Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common ‘core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain ‘super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

FLT3 internal tandem duplication (FLT3ITD) are common mutations in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide CRISPR/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations, and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI, and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation driven leukemias.

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The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of the 'cell of origin' from which the leukemia develops. Here we show that depending on extrinsic cues, human neonatal CD34(+) cells are readily immortalized along either the myeloid or lymphoid lineage upon MLL-AF9 expression and give rise to mainly lymphoid leukemia in immunocompromised mice. In contrast, immortalization of adult bone marrow CD34(+) cells is more difficult to achieve and is myeloid-biased, even when MLL-AF9 is expressed in purified hematopoietic stem cells (HSCs). Transcriptome analysis identified enrichment of HSC but not progenitor gene signatures in MLL-AF9-expressing cells. Although not observed in adult cells, neonatal cells expressing MLL-AF9 were enriched for gene signatures associated with poor prognosis, resistance to chemotherapeutic agents and MYC signaling. These results indicate that neonatal cells are inherently more prone to MLL-AF9-mediated immortalization than adult cells and suggest that intrinsic properties of the cell of origin, in addition to extrinsic cues, dictate lineage of the immortalized cell.

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis, fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro, preferentially promoting B-lymphocyte development, enhancing self-renewal of B-cell precursors, and leading to the establishment of long-term growth factor-dependent pre-Bcell lines. However, it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineagespecific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis, TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. IntroductionThe t(12;21)(p13;q22) translocation is present in up to 25% of children with pre-B-cell acute lymphoblastic leukemia (ALL). 1,2 This translocation results in the fusion of the AML1 (acute myeloid leukemia-1) gene with the TEL (translocation-Ets-leukemia) gene and generates a TEL-AML1 fusion transcription factor. 1,3 Both the AML1 and TEL genes are frequently rearranged in human myeloid and lymphoid leukemias, suggesting that they may be key regulators of hematopoiesis. Indeed, inactivation of these genes by homologous recombination established that AML1 is necessary for definitive hematopoiesis of all lineages 4,5 and that TEL is required for hematopoiesis of all lineages in the bone marrow. 6,7 Two recent studies have examined whether TEL-AML1 can induce leukemia. Transgenic mice in which expression of TEL-AML1 was driven by the immunoglobulin heavy gene enhancer/promoter failed to develop leukemia or any signs of hematologic disorder. 8 However, transfer of adult bone marrow infected with TEL-AML1-expressing retrovirus was found to induce ALL in 2 of 9 irradiated syngeneic mice after a long latency. 9 Furthermore, loss of the p16 INK4a p19 ARF genes, which encode cyclin-dependent kinase inhibitors, was found to cooperate with TEL-AML1 in this model of leukemogenesis. Taken together with the relatively low incidence and long latency of leukemia induced by TEL-AML1 alone, this suggests that secondary tumorigenic mutations are necessary for leukemia associated with TEL-AML1. This conclusion also can be drawn from studies on the occurrence of the TEL-AML1 translocation in the human population. Screening of unselected human cord blood samples established that approximately 1% have a leukemic TEL-AML1 gene fusion. 10 This frequency is 100 times the incidence rate of ALL with TEL-...

IntroductionChronic myeloid leukemia (CML) was the first cancer to be associated with a defined chromosomal abnormality, the Philadelphia chromosome (Ph), which occurs as a consequence of reciprocal translocation of chromosomes 9 and 22 (t9;22)(q34;q11). [1][2][3] In the pre-tyrosine kinase inhibitor (TKI) era, CML was characterized by distinct clinical stages evolving from a chronic phase (CP) through an accelerated phase (AP) into blast crisis (BC). 4,5 The BCR-ABL oncoprotein is necessary and sufficient for initiating the chronic phase of the disease when preferential expansion of myeloid progenitors and differentiated progeny is observed. [6][7][8] While overexpression of BCR-ABL in murine bone marrow (BM) was sufficient to induce transplantable leukemias in almost all recipient mice, 9 attempts to develop human CML models did not result in overt hematologic disease. When cord blood CD34 ϩ cells were retrovirally transduced with p210 BCR-ABL and transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/ SCID) mice, enhanced numbers of erythroid and megakaryocytic cells were observed, but after 5 months, only a few mice developed signs of a myeloproliferative disease. 10 When primary CML samples were injected into NOD/SCID mice in an attempt to model the disease, recipients showed an accumulation of abnormal populations of cells that recapitulated the onset of the disease, rather than the blast crisis, and no reproducible human hematopoietic malignancies were generated, so far. 11 Various pathways downstream of BCR-ABL have been implicated in the transformation process, including the activation of ␤-catenin, 12,13 signal transducer and activator of transcription 5 (STAT5), [14][15][16] Rac GTPases, 17,18 and mitogen-activated protein kinase/extracellular signal-related kinase (MEK/ERK). 19 Even though many of these pathways are activated by BCR-ABL and are required for the onset of disease, little is known about the molecular mechanisms that cooperate with BCR-ABL in the transition from CP-to BC-CML. Pathways that might cooperate with BCR-ABL in the transition from CP-CML into BC-CML include the Wnt and Hedgehog pathways. 12,20,21 Recent findings report that the expression of the polycomb gene, BMI1, which is implicated in normal and leukemic stem cell proliferation, 22,23 is significantly more highly expressed in patients with advanced disease than in patients in CP. 24 BMI1 has been linked to leukemogenesis since its discovery as a cooperating partner of c-Myc in the induction of B-cell lymphomas. 25 When HOXA9 and MEIS1 were coexpressed in bmi1 Ϫ/Ϫ cells, leukemia did not occur in secondary transplanted animals, suggesting that BMI1 has an important role in the maintenance of leukemic stem cells in this model. 22 We have classified BMI1 as a strong intrinsic regulator of human stem cells, as BMI1-expressing cells engrafted efficiently in the NOD/SCID mice even after in vitro culturing. 26 However, recipient mice did not show signs of disease, documenting that BMI1 by itself is not sufficien...

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.