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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.

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.

FLT3 internal tandem duplication (FLT3

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.

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.

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.

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.

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.