Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress.

ABSTRACT: Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL), but pan-Notch inhibitors showed excessive toxicity in clinical trials. To find alternative ways to target Notch signals, we investigated cell division cycle 73 (Cdc73), which is a Notch cofactor and key component of the RNA polymerase-associated transcriptional machinery, an emerging target in T-ALL. Although we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, chromatin and nascent gene expression profiling showed that Cdc73 intersects with Ets1 and Notch at chromatin within enhancers to activate expression of known T-ALL oncogenes through its enhancer functions. Cdc73 also intersects with these factors within promoters to activate transcription of genes that are important for DNA repair and oxidative phosphorylation through its gene body functions. Consistently, Cdc73 deletion induced DNA damage and apoptosis and impaired mitochondrial function. The CDC73-induced DNA repair expression program co-opted by NOTCH1 is more highly expressed in T-ALL than in any other cancer. These data suggest that Cdc73 might induce a gene expression program that was eventually intersected and hijacked by oncogenic Notch to augment proliferation and mitigate the genotoxic and metabolic stresses of elevated Notch signaling. Our report supports studying factors such as CDC73 that intersect with Notch to derive a basic scientific understanding on how to combat Notch-dependent cancers without directly targeting the Notch complex.

SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation.

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. Deregulation, including epigenetic deregulation, of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia. We recently showed that the Histone 3 Lysine 9 methyltransferase SETDB1 negatively regulates the expression of the pro-leukemic genes Hoxa9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation in MLL-AF9 leukemia cells. Here, we investigated the biological impact of altered SETDB1 expression and changes in H3K9 methylation on acute myeloid leukemia. We demonstrate that SETDB1 expression is correlated to disease status and overall survival in acute myeloid leukemia patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of leukemia cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While myeloid leukemia cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of cKit+ hematopoietic stem and progenitor cells. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of leukemia cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with acute myeloid leukemia, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in leukemia that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing acute myeloid leukemia.

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

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.

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

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.

MLL fusion protein-driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction.

MLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target.

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.

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.

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

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.

The YEATS domain epigenetic reader proteins ENL and AF9 and their therapeutic value in leukemia.

Recent studies have uncovered similarities and differences between 2 highly homologous epigenetic reading proteins, namely, ENL (MLLT1) and AF9 (MLLT3) with therapeutic implications. The importance of these proteins has traditionally been exemplified by their involvement in chromosomal translocations with the mixed-lineage leukemia gene (MLL; aka KMT2a). MLL rearrangements occur in a subset of acute leukemias and generate potent oncogenic MLL-fusion proteins that impact epigenetic and transcriptional regulation. Leukemic patients with MLL rearrangements display intermediate-to-poor prognoses, necessitating further mechanistic research. Several protein complexes involved in regulating RNA polymerase II transcription and the epigenetic landscape are hijacked in MLL-r leukemia, which include ENL and AF9. Recent biochemical studies have defined a highly homologous YEATS domain in ENL and AF9 that binds acylated histones, which aids in the localization and retention of these proteins to transcriptional targets. In addition, detailed characterization of the homologous ANC-1 homology domain (AHD) on ENL and AF9 revealed differential association with transcriptional activating and repressing complexes. Importantly, CRISPR knockout screens have demonstrated a unique role for wild-type ENL in leukemic stem cell function, whereas AF9 appears important for normal hematopoietic stem cells. In this perspective, we examine the ENL and AF9 proteins with attention to recent work characterizing the epigenetic reading YEATS domains and AHD on both wild-type proteins and when fused to MLL. We summarized the drug development efforts and their therapeutic potential and assess ongoing research that has refined our understanding of how these proteins function, which continues to reveal new therapeutic avenues.

The ENL YEATS epigenetic reader domain critically links MLL-ENL to leukemic stem cell frequency in t(11;19) Leukemia.

MLL (KMT2a) translocations are found in ~10% of acute leukemia patients, giving rise to oncogenic MLL-fusion proteins. A common MLL translocation partner is ENL and associated with a poor prognosis in t(11;19) patients. ENL contains a highly conserved N-terminal YEATS domain that binds acetylated histones and interacts with the PAF1c, an epigenetic regulator protein complex essential for MLL-fusion leukemogenesis. Recently, wild-type ENL, and specifically the YEATS domain, was shown to be essential for leukemic cell growth. However, the inclusion and importance of the YEATS domain in MLL-ENL-mediated leukemogenesis remains unexplored. We found the YEATS domain is retained in 84.1% of MLL-ENL patients and crucial for MLL-ENL-mediated leukemogenesis in mouse models. Mechanistically, deletion of the YEATS domain impaired MLL-ENL fusion protein binding and decreased expression of pro-leukemic genes like Eya1 and Meis1. Point mutations that disrupt YEATS domain binding to acetylated histones decreased stem cell frequency and increased MLL-ENL-mediated leukemia latency. Therapeutically, YEATS containing MLL-ENL leukemic cells display increased sensitivity to the YEATS inhibitor SGC-iMLLT compared to control AML cells. Our results demonstrate that the YEATS domain is important for MLL-ENL fusion protein-mediated leukemogenesis and exposes an "Achilles heel" that may be therapeutically targeted for treating t(11;19) patients.

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress.

Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL) but pan-Notch inhibitors were toxic in clinical trials. To find alternative ways to target Notch signals, we investigated Cell division cycle 73 (Cdc73), which is a Notch cofactor and component of transcriptional machinery, a potential target in T-ALL. While we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the lymphoid transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, Cdc73, Ets1, and Notch intersect chromatin at promoters and enhancers to activate oncogenes and genes that are important for DNA repair and oxidative phosphorylation. Consistently, Cdc73 deletion in T-ALL cells induced DNA damage and impaired mitochondrial function. Our data suggests that Cdc73 might promote a gene expression program that was eventually intersected by Notch to mitigate the genotoxic and metabolic stresses of elevated Notch signaling. We also provide mechanistic support for testing inhibitors of DNA repair, oxidative phosphorylation, and transcriptional machinery. Inhibiting pathways like Cdc73 that intersect with Notch at chromatin might constitute a strategy to weaken Notch signals without directly targeting the Notch complex.

Insight into the multi-faceted role of the SUV family of H3K9 methyltransferases in carcinogenesis and cancer progression.

Growing evidence implicates histone H3 lysine 9 methylation in tumorigenesis. The SUV family of H3K9 methyltransferases, which include G9a, GLP, SETDB1, SETDB2, SUV39H1 and SUV39H2 deposit H3K9me1/2/3 marks at euchromatic and heterochromatic regions, catalyzed by their conserved SET domain. In cancer, this family of enzymes can be deregulated by genomic alterations and transcriptional mis-expression leading to alteration of transcriptional programs. In solid and hematological malignancies, studies have uncovered pro-oncogenic roles for several H3K9 methyltransferases and accordingly, small molecule inhibitors are being tested as potential therapies. However, emerging evidence demonstrate onco-suppressive roles for these enzymes in cancer development as well. Here, we review the role H3K9 methyltransferases play in tumorigenesis focusing on gene targets and biological pathways affected due to misregulation of these enzymes. We also discuss molecular mechanisms regulating H3K9 methyltransferases and their influence on cancer. Finally, we describe the impact of H3K9 methylation on therapy induced resistance in carcinoma. Converging evidence point to multi-faceted roles for H3K9 methyltransferases in development and cancer that encourages a deeper understanding of these enzymes to inform novel therapy.

Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.

MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9-DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

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.

Epigenetic dysregulation in cancer.

One of the great paradoxes in cellular differentiation is how cells with identical DNA sequences differentiate into so many different cell types. The mechanisms underlying this process involve epigenetic regulation mediated by alterations in DNA methylation, histone posttranslational modifications, and nucleosome remodeling. It is becoming increasingly clear that disruption of the "epigenome" as a result of alterations in epigenetic regulators is a fundamental mechanism in cancer. This has major implications for the future of both molecular diagnostics as well as cancer chemotherapy.

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.

The PAF1c Subunit CDC73 Is Required for Mouse Hematopoietic Stem Cell Maintenance but Displays Leukemia-Specific Gene Regulation.

The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is required for MLL fusion-driven acute myeloid leukemia (AML) through direct regulation of pro-leukemic target genes such as Hoxa9 and Meis1. However, the role of the PAF1c in normal hematopoiesis is unknown. Here, we discovered that the PAF1c subunit, CDC73, is required for both fetal and adult hematopoiesis. Loss of Cdc73 in hematopoietic cells is lethal because of extensive bone marrow failure. Cdc73 has an essential cell-autonomous role for adult hematopoietic stem cell function in vivo, and deletion of Cdc73 results in cell-cycle defects in hematopoietic progenitors. Gene expression profiling indicated a differential regulation of Hoxa9/Meis1 gene programs by CDC73 in progenitors compared with AML cells, suggesting disease-specific functions. Thus, the PAF1c subunit, CDC73 is essential for hematopoietic stem cell function but exhibits leukemia-specific regulation of self-renewal gene programs in AML cells.

PAF1 complex interactions with SETDB1 mediate promoter H3K9 methylation and transcriptional repression of Hoxa9 and Meis1 in acute myeloid leukemia.

The Polymerase Associated Factor 1 complex (PAF1c) is an epigenetic co-modifying complex that directly contacts RNA polymerase II (RNAPII) and several epigenetic regulating proteins. Mutations, overexpression and loss of expression of subunits of the PAF1c are observed in various forms of cancer suggesting proper regulation is needed for cellular development. However, the biochemical interactions with the PAF1c that allow dynamic gene regulation are unclear. We and others have shown that the PAF1c makes a direct interaction with MLL fusion proteins, which are potent oncogenic drivers of acute myeloid leukemia (AML). This interaction is critical for the maintenance of MLL translocation driven AML by targeting MLL fusion proteins to the target genes Meis1 and Hoxa9. Here, we use a proteomics approach to identify protein-protein interactions with the PAF1c subunit CDC73 that regulate the function of the PAF1c. We identified a novel interaction with a histone H3 lysine 9 (H3K9) methyltransferase protein, SETDB1. This interaction is stabilized with a mutant CDC73 that is incapable of supporting AML cell growth. Importantly, transcription of Meis1 and Hoxa9 is reduced and promoter H3K9 trimethylation (H3K9me3) increased by overexpression of SETDB1 or stabilization of the PAF1c-SETDB1 interaction in AML cells. These findings were corroborated in human AML patients where increased SETDB1 expression was associated with reduced HOXA9 and MEIS1. To our knowledge, this is the first proteomics approach to search for CDC73 protein-protein interactions in AML, and demonstrates that the PAF1c may play a role in H3K9me3-mediated transcriptional repression in AML.

CYR61 (CCN1) is essential for placental development and vascular integrity.

CYR61 (CCN1) is a member of the CCN family of secreted matricellular proteins that includes connective tissue growth factor (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a growth factor-inducible immediate-early gene, CYR61 is an extracellular matrix-associated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR61 during development, we have disrupted the Cyr61 gene in mice. We show here that Cyr61-null mice suffer embryonic death: approximately 30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR61 as a novel and essential regulator of vascular development. CYR61 deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting differentiation of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR61-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.

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.