MLL fusion proteins resistant to ASB2-mediated degradation and may contribute to leukemogenesis.

C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis.

Significance Acute myeloid leukemia (AML) is a highly heterogeneous form of cancer that results from the uncontrolled proliferation of primitive immune cells. Homeobox A9 (HOXA9) is an evolutionarily conserved transcription factor that is overexpressed in a large percentage of AML cases and is associated with a poor prognosis. Here, we show that CCAAT/enhancer binding protein alpha (C/EBPα), a transcription factor involved in immune cell development that is commonly mutated in AML, is a critical collaborator required for HOXA9-mediated leukemic transformation. We also establish that the cell cycle regulator cyclin-dependent kinase inhibitors Cdkn2a/b are corepressed by the Hoxa9–C/EBPα complex. These findings suggest a novel functional interaction between two leukemic transcription factors, HOXA9 and C/EBPα, that is altered in a large percentage of AML cases. Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

Transcription factor GATA-1 and Down syndrome leukemogenesis.

Mutations in transcription factors constitute one means by which normal hematopoietic progenitors are converted to leukemic stem cells. Recently, acquired mutations in the megakaryocytic regulator GATA1 have been found in essentially all cases of acute megakaryoblastic leukemia (AMkL) in children with Down syndrome and in the closely related malignancy transient myeloproliferative disorder. In all cases, mutations in GATA1 lead to the expression of a shorter isoform of GATA-1, named GATA-1s. Because GATA-1s retains both DNA binding zinc fingers, but is missing the N-terminal transactivation domain, it has been predicted that the inability of GATA-1s to regulate its normal class of megakaryocytic target genes is the mechanism by which mutations in GATA1 contribute to the disease. Indeed, several recent reports have confirmed that GATA-1s fails to properly regulate the growth of megakaryocytic precursors, likely through aberrant transcriptional regulation. Although the specific target genes of GATA-1 mis-regulated by GATA-1s that drive this abnormal growth remain undefined, multiple candidate genes have been identified via gene array studies. Finally, the inability of GATA-1s to promote expression of important metabolic genes, such as cytadine deaminase, likely contributes to the remarkable hypersensitivity of AMkL blasts to cytosine arabinoside. Future studies to define the entire class of genes dysregulated by mutations in GATA1 will provide important insights into the etiology of these malignancies.

A subset of mixed lineage leukemia proteins has plant homeodomain (PHD)-mediated E3 ligase activity

Background: MLL1 contains four PHD fingers that are deleted in leukemogenic MLL1 fusion proteins. Results: The second PHD finger of MLL1 exhibits E3 ubiquitin ligase activity, and this activity is conserved in MLL4. Conclusion: PHD2 ligase activity potentially regulates MLL1 levels and activity. Significance: This study reveals a novel activity of MLL PHD fingers that may have important roles in gene regulation and carcinogenesis. The mixed lineage leukemia protein MLL1 contains four highly conserved plant homeodomain (PHD) fingers, which are invariably deleted in oncogenic MLL1 fusion proteins in human leukemia. Here we show that the second PHD finger (PHD2) of MLL1 is an E3 ubiquitin ligase in the presence of the E2-conjugating enzyme CDC34. This activity is conserved in the second PHD finger of MLL4, the closest homolog to MLL1 but not in MLL2 or MLL3. Mutation of PHD2 leads to MLL1 stabilization, as well as increased transactivation ability and MLL1 recruitment to the target gene loci, suggesting that PHD2 negatively regulates MLL1 activity.