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HOXA9 is commonly upregulated in acute myeloid leukemia (AML), in which it confers a poor prognosis. Characterizing the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro, and prolong survival in vivo. Integrated genomic, transcriptomic, and proteomic analyses further demonstrated that the HOXA9-SAFB (H9SB)–chromatin complex associates with nucleosome remodeling and histone deacetylase (NuRD) and HP1γ to repress the expression of factors associated with differentiation and apoptosis, including NOTCH1, CEBPδ, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1γ–associated catalytic activity also triggered differentiation, apoptosis, and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates the active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML that may be amenable to therapeutic intervention by targeting the H9SB interface and/or NuRD and HP1γ activity.

HOXA9is commonly upregulated in acute myeloid leukemia (AML), where it confers poor prognosis. Characterising the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein-SAFB.SAFBperturbation phenocopiedHOXA9knockout to decrease AML proliferation, increase differentiation and apoptosisin vitroand prolonged survivalin vivo. Integrated genomic, transcriptomic and proteomic analyses further demonstrated that the HOXA9-SAFB-chromatin complex associates with NuRD and HP1γ to repress the expression of factors associated with differentiation and apoptosis, includingNOTCH, CEBPδ,S100A8, andCDKN1A. Chemical or genetic perturbation of NuRD and HP1γ catalytic activity also triggered differentiation, apoptosis and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates activeHOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML, that may be amenable to therapeutic intervention via therapies targeting the HOXA9/SAFB interface and/or NuRD and HP1γ activity.Key Points-Identification of the endogenous human HOXA9 protein interactome in AML-HOXA9 forms a repressive complex with S/MAR binding protein (SAFB) that is critical for the maintenance of AML by facilitating proliferation and preventing differentiation and cell death.-The HOXA9/SAFB (H9SB) complex represses gene expression via recruitment of NuRD and HP1γ.

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.

Initial clinical trials with drugs targeting epigenetic modulators - such as bromodomain and extraterminal (BET) inhibitors - demonstrate modest results in acute myeloid leukemia (AML). The main reason for this involves an increased transcriptional plasticity within AML, which allows cells to escape the therapeutic pressure. To study mechanisms of resistance, we investigated immediate epigenetic and transcriptional responses following BET inhibition, and could demonstrate that BET inhibitor-mediated release of BRD4 from chromatin is accompanied by an acute compensatory feedback loop that attenuates inhibition, or even increases expression, of specific transcriptional modules. This adaptation is most marked at key AML maintenance genes and is mediated by p300, suggesting a rational therapeutic opportunity by combining BET- and p300- inhibition. p300 activity is required during all steps of adaptation. However, the transcriptional programs that p300 regulates to induce resistance to BETi differ between AML subtypes. Remarkably, in some AMLs, p300 regulates a series of transitional transcriptional patterns that allow homeostatic adjustments during earlier stages of resistance to BET-inhibitors. In consequence, p300 remains crucial throughout all stages of resistance in sensitive AML-subtypes, although its importance declines following the development of chronic resistance to BET inhibitors in some other AMLs. Altogether, our study elucidates the mechanisms that underlie an “acute” state of resistance to BET inhibition, achieved through p300 activity, and how these mechanisms remodel to become “chronic”. Importantly, however, our data also suggest that a sequential treatment with BET- and p300 inhibition may prevent resistance development, thereby improving outcomes.Key pointsA mechanistic feedback to p300 enables acute tolerance to BET inhibition.p300 regulates transcriptional networks that lead to chronic resistance to BET inhibition.Sequential BET-, followed by p300-inhibition, is synthetically lethal in AML, and is optimally deployed during earlier stages of resistance to BET inhibitors.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. scRNA-seq and bulk RNA-seq data were deposited in ArrayExpress with accession number E-MTAB-10853 and ERP006862, respectively. Code used for single-cell RNA-seq data analysis was deposited in Zenodo and can be accessed as G. Shikha and P. Cristina (2022). scRNA-seq analysis of RUNX1-RUNX1T1(9a) preleukemia, Zenodo; https://doi.org/10.5281/zenodo.6584118. Supplementary Materials: This PDF file includes: Figs. S1 to S7 avilable at: https://www.science.org/doi/suppl/10.1126/sciadv.abn4886/suppl_file/sciadv.abn4886_sm.pdf (223.69 MB). Other Supplementary Material for this manuscript includes the following: Supplementary Files S1 to S9 available at: https://www.science.org/doi/suppl/10.1126/sciadv.abn4886/suppl_file/sciadv.abn4886_supplementary_files_s1_to_s9.zip (2.34 MB). View/request a protocol for this paper from Bio-protocol available at: https://en.bio-protocol.org/cjrap.aspx?eid=10.1126/sciadv.abn4886 Copyright © 2022 The Authors. Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of premalignancy to cancer. To investigate this, we initiated preleukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA sequencing of preleukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of preleukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. That is, transcriptional variability confers forward momentum to cell fate systems, with differential multistage impact throughout cancer evolution. Wellcome: WT098051; Leuka: John Goldman Fellowship; Lady Tata Memorial Trust: International Scholarship 2017-2021; Cancer Research UK: C22324/A23015; Kay Kendall Leukaemia Fund: KKL888; Wellcome Trust / University of Cambridge ISSF: Bridging Funding 2019.

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a novel connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.

Key Points KAT2A is required at different stages of human cord blood erythroid development with ATAC and SAGA complex specificity.KAT2A regulates leukemia cell maintenance and identity through specific participation in ATAC and SAGA complexes., Visual Abstract, Epigenetic histone modifiers are key regulators of cell fate decisions in normal and malignant hematopoiesis. Their enzymatic activities are of particular significance as putative therapeutic targets in leukemia. In contrast, less is known about the contextual role in which those enzymatic activities are exercised and specifically how different macromolecular complexes configure the same enzymatic activity with distinct molecular and cellular consequences. We focus on KAT2A, a lysine acetyltransferase responsible for histone H3 lysine 9 acetylation, which we recently identified as a dependence in acute myeloid leukemia stem cells and that participates in 2 distinct macromolecular complexes: Ada two-A-containing (ATAC) and Spt-Ada-Gcn5-Acetyltransferase (SAGA). Through analysis of human cord blood hematopoietic stem cells and progenitors, and of myeloid leukemia cells, we identify unique respective contributions of the ATAC complex to regulation of biosynthetic activity in undifferentiated self-renewing cells and of the SAGA complex to stabilization or correct progression of cell type–specific programs with putative preservation of cell identity. Cell type and stage-specific dependencies on ATAC and SAGA-regulated programs explain multilevel KAT2A requirements in leukemia and in erythroid lineage specification and development. Importantly, they set a paradigm against which lineage specification and identity can be explored across developmental stem cell systems.

The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody–drug conjugate in vitro and in vivo.

Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of pre-malignancy to cancer. To investigate this, we initiated pre-leukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA-sequencing of pre-leukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of pre-leukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. In other words, transcriptional variability confers forward momentum to cell fate systems, with differential multi-stage impact throughout cancer evolution.One-sentence summaryLoss of Kat2a enhances transcriptional variability of ribosome biosynthetic programs and transiently accelerates pre-leukemia

Altered transcription is a cardinal feature of acute myeloid leukemia (AML), however, exactly how mutations synergize to remodel the epigenetic landscape and rewire 3-Dimensional (3-D) DNA topology is unknown. Here we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML, Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. These analyses allow the identification of long-range cis-regulatory circuits, including a novel super-enhancer of the Hoxa locus, as well as larger and more detailed gene-regulatory networks, whose importance we demonstrate through perturbation of network members.

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets., The oncogene MLL is frequently translocated in leukemia, resulting in oncogenic fusion proteins. Here, the authors report a temporally controlled mouse model of MLL-ENL driven leukemia AND identify therapeutic targets associated with early MLL-ENL driven leukaemogenesis.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective white blood cell differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity, that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics of a conditional knockout mouse, we demonstrate that Kat2a contributes to leukemia propagation through preservation of leukemia stem-like cells. Kat2a loss impacts transcription factor binding and reduces transcriptional burst frequency in a subset of gene promoters, generating enhanced variability of transcript levels. Destabilization of target programs shifts leukemia cell fate out of self-renewal into differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and distinct stages of cancer evolution., eLife digest Less than 30% of patients with acute myeloid leukaemia – an aggressive cancer of the white blood cells – survive five years post-diagnosis. This disease disrupts the maturation of white blood cells, resulting in the accumulation of immature cells that multiply and survive but are incapable of completing their maturation process. Amongst these, a group of cancer cells known as leukemic stem cells is responsible for continually replenishing the leukaemia, thus perpetuating its growth. Cancers develop when cells in the body acquire changes or mutations to their genetic makeup. The mutations that lead to acute myeloid leukaemia often affect the activity of genes known as epigenetic regulators. These genes regulate which proteins and other molecules cells make by controlling the way in which cells ‘read’ their genetic instructions. The epigenetic regulator Kat2a is thought to ‘tune’ the frequency at which cells read their genetic instructions. This tuning mechanism decreases random fluctuations in the execution of the instructions cells receive to make proteins and other molecules. In turn, this helps to ensure that individual cells of the same type behave in a similar way, for example by keeping leukaemia cells in an immature state. Here, Domingues, Kulkarni et al. investigated whether interfering with Kat2a can make acute myeloid leukaemia less aggressive by allowing the immature white blood cells to mature. Domingues, Kulkarni et al. genetically engineered mice to remove Kat2a from blood cells on demand and then inserted a mutation that causes acute myeloid leukaemia. The experiments showed that the loss of Kat2a delayed the development of leukaemia in the mice and progressively depleted leukaemia stem cells, causing the disease to become less aggressive. The results also showed that loss of Kat2a caused more fluctuations in how the white blood cells read their genetic code, which resulted in more variability in the molecules they produced and increased the tendency of the cells to mature. These findings establish that loss of Kat2a causes leukaemia stem cells to mature and stop multiplying by untuning the frequency at which the cells read their genetic instructions. In the future, it may be possible to develop drugs that target human KAT2A to treat acute myeloid leukaemia.

Summary DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

Acute myeloid leukemia (AML) is a heterogenous clonal disorder of hematopoietic progenitor cells with a dismal survival. It has a strong reliance on epigenetic and transcriptional factors for disease progression. Accordingly, we have previously identified KAT2A, a histone acetyl-transferase, as a requirement for AML maintenance; where chemical inhibition of KAT2A promotes differentiation of AML cell lines (Tzelepis et al., 2016, Cell Reports 17, 1193-1205). More recently, using a conditional knockout mouse model for Kat2a we showed that it sustains KMT2A/MLLT3 AML stem cells. Kat2a is a classical regulator of transcriptional variability, it's loss leads to cell-to-cell heterogeneity in transcription levels specifically from genes involved in ribosomal biogenesis and translation (Domingues et al., 2020, eLife 9:e51754). No recurrent mutations in the KAT2A gene have been described in AML, and it is unclear if and how it participates in pre-leukemia-to-AML progression. Herein, we use our conditional Kat2a knockout mouse model to analyze the effects of Kat2a loss in biology of RUNX1-RUNX1T1(9a) and Idh1R132H-initiated AML. These models represent forms of human disease with a prolonged pre-leukemia phase that typically require additional mutations for leukemia progression. We observed that loss of Kat2a accelerates leukemia initiation and progression in vivo. This acceleration was a consequence of fixation of transformed Kat2a KO cells in vivo which reflects as enhanced self-renewal capacity in vitro as measured by serial re-plating colony forming assay. Given the central role of Kat2a in limiting cell-to-cell transcription heterogeneity, we interrogated a potential link between loss of Kat2a, its consequent increase in transcriptional heterogeneity and pre-leukemia progression. For this, we performed single-cell RNA sequencing (scRNA-seq) of early-stage Kat2a WT and Kat2a KO RUNX1-RUNX1T1(9a) pre-leukaemia. Compatible with our previous observation, we observed that Kat2a KO cells were more heterogenous transcriptionally. Interestingly, this was accompanied by diversification of cell fates towards B-lymphocytes and monocytes. Furthermore, pseudo-temporal ordering of single Kat2a KO cells revealed highly branched trajectory heavily populated with intermediate stages of transformation; including accumulation of leukemia progenitors with RUNX1-RUNX1T1 signature. In contrast, Kat2a WT cells have linear normal hematopoiesis trajectory with minimal branching and an abrupt transition towards candidate leukemia progenitor state. Pathway analysis of Kat2a KO leukemia progenitor cells indicated perturbation of ribosomal biogenesis and translation associated genes. In order to test how these changes contributed to transformation, we performed S6K1 inhibition on Kat2a WT cells which transiently promoted transformation in vitro in both RUNX1-RUNX1T1(9a) and Idh1R132H cells, thus, phenocopying the effects of Kat2a loss. This suggested a mechanistic contribution of observed transcriptional changes in protein synthesis machinery towards leukemia progression. Taken together, our work suggests that loss of Kat2a results in diversification of cell fates, including with increased accessibility to cell states prone to transformation. Furthermore, these cells, prone to transformation, may benefit from a low biosynthetic activity that promotes their progression to leukemia state. We hypothesize that Kat2a loss may function similarly in the context of other malignancies. In the future, this knowledge may aid in development of early diagnostic tools and suggest bespoke therapeutic interventions. Figure Disclosures Prabakaran: Noncodomics: Consultancy. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

In this issue of Cancer Cell, de Boer et al. refine a set of acute myeloid leukemia (AML)-enriched plasma membrane markers that can be used to identify, prospectively isolate, and longitudinally track leukemic subclones within individual AML patients, correlating immunophenotypic profiles with specific mutational signatures, transcription, functional behavior, and therapeutic outcomes.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective myelo-monocytic differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics, we demonstrate that Kat2a contributes to leukemia propagation through homogeneity of transcriptional programs and preservation of leukemia stem-like cells. Kat2a loss reduces transcriptional bursting frequency in a subset of gene promoters, generating enhanced variability of transcript levels but minimal effects on mean gene expression. Destabilization of target programs shifts cellular equilibrium out of self-renewal towards differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and at distinct stages of cancer evolution. This work was funded by a Kay Kendall Leukaemia Fund Intermediate Fellowship (KKL888) and by a Leuka John Goldman Fellowship for Future Science (2017) to C.P.. S.P. is funded through a Cambridge-DBT Lectureship; R.K. was funded by an Isaac Newton Trust (INT) Research Grant and a Wellcome Trust ISSF/INT/University of Cambridge Joint Research Grant to C.P.; S.G. is funded by a Lady Tata Memorial Trust PhD Studentship, a Trinity Henry Barlow Trust Scholarship, and the Cambridge Trust; K.Z. received funding from AIRC (Italian Association for Cancer Research) and is the current recipient of a European Commission Horizon 2020 Marie Sklodowska Curie Post-Doctoral Fellowship.

Acute myeloid leukemia (AML), the most prevalent acute leukemia in adults, is an aggressive hematological malignancy arising in hematopoietic stem and progenitor cells. With the exception of a few specific AML subtypes, the mainstays of treatment have not significantly changed over the last 20 years, and are still based on standard cytotoxic chemotherapy. As a result, clinical outcome remains poor for the majority of patients, with overall long-term survival in the region of 20–30%. Recent successes in characterizing the genetic landscape of AML have highlighted that, despite its heterogeneity, many cases of AML carry recurrent mutations in genes encoding epigenetic regulators. Transcriptional dysregulation and altered epigenetic function have therefore emerged as exciting areas in AML research and it is becoming increasingly clear that epigenetic dysfunction is central to leukemogenesis in AML. This has subsequently paved the way for the development of epigenetically targeted therapies. In this review, we will discuss the most recent advances in our understanding of the role of epigenetic dysregulation in AML pathobiology. We will particularly focus on those altered epigenetic programs that have been shown to be central to the development and maintenance of AML in preclinical models. We will discuss the recent development of therapeutics specifically targeting these key epigenetic programs in AML, describe their mechanism of action and present their current clinical development. Finally, we will discuss the opportunities presented by epigenetically targeted therapy in AML and will highlight future challenges ahead for the AML community, to ensure that these novel therapeutics are optimally translated into clinical practice and result in clinical improvement for AML patients.

FLT3 internal tandem duplication (FLT3

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

Aberrant transcriptional programs are cardinal features of Acute Myeloid Leukemia (AML). Recently, it has been shown that specific distal cis-regulatory elements called enhancers communicate with promoters through 3-D DNA looping to regulate tissue-specific gene expression. Recurrent mutations in epigenetic regulators that modify enhancers, transcription factors that bind enhancers and the structural proteins that promote DNA looping, such as the Cohesin complex and its major binding partner CTCF have been demonstrated in AML. However, how these mutations regulate chromatin and alter 3D-DNA topology and communication between enhancers and promoters to generate leukemia-specific transcriptional programs remains poorly understood. In addition, many AML cases lack mutations in epigenetic regulators, transcription factors or DNA structural proteins, yet still demonstrate aberrant transcription, suggesting indirect effects of other mutations on enhancer function and the epigenetic landscape. To address these questions, we have utilized an allelic series of mice carrying the most common mutations in AML, namely Flt3-ITD and Npm1c (co-mutated in ~15% of all AMLs). These model different "transition states" (normal: wild type (WT), Pre-Malignant: single mutant (SM) with either Flt3-ITD or Npm1c mutations and Malignant: double mutant (DM)) during AML induction. Moreover, our design allows analysis of the SM mice to deconvolute the contribution of individual mutations to altered chromatin regulation. We have analyzed hematopoietic stem and progenitor cells (HSPCs) from WT and mutant mice for gene expression (RNA-seq), chromatin activation states (ChIP-seq for H3K4me1, H3K4me3, H3K27ac and H3K27me3), chromatin accessibility (ATAC-seq), and promoter-anchored 3-D chromatin interaction (promoter capture HiC, pCHiC)(Figure 1) and have integrated these analyses to determine the transcriptional, epigenetic and DNA-topological evolution of AML. Through pairwise comparisons between mutant and WT HSPCs, our data demonstrated that SM cells, with either Flt3-ITDor Npm1c mutations, alter gene expression only very modestly. However, when both mutations are present in DM cells, much larger gene programs that drive leukemia are both up- and downregulated. To examine the epigenetic regulation of these programs, we next built an enhancer compendium across all 4 allelic states using the H3K4me1 mark. Layering on H3K27ac activation, our data demonstrated that, in contrast to gene expression, significant alterations in enhancer specification and activation occur in advance of gene expression changes, to "prime" critical genes in Flt3-ITD, but not in Npm1c HSPCs. By contrast, Flt3-ITD and Npm1c mutations both altered global chromatin accessibility, with losses and gains evident at multiple critical genes. Similarly, our pCHiC data demonstrated significant alterations in DNA topology in mutant HSPCs that culminate in alterations in DNA "compartments" in DM HSPC. Moreover, they identified "hardwired" and "rewired" interactions between promoters and enhancers important for expression of critical leukemia programs. Analyses of all of these separate layers demonstrated a uniform pattern; progressive alterations in the transition from SM to DM HSPCs. Integrating these layers of analysis clearly demonstrated synergy between the mutations and a correlation between gene expression changes and chromatin dynamics in mutant cells. Furthermore, performing de novo motif analysis suggested a signal-specific transcription factor (TF) network downstream of Flt3-ITD that was amplified in the DM HSPC and that was corroborated by GSEA analysis. Our data had identified long-range regulatory control regions at the Spi1/PU.1 and Hoxa cluster loci amongst many others, and motif analysis had suggested Hox and Pu.1 to be important TFs in our malignant networks. Using these as examplars, we then perturbed the genes and regulatory elements at these loci by shRNA knockdown and CRISPR-mediated excision and could abrogate leukemic growth, validating the importance of our proposed networks. Taken together, these integrated analyses demonstrate a highly dynamic and coordinated process, where the effects of individual mutations synergize to remodel the chromatin landscape and 3D-DNA topology to generate networks that initiate and maintain AML transcriptional programs. Figure Disclosures Vassiliou: Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding.

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies(1). Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction(2,3). Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC(2,3) and the polymerase-associated factor complex (PAFc)(4,5), are associated with the BET family of acetyl-lysine recognizing, chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias.

The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Differences in the number of functionally and/or phenotypically defined bone marrow cells in inbred mouse strains have been exploited to map quantitative trait loci (QTL) that determine the variation in cell frequency. To extend this approach to the differences in the stem/progenitor cell compartment in CBA/H and C57BL/6 mice, we have exploited the resolution of flow cytometry and the power of QTL analyses in 124 F(2) mice to analyze lineage-negative (Lin(-)) bone marrow cells according to the intensity of labeling with Sca-1 and c-Kit. In the Lin(-) Sca-1(+) c-Kit(+) enriched population, six QTL were identified: one significant and five suggestive. Whereas previous in vitro clonogenic, LTC-IC, day 35 CAFC, and flow cytometry each identified different QTL, our approach identified the same or very similar QTL at all three loci (chromosomes 1, 17, and 18) as well as QTL on chromosomes 6 and 10. In silico analyses implicate hematopoietic stem cell homing involving Cxcr4 and Cxcl12 as being the determining pathway. The mapping of the same or very similar QTL in independent studies using different assay(s) suggests a common genetic determinant, and thus reinforces the biological and genetic significance of the QTL. These data also suggest that mouse bone marrow cell subpopulations can be functionally, phenotypically, and genetically defined.

The relationship between late normal tissue radiation injury phenotypes in 167 breast cancer patients treated with radiotherapy and: (i) radiotherapy dose (boost); (ii) an early acute radiation reaction and (iii) genetic background was examined. Patients were genotyped at single nucleotide polymorphisms (SNPs) in eight candidate genes. An early acute reaction to radiation and/or the inheritance of the transforming growth factor-beta1 (TGFbeta1 -509T) SNP contributed to the risk of fibrosis. In contrast, an additional 15 Gy electron boost and/or the inheritance of X-ray repair cross-complementing 1 (XRCC1) (R399Q) SNP contributed to the risk of telangiectasia. Although fibrosis, telangiectasia and atrophy, all contribute to late radiation injury, the data suggest that they have distinct underlying genetic and radiobiological causes. Fibrosis risk is associated with an inflammatory response (an acute reaction and/or TGFbeta1), whereas telangiectasia is associated with vascular endothelial cell damage (boost and/or XRCC1). Atrophy is associated with an acute response, but the genetic predisposing factors that determine the risk of an acute response or atrophy have yet to be identified. A combined analysis of two UK breast cancer patient studies shows that 8% of patients are homozygous (TT) for the TGFbeta1 (C-509T) variant allele and have a 15-fold increased risk of fibrosis following radiotherapy (95% confidence interval: 3.76-60.3; P=0.000003) compared with (CC) homozygotes.

Background B-cell precursor acute lymphoblastic leukemia (B-ALL) is amongst the leading causes of childhood cancer-related mortality. Its most common chromosomal aberration is the ETV6-RUNX1 fusion gene, with ~25 % of ETV6-RUNX1 patients also carrying PAX5 alterations. Methods We have recreated this mutation background by inter-crossing Etv6-RUNX1 (Etv6RUNX1-SB) and Pax5+/− mice and performed an in vivo analysis to find driver genes using Sleeping Beauty transposon-mediated mutagenesis and also exome sequencing. Results Combination of Etv6-RUNX1 and Pax5+/− alleles generated a transplantable B220 + CD19+ B-ALL with a significant disease incidence. RNA-seq analysis showed a gene expression pattern consistent with arrest at the pre-B stage. Analysis of the transposon common insertion sites identified genes involved in B-cell development (Zfp423) and the JAK/STAT signaling pathway (Jak1, Stat5 and Il2rb), while exome sequencing revealed somatic hotspot mutations in Jak1 and Jak3 at residues analogous to those mutated in human leukemias, and also mutation of Trp53. Conclusions Powerful synergies exists in our model suggesting STAT pathway activation and mutation of Trp53 are potent drivers of B-ALL in the context of Etv6-RUNX1;Pax5+/−. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1586-1) contains supplementary material, which is available to authorized users.

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks1,2. Early clinical trials have shown promise, especially in acute myeloid leukaemia3, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.

Giotopoulos et al. report a novel mouse model that closely mimics the natural progression of human chronic myeloid leukemia to blast crisis, and use this model to identify novel candidate genes and pathways that, in combination with BCR-ABL, drive disease progression., The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease.

Loss-of-function mutations of the cyclic-AMP response element binding protein, binding protein (CREBBP) gene have recently been described at high frequencies across a spectrum of lymphoid malignancies, particularly follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). The multiple effects of this epigenetic regulator on developmental and homeostatic processes have been extensively studied, however, exactly how CREBBP functions as a tumor suppressor and the reasons for its particular predilection for suppression of lymphoid tumors remains unclear. In addition, for many mature lymphoid malignancies, the existence of cancer stem cells is unproven and their provenance and the initial target cell for transformation a source of ongoing debate. Here we use multiple mouse strains to model loss of Crebbp in different lymphoid compartments to address these questions. We demonstrate that early loss of Crebbp in hematopoietic stem and progenitor cells (HSPC), through disruption by the Mx1-Cre recombinase, leads to increased development of hematological malignancies, particularly of the B-lymphoid lineage that mimic features of human lymphomas. Theses B-cell malignancies are of long latency and are preceded by significant alterations in the proliferation, self-renewal and differentiation of lymphoid progenitors, allowing hyperproliferative lymphoid progenitors whose differentiation is blocked to accumulate. Using an aberrant surface phenotype that resembles the eventual tumor, we identify a pre-malignant population in the peripheral blood of animals that is often evident many months before any disease characteristics. We demonstrate pre-malignant stem cell characteristics for this population in functional experiments, where it generates high level reconstitution of peripheral blood in transplant recipients, but only gives rise to disease in these animals after a long latency. We also utilize this unique cellular population in longitudinal genome scale analyses (clonality, RNA-Seq, ChIP-Seq and exome sequencing) to document the mechanisms of malignant evolution. Linking the increased rate of mutation we describe to Crebbp loss, we also demonstrate increased DNA damage and an altered DNA-damage response in premalignant lymphoid progenitors. Importantly, using a Cd19-Cre recombinase that excises only within committed lymphoid cells, we are able to demonstrate that when Crebbp is lost at a later stage of lymphoid development, the marked cellular abnormalities described above are completely lost and the development of tumors is no different from normal (Figure, below). Taken together, these findings define the developmental stage-specific tumor suppressor functions of Crebbp and shed light on the cellular origins and subsequent evolution of lymphoid malignancies. In addition, the altered response to DNA damage that we demonstrate upon loss of Crebbp, allied to the increased exposure to physiological DNA-damage during lymphoid ontogeny offers an explanation for the high incidence of CREBBP mutations in mature lymphoid malignancies. Figure Left panel, Kaplan Meier graph for Mx1-Cre Crebbp mice with loss of Crebbp in the HSPC compartment demonstrates significantly shorter survival vs WT littermates with intact expression of Crebbp. In contrast, when Crebbp is excised in a later lymphoid compartment through Cd19-Cre mediated recombination, right panel, no difference in survival is noted from WT littermate controls. Figure. Left panel, Kaplan Meier graph for Mx1-Cre Crebbp mice with loss of Crebbp in the HSPC compartment demonstrates significantly shorter survival vs WT littermates with intact expression of Crebbp. In contrast, when Crebbp is excised in a later lymphoid compartment through Cd19-Cre mediated recombination, right panel, no difference in survival is noted from WT littermate controls. Disclosures Huntly: Novartis: Speakers Bureau; BMS: Speakers Bureau; Ariad: Speakers Bureau; Pfizer: Speakers Bureau.

The t(12;21) translocation that generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. Single-nucleotide polymorphism array analysis of ETV6-RUNX1 patient samples has identified multiple additional genetic alterations; however, the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that cooperate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to coexpress the Etv6-RUNX1 fusion and Sleeping Beauty transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a Sleeping Beauty transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insight into the cooperating genetic alterations in ETV6-RUNX1 leukemia.

Whole body exposure to ionizing radiation increases the risk of radiation-induced acute myeloid leukaemia (r-AML). r-AML is the result of the accumulation of mutations in a single haemopoietic stem cell, so risk is therefore a function of the number of mutations required to transform the stem cell and the mutation rate. There is a genetic component to the risk of AML within the general population, and low penetrance variant alleles encoding DNA repair enzymes have been genetically implicated in therapy-related AML susceptibility. However, what is largely ignored is that target cell number, which defines the number of genomes at risk from DNA damaging agents, is also part of the equation that defines risk. We will review the evidence from genetic studies of inbred mouse models that target cell frequency is a risk factor in radiation leukaemogenesis. Inbred mouse strains that differ in their susceptibility to radiation-induced r-AML and thymic lymphoma (r-TL), spontaneous TL and pristane-induced plasmacytoma (PCT) have been exploited to identify susceptibility loci. The target cell in AML is the haemopoietic stem cell, whereas TLs and PCT arise from more mature lymphoid progenitor cells. Inbred mice also differ significantly in all aspects of haemopoiesis, and these differences have been used to identify quantitative trait loci (QTL) that determine the frequency of specific haemopoietic stem, progenitor or mature blood cells. The co-localization of QTL that determine risk and target cell frequency in all three haemopoietic malignancies is strong evidence that target cell frequency is a risk factor in radiation leukaemogenesis.

Erythropoiesis is under fine control and genetic loci that affect it are likely to be important in a range of conditions. To assess the relative contributions of different genetic loci to parameters of erythropoiesis, we have measured RBC counts in the peripheral circulation and committed erythroid cells (RBC and small normoblasts) in the bone marrow in a cohort of (CBA/H x C57BL/6) F2 mice to map quantitative trait loci (QTL). Candidate genes were assessed using bioinformatics and DNA sequencing. Different autosomal loci affect bone marrow (chromosomes 5, 11 and 19) and peripheral blood (chromosome 4) erythroid cell counts but there may be a common chromosome X locus. Spleen weight QTL were found on chromosomes 3, 15 and 17. Surprisingly, erythropoietin (Epo) is the best candidate quantitative trait gene (QTG) in the chromosome 5 locus that affects bone marrow but not peripheral blood erythroid cell counts. Epo gene expression is known to be genetically regulated in mice, but our data suggest a tissue-specific role for epo in mouse erythropoiesis that is also genetically determined. The identity of the other QTG will be important both to further knowledge of the control of erythropoiesis and as potential modifier genes for haematological disorders.

Objective To examine DNA methylation in mouse hemopoiesis before and after in vivo exposure to a leukemogenic dose of x-rays, and address whether methylation levels are associated with the relative radiosensitivity of tissues in vivo. Methods The methylation status of control CBA/H and C57BL/6 mouse tissues before and after exposure to 3-Gy x-rays, and myeloid and lymphoid leukemias and lymphomas, was assessed by the direct analysis of the 5-methylcytosine (5- Me C) content of DNA, and by Southern blot analysis of genomic repeat sequences. Results The DNA 5- Me C content of bone marrow is 15% lower than spleen. Together with the analyses of stem (myeloid) and progenitor (lymphoid) leukemias and lymphomas, we found a trend of increasing methylation during hemopoietic differentiation. Exposure to x-rays induced greater cell death in the hypomethylated bone marrow (>80%) than spleen (50%) in vivo, supporting the observed correlation found between methylation status and radiosensitivity of other high-turnover hierarchical tissues. Furthermore, there was an 8% DNA 5- Me C content decrease in bone marrow after in vivo exposure to 3-Gy x-rays, but this was genotype dependent, being observed in AML-susceptible (CBA/H) but not AML-resistant (C57BL/6) inbred mice. Conclusion Together these data suggest that methylation status may be related to the relative radiosensitivity of high-turnover hierarchical tissues such as bone marrow and that radiation-induced DNA hypomethylation has a role in radiation leukemogenesis.

The BET inhibitors are first-in-class, epigenetic targeted therapies that deliver a new therapeutic paradigm by directly targeting protein-protein interactions at chromatin. Early clinical trials have shown significant promise, especially in AML, suggesting that these compounds are likely to form an important component of future anti-cancer regimens. Therapeutic resistance is an inevitable consequence of most cancer therapies, therefore the evaluation of resistance mechanisms is of utmost importance in order to optimize the clinical utility of this novel class of drugs. Using primary murine stem and progenitor cells immortalized with MLL-AF9, we have developed a novel approach to generate over 20 clones stably resistant to the prototypical BET inhibitor, IBET. Resistance has been established at >IC90 of the parental cell line. In parallel, we have maintained matched vehicle treated clones in addition to the parental cell line. Resistant clones maintain their clonogenic capacity in IBET and are also impervious to IBET induced cell-cycle arrest and apoptosis. Resistance to IBET confers cross-resistance to other chemically distinct BET inhibitors such as JQ1 and also resistance to genetic knockdown of BET proteins. Moreover, resistance is stably maintained across subsequent cell generations in the absence of ongoing selective pressure. Resistance is not mediated through increased drug efflux or metabolism but is demonstrated to emerge from the leukemia stem cell (LSC) compartment. Resistant clones display an immature phenotype (c-kithi/Gr1-/CD11b-) and functionally, exhibit increased clonogenic capacity in vitro and markedly shorter disease latency following primary syngeneic transplantation (Figure A, B and C). Importantly, resistant clones maintain their resistance to IBET therapy in vivo. We will present data gleaned from exome capture sequencing, ChIP-seq and RNA-seq, to demonstrate the underlying molecular mechanisms of resistance to epigenetic therapies, including genetic changes, molecular events at chromatin and the upregulation of compensatory pathways that will inform future combination therapies to obviate and/or overcome BET inhibitor resistance. In summary, we have utilized a primary murine model of MLL leukemia to derive over 20 individual clones that are resistant to BET inhibition. Our data is consistent with resistance emerging from the LSC population. This data will allow us to develop rational drug combinations to overcome resistance and enhance the therapeutic efficacy of emerging epigenetic therapies. Furthermore, our data provides novel insights into the biology of AML and provides an unprecedented opportunity to study leukemia stem cells and develop therapeutic strategies to eradicate them. Figure 1 Figure 1. Disclosures Lugo: GlaxoSmithKline: Employment. Jeffrey:GlaxoSmithKline: Employment. Gregory:GlaxoSmithKline: Employment. Prinjha:GlaxoSmithKline: Employment.

Acute leukemia is the most common malignancy of childhood. The pattern of incidence of childhood leukemia reveals how age at diagnosis is associated with particular molecular subtypes, responsiveness to treatment and outcome. Translocations involving the Mixed Lineage Leukemia gene (MLL) in particular are associated with the diagnosis of acute leukemia in infancy. As with other forms of childhood leukemia, evidence from retrospective cohort analysis of neonatal blood spots suggests that MLL translocations are present at birth in many of these cases, indicating that the initial steps towards leukemia development occurred in utero (Greaves Early Human Development 2005). Concurrently there has been increasing recognition of the differences between the foetal and adult hematopoietic cells and supportive niches. These differences might have profound effects on a cell’s ability to undergo malignant transformation and its subsequent properties. Unlike mouse models of myeloid leukemia associated with MLL fusions, previous attempts to mouse model acute lymphoid leukemia of infancy associated with the MLL-AF4 fusion gene have had mixed results, with unsatisfactory disease latencies and phenotypes (Stam Cell Research 2012). However, many of these models have introduced the translocation only within the adult hematopoietic system – discounting the possible effect of this mutation in utero. The aim of this study was to examine the effect of MLL-AF4 within the context of the varied hematopoietic sites of the developing embryo, and how the induction of MLL-AF4 within these niches might influence subsequent disease development. For this, we utilised the Cre inducible Mll-AF4 “invertor mouse” that has previously been described (Metzler et al Oncogene 2006). As embryonic stem cell work suggests MLL-AF4 may alter the emergence of Hematopoietic Stem Cells (HSCs) from the hemogenic precursors (Bueno Cell Research 2012), we crossed the invertor mice with Ve-cadherin-Cre and Vav-Cre murine lines to induce Mll-AF4 expression in (pre)HSCs before and after this critical time point. HSC and progenitor populations at several established hematopoietic sites through ontogeny were assessed by flow cytometry and functional assays, including in vitro methylcellulose based colony forming assays and in vivo transplantation assays. Mice were also bred to adulthood and observed for the development of disease. Despite no statistically significant changes in HSC and progenitor populations within in utero HSC niches, functional assays reveal marked differences in both myeloid and lymphoid colony forming assays with a particularly strong effect seen in lymphoid assays; however, despite evidence of a limited increase in serial repeatability, these cells did not appear to be fully transformed and could not be re-plated successfully beyond the third round. In vivo transplantation assays also showed enhanced multi-lineage engraftment by foetal liver cells carrying the Mll-AF4 fusion gene. Adult mice developed both B and T cell malignancies with long latencies and median survival of 437 day to 551 days for Mll-AF4xVeCadherin-Cre and Mll-AF4xVav-Cre lines, respectively. We have thus shown for the first time that the induction of Mll-AF4 has an effect on the myeloid and lymphoid output of foetal derived HSCs. Although this effect appears to fall short of full transformation, the development of lymphoid malignancies in adult mice suggests that such cells have pre-malignant potential. We continue to explore the mechanism of these early pre-malignant effects and aim to explore how additional factors may unlock the true malignant potential of MLL-AF4 in murine model systems. Disclosures No relevant conflicts of interest to declare.

62 16 0.81 (0.38–1.72) 0.70 (0.43–1.16) TT 17 4 0.71 (0.21–2.43) 0.80 (0.40–1.62) GT 1 TT 79 20 0.79 (0.39–1.60) 0.73 (0.46–1.15) p Value 0.03 1 0.38 2

Although molecularly and clinically heterogeneous, AML is characterised by aberrant transcription and abnormal epigenetic regulation. The transcriptional co-activators CBP and p300 have well characterised roles in hematopoiesis and hematopoietic stem cell (HSC) function. These are mutated in AML and also bind a number of AML-associated oncogenes. However, despite this, their roles in the induction and maintenance of AML are poorly understood. To address this question, we have combined genetic and pharmacological inhibition of CBP and p300 in AML and normal hematopoiesis. Using a murine model where Cbp was conditionally deleted from murine hematopoietic stem and progenitor cells (HSPC), either prior to, or following expression of a number of AML-associated oncogenes, we assessed the role of Cbp in the induction and maintenance of AML. We demonstrated that although not an absolute requirement, Cbp confers a selective advantage for robust immortalisation in vitro, and that Cbp is also an important requirement for the generation and maintenance of AML in vivo. Furthermore, redundancy between Cbp and p300 in myeloid transformation was demonstrated, as p300 knockdown further decreased proliferation in Cbp-/- AML cells. We next validated CBP/p300 as potential therapeutic targets, using a pharmacological strategy. Using a selective small molecule inhibitor (C646) of the lysine acteyltranferase (KAT) activity of CBP/p300, we demonstrated a significant decrease in growth and clonogenic potential across multiple AML subtypes in vitro. This was mediated through induction of apoptosis and cell cycle arrest. Importantly, no alteration in the growth of normal murine and human hematopoietic progenitors was detected at similar doses. We further demonstrated that inhibition of CBP/p300 KAT activity in human leukemia cells alters a transcriptional programme associated with genomic integrity, linking transcriptional changes to the cellular phenotype. Finally, we demonstrated the efficacy of the HAT inhibitors to decrease clonogenic growth across a panel of primary AML patient samples, representing multiple genetic subtypes. Taken together, these data suggest that CBP/p300 are involved in leukemogenesis across multiple subtypes in AML and that targeting these proteins may be possible with an acceptable side-effect profile. Disclosures: No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, caused by a reciprocal chromosomal translocation that generates the BCR-ABL fusion protein, a constitutively activated tyrosine kinase. Patients with CML usually present in an indolent chronic phase (CP), however, if left untreated, they irrevocably progress to an aggressive form of acute leukemia, termed blast crisis (BC) that is usually fatal. Tyrosine kinase inhibitor (TKI) (e.g. Imatinib) treatment has revolutionised the treatment of CML CP. However, ∼5-10% of CP patients will progress to BC despite TKI treatment, and an additional 10-15% of patients are beyond CP at initial presentation. Upon disease progression, treatment options are very limited and prognosis is dismal. Hence, understanding the events that drive disease progression and identifying potential therapeutic targets remains an unmet clinical need. The mechanisms of BC transformation are poorly understood, but it is generally accepted that additional somatic mutations are required. To date, a small number of recurrent mutations have been reported, but these only account for a relatively small number of cases and their exact nature is not fully understood. In order to study the mechanisms of BC progression and to identify the co-operating mutations that drive this, we have utilised a published transgenic murine model of chronic phase CML (Koschmieder et al., 2005) and performed a transposon-based forward insertional mutagenesis study. In our mouse model, expression of BCR-ABL was driven in the hematopoietic stem and progenitor cell compartment (HSPC) by an SCL enhancer in a tetracycline dependant manner. Following BCR-ABL expression we conditionally induced ongoing mutations via a transposon-transposase system (SB) within HSPC and monitored disease progression from the chronic/BCR-ABL dependant phase to the transposon-mediated BC. Utilising the design of the transposon based system, it was then possible to identify these mutations by multiplexed next generation sequencing (NGS). Our experimental cohort was comprised of BC mice (which expressed BCR-ABL and transposon/transposase mediated mutation induction), CML mice (BCR-ABL only) and SB mice (mutation induction only). BC mice demonstrated a significantly shorter survival (p Disclosures: No relevant conflicts of interest to declare.

FLT3 internal tandem duplication (FLT3ITD) mutations are common 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 clustered regularly interspaced short palindromic repeats (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.

Lympho-myeloid restricted early thymic progenitors (ETPs) are postulated to be the cell of origin for ETP leukemias, a therapy-resistant leukemia associated with frequent co-occurrence of EZH2 and RUNX1 inactivating mutations, and constitutively activating signaling pathway mutations. In a mouse model, we demonstrate that Ezh2 and Runx1 inactivation targeted to early lymphoid progenitors causes a marked expansion of pre-leukemic ETPs, showing transcriptional signatures characteristic of ETP leukemia. Addition of a RAS-signaling pathway mutation (Flt3-ITD) results in an aggressive leukemia co-expressing myeloid and lymphoid genes, which can be established and propagated in vivo by the expanded ETPs. Both mouse and human ETP leukemias show sensitivity to BET inhibition in vitro and in vivo, which reverses aberrant gene expression induced by Ezh2 inactivation.