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Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD–positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.

Purpose of review In recent years, the N6-methyladenosine (m6A) modification of RNA has been shown to play an important role in the development of acute myeloid leukemia (AML) and the maintenance of leukemic stem cells (LSCs). In this review we summarise the recent findings in the field of epitranscriptomics related to m6A and its relevance in AML. Recent findings Recent studies have focused on the role of m6A regulators in the development of AML and their potential as translational targets. The writer Methyltransferase Like 3 and its binding partner Methyltransferase Like 14, as well as the reader YTH domain-containing family protein 2, were shown to be vital for LSC survival, and their loss has detrimental effects on AML cells. Similar observations were made with the demethylases fat mass and obesity-associated protein and AlkB homologue 5 RNA demethylase. Of importance, loss of any of these genes has little to no effect on normal hemopoietic stem cells, suggesting therapeutic potential. Summary The field of epitranscriptomics is still in its infancy and the importance of m6A and other RNA-modifications in AML will only come into sharper focus. The development of therapeutics targeting RNA-modifying enzymes may open up new avenues for treatment of such malignancies.

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.

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.

To evaluate maternal serum pregnancy-associated plasma protein-A (PAPP-A) levels at 11-14 weeks of gestation and preeclampsia risk in women with common congenital anatomic uterine abnormalities (AUAs). First trimester screening markers were compared between 12 AUA pregnancies, 60 age matched controls and 12 cases of early preeclampsia. PAPP-A level and birth weight were significantly lower in AUA compared to control and early preeclampsia group (

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3–METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5–7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3–METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy. Treatment with a specific inhibitor of the N6-methyladenosine methyltransferase METTL3 leads to reduced growth of cancer cells, indicating the potential of approaches targeting RNA-modifying enzymes for anticancer therapy.

© 2020 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited., The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2'-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner. Here we describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, we have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N6-methyladenosine (m6A), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we show that METTL16 deposits m6A to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation., We thank UKRI (BBSRC DTP scholarships to S.M. and H.K.C) and the Jardine Foundation and Cambridge Trust (Ph.D. scholarship to M.E.H.). K.T. was funded by a Wellcome Trust Sir Henry Wellcome Fellowship (grant reference RG94424). G.S.V. is funded by a Cancer Research UK Senior Cancer Fellowship (C22324/A23015). Research in the Shi lab is supported by Ludwig Institute for Cancer Research. Y.S. is American Cancer Society Research Professor. G.J.L.B. is a Royal Society University Research Fellow (URF\R\180019) and an FCT Investigator (IF/00624/2015).

SUMMARYThe global outbreak of SARS-CoV-2 necessitates the rapid development of new therapies against COVID-19 infection. Here, we present the identification of 200 approved drugs, appropriate for repurposing against COVID-19. We constructed a SARS-CoV-2-induced protein (SIP) network, based on disease signatures defined by COVID-19 multi-omic datasets(Bojkova et al., 2020; Gordon et al., 2020), and cross-examined these pathways against approved drugs. This analysis identified 200 drugs predicted to target SARS-CoV-2-induced pathways, 40 of which are already in COVID-19 clinical trials(Clinicaltrials.gov, 2020) testifying to the validity of the approach. Using artificial neural network analysis we classified these 200 drugs into 9 distinct pathways, within two overarching mechanisms of action (MoAs): viral replication (130) and immune response (70). A subset of drugs implicated in viral replication were tested in cellular assays and two (proguanil and sulfasalazine) were shown to inhibit replication. This unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials.

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukaemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3 and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.

Dovey, Oliver M. et al., NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandemduplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis showthatcompoundNpm1cA/1;NrasG12D/1orNpm1cA;Flt3ITD share anumber of features:Hox gene overexpression, enhancedself-renewal, expansionof hematopoietic progenitors,and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higherperipheral leukocytecounts, earlydepletionofcommonlymphoidprogenitors, anda monocytic bias in comparison with the granulocytic bias in Npm1cA/1;NrasG12D/1 mutants. Underlying thiswas a strikingmolecular synergymanifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD, but not Npm1cA/1;NrasG12D/1, progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/1;NrasG12D/1. During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/1;NrasG12D/1 mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRASG12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML., O.M.D., J.L.C., and G.S.V. are funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA). A.M. was funded by the Kay Kendall Leukaemia Fund project grant (KKL634). C.S.G. was funded by a Bloodwise Clinical Research Training Fellowship. I.V. is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal.

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.

Setbp1 overexpression acts in the place of class-defining somatic mutations to drive mouse and human FLT3-ITD-mutant AMLs Suruchi Pacharne,1,2 Oliver M. Dovey,1 Jonathan L. Cooper,1 Muxin Gu,1,2 MS Vijaybaskar,1,2 Mathias J. Friedrich,1,5 Malgorzata Gozdecka,1,2 Sandeep S. Rajan,1,4, Etienne De Braekeleer,1,2 Maxim Barenboim,5,6 Grace Collord,1,2 Hannes Ponstingl,1 Ruben Bautista,1 Milena Mazan,1,8 Roland Rad,5,6 Konstantinos Tzelepis,1,7 Penny Wright,3 and George S. Vassiliou1,2,9* Abstract Internal tandem duplications in FLT3 (FLT3-ITD) are found in 30% of acute myeloid leukemia (AML) cases and impart a poor prognosis. FLT3-ITD commonly synergizes with class-defining mutations such as chimeric fusion genes or mutations in NPM1, RUNX1, CEBPA or MLL to drive AML. However, 20% of FLT3-ITD-mutant AMLs bare no class-defining mutations and the mechanisms of acute leukemic transformation in these cases are unknown. To identify pathways that can drive FLT3-ITD-mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screen in Flt3-ITD mice using the Sleeping Beauty transposon system, activated by the Mx1-Cre recombinase in hematopoietic stem cells. All mice developed acute leukemia, predominantly AML, after a median latency of 73 days (Figure A). Analysis of transposon insertions in 38 Flt3-ITD/IM leukemias identified common integration sites (CISs) in 22 loci (Figure B). The most commonly "hit" genes were Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), Flt3 (6/38) and Runx1 (5/38) (Figure B). Of these, Setbp1 and Runx1 were unique to Flt3-ITD and not identified as CISs in insertional mutagenesis screens of wild type, Npm1c or BCR-ABL-expressing mice. Transposon insertions in Setbp1, primarily located upstream of its first coding exon, were associated with Setbp1 and Hoxa mRNA overexpression and were invariably associated with AML development (Figure B). These findings propose that overexpression of wild type SETBP1 may collaborate with FLT3-ITD to drive leukemogenesis in human AMLs lacking mutations known to collaborate with mutant FLT3. Corroborating this, we found that SETBP1 expression was higher in human FLT3-ITD-mutant AMLs lacking class-defining mutations and in those with RUNX1 mutations (Figure C). We go on to show that Setbp1 insertions activate a Hoxa gene signature such that shares significant similarities, but also specific differences to those driven by mutant Npm1 and MLL fusion genes. We go on to show, using CRISPR-gRNA, that whilst Flt3ITD/+/SETBP1IM+AMLs are entirely dependent on Setbp1 expression, Flt3ITD/+/Npm1cA/+AMLs are not, but do depend on the expression of the homebox gene Nkx2.3. Our findings propose that SETBP1 overexpression activates a gene expression pattern that collaborates with FLT3-ITD to drive many human AMLs and that this combination represents a specific subtype of AML amongst AMLs lacking class-defining mutations. To identify genetic vulnerabilities of this AML subtype, we performed genome-wide CRISPR-Cas9 recessive screens in primary murine Flt3ITD/+SETBP1IM+AMLs and identified more than 2000 genetic vulnerabilities, of which 677 were not required for the survival of HPC7 non-leukemic hematopoietic cells including >100 "druggable" genes such as Brd3, Ezh2 and Hmgcr (Figure D). Collectively our study: i) identifies SETBP1overexpression as a non-genetic alteration driving a subgroup of FLT3-ITD mutant AMLs lacking class-defining somatic mutations and ii) goes on to define the genetic vulnerabilities of such AMLs as a starting point for the development of targeted therapies. Figure Disclosures Vassiliou: Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

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.

Exciting research has connected specific RNA modifications to chromatin, providing evidence for co-transcriptional deposition and function in gene regulation. Here we review insights gained from studying the co-transcriptional roles of RNA modifications, and their influence in normal and disease contexts. We also discuss how the availability of novel technical approaches could raise the translational potential of targeting RNA-modifying enzymes for the treatment of disease. Tzelepis, Rausch and Kouzarides review the action of RNA modifications in the context of chromatin and discuss the emerging potential of RNA-modifying enzymes as new drug targets.

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3′ splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia.

The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs., This study was primarily funded by a joint Bloodwise Program Grant (17006) to B.J.P.H. and G.S.V. Work in the laboratory of B.J.P.H. is also funded by an ERC consolidator award (grant 647685 COMAL), a Cancer Research UK program award, the Medical Research Council, (MRC) the Wellcome Trust (WT) and the Cambridge NIHR BRC. We acknowledge the WT/MRC Center grant (097922/Z/11/Z) and support from WT strategic award 100140. G.S.V. is funded by a Cancer Research UK Senior Cancer Research Fellowship (C22324/A23015). The laboratory of G.S.V. is also supported by the Kay Kendall Leukemia Fund and core funding from the Sanger Institute (WT098051).

FLT3 internal tandem duplication (FLT3

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. New evidence has revealed an important role for RNA modifications in cancer development and maintenance via the catalytic function of RNA-modifying enzymes. We and others have recently shown that METTL3, the RNA methyltransferase responsible for the deposition of N-6-methyl groups on adenosine (m6A) in mRNA, is a promising therapeutic target for AML1,2. Here we present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3 as an effective therapeutic strategy in AML. Recently, we generated a comprehensive catalogue of RNA-modifying enzymes that are essential for AML cells using CRISPR-Cas9 recessive screens and characterised METTL3 as a novel therapeutic candidate through its effects on mRNA translational efficiency of key leukemia oncogenes1. Using a structure-guided medicinal chemistry platform we developed and optimised small molecule inhibitors of METTL3 from 2 distinct chemical series. Here we demonstrate that compounds 1 and 2 show biochemical inhibition of METTL3 enzyme with single digit nanomolar potency, while direct binding to METTL3 was confirmed by Surface Plasmon Resonance (SPR) analysis with comparable potency between compounds. Additionally, we developed compound 3 as an inactive analog which was confirmed inactive in enzyme assays (>50 µM IC50). Importantly, we verified that compounds 1 and 2 are selective for METTL3 and do not inhibit a panel of other RNA, DNA or protein methyltransferases tested (>10 µM IC50). Cellular target engagement was confirmed by demonstrating that compounds 1 and 2 reduced m6A levels and inhibited the protein expression of METTL3-dependent m6A substrates in mouse and human AML models, including SP1, with nanomolar potency. Furthermore, treatment of MOLM13 cells with compounds 1 and 2 inhibited their proliferation with comparable potency to SP1 inhibition. The same anti-proliferative effect was observed using a large panel of human AML cell lines. In addition, polyribosome profiling in MOLM13 cells treated with compounds 1 and 2 revealed enhanced blocking of mRNA translation, mirroring the effects derived from the genetic inhibition of METTL3. Notably, all of the above effects were not observed when the inactive analog (compound 3) was used, further highlighting the specificity and sensitivity of our active candidates. We subsequently performed in vivo characterisation of compound 1. This compound exhibited excellent bioavailability after oral or intraperitoneal administration with good dose-proportional exposure in mice and a half-life of 3.5 hours. It also appeared to be well-tolerated with no body weight loss or clinical signs of toxicity. We also evaluated its anti-tumor effects in patient derived xenotransplantation experiments (PDX) as well as transplantation experiments using an MLL-AF9 driven primary murine AML model. Daily dosing of 30 mg/kg significantly inhibited AML expansion and reduced spleen weight compared to vehicle control, indicating a pronounced anti-tumor effect in vivo. Target engagement was confirmed in bone marrow and spleen as measured by the reduction of METTL3-dependent m6A targets. Importantly, we went on to demonstrate that, while the pharmacological inhibition of METTL3 is required for AML cell survival, it was dispensable for normal hematopoiesis. Collectively, we describe the detailed characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrate their activity and utility using biochemical, cellular and in vivo systems. We show that inhibition of METTL3 by small molecules in vivo leads to strong anti-tumor effects in physiologically and clinically relevant models of AML. To our knowledge, this is the first study demonstrating in vivo activity of inhibitors of an RNA methyltransferase, hence providing proof of concept that RNA modifying enzymes represent a new target class for anti-cancer therapeutics. References Barbieri, I. et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature552, 126-131, doi:10.1038/nature24678 (2017). Vu, L. P. et al. The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med23, 1369-1376, doi:10.1038/nm.4416 (2017). Disclosures Yankova: STORM THERAPEUTICS: Employment. Fosbeary:STORM THERAPEUTICS: Employment. Hendrick:STORM THERAPEUTICS: Employment. Leggate:STORM THERAPEUTICS: Employment. Ofir-Rosenfeld:STORM THERAPEUTICS: Employment. Sapetschnig:STORM THERAPEUTICS: Employment. Albertella:STORM THERAPEUTICS: Employment. Blackaby:STORM THERAPEUTICS: Employment. Rausch:STORM THERAPEUTICS: Employment. Vassiliou:Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding. Kouzarides:STORM THERAPEUTICS: Equity Ownership.

Mutations affecting the gene for the de novo DNA Methyltransferase 3A (DNMT3A) are the most common drivers of clonal hematopoiesis (CH) and amongst the most common somatic events in acute myeloid leukaemia (AML). Approximately two thirds of AML-associated DNMT3A mutations are heterozygous substitutions affecting codon R882, located within the methytransferase domain and are correlated with global hypomethylation. DNMT3A mutations are initiating events in leukemogenesis and leukemic progression relies on the acquisition of additional mutations in genes such as NPM1, TET2, IDH1/2, FLT3 andNRAS. AMLs harboring DNMT3A-R882 mutations display increased resistance to chemotherapy and carry a worse prognosis for patients, but the molecular basis of this is not well understood. To improve our understanding of the molecular effects of mutant DNMT3A, we developed a conditional model of DNMT3A-R882H. Our conditional Dnmt3a-flox-R882H allele retains the native locus intact, but generates the Dnmt3a-R882H mutant upon Cre-loxP recombination (Mx1-Cre). We observed that Dnmt3aR882H/+ mutant bone marrow (BM) cells had markedly enhanced self-renewal potential in serial re-plating assays and increased BM repopulation ability in competitive transplants. Approximately 30% of Dnmt3aR882H/+ mice developed leukaemia after a long latency (median of 532 days). Introduction of Flt3-ITD mutation significantly accelerated spontaneous disease, in agreement with other published reports, with majority of mice succumbing to AML after a median of 192 days. Due to the frequent co-occurrence and strong cooperation between Dnmt3aR882H/+ with Flt3ITD/+, we sought to identify potential genetic vulnerabilities of this combination, to facilitate patient treatment. We therefore generated Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+ mice, induced Dnmt3aR882H mutation with pIpC and isolated hematopoietic stem and progenitor cells (HSPCs). We cultured HSPCs in vitro and performed whole-genome CRISPR dropout screens to identify genetic vulnerabilities. This identified 526 dropout genes at a genome-wide FDR of 20%, including pan-essential genes such as components of the ribosome, proteasome or spliceosome. To identify essential genes specific to the Dnmt3aR882H/+/Flt3ITD/+ genotype, we performed analogous screens in the non-leukemic mouse hematopoietic precursor cell-7 (HPC-7) and used published data indicating essential genes for normal human CD34 cells. Overlap of these screens revealed 196 dropouts present only in Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+ cells. Among these were genes coding for "druggable" candidates such as kinases and histone modifying enzymes, including SET Domain Containing 1B (Setd1b), one of the six mammalian proteins (also Setd1a and Mll1-4) that can catalyze methylation of H3K4. Each of these six proteins can interact with obligate proteins as a part of the methyltransferase complex, but also form distinct complexes with non-redundant function. We firstly validated that the knockout of Setd1b induced by gRNA significantly reduced the growth of pre-leukemic and leukemic HSPCs from both Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+ and Dnmt3aR882H/+/Mx1-Cre+/Rosa-Cas9+ mice. Importantly, Setd1b deletion in normal HSPC had no major effect on cell growth. To investigate the molecular role of Setd1b in Dnmt3aR882H context, we asked if Setd1b catalytic activity and linked transcriptional programs were responsible for the observed phenotypes. For this, we performed histone profiling and RNA-sequencing in Dnmt3aR882H/+/Flt3ITD/+/Mx1-Cre+/Rosa-Cas9+HSPC upon Setd1b KO vs control and observed a striking reduction of H3K4me3 marks at particular loci in association with loss of expression of corresponding genes. These included several known to be required for cellular proliferation and transformation, some of which were themselves dropouts in our original screen. Importantly the majority of genes were also significantly upregulated when gene expression from Dnmt3aR882H/+/Flt3ITD/+ HSPC were compared with Flt3ITD/+ HSPC. Our data propose that the catalytic activity of Setd1b plays a mechanistic role in driving the growth/fitness-promoting effects of Dnmt3aR882H/+. Blocking the catalytic activity of SETD1B may therefore represent a plausible therapeutic option for the treatment of AML of even CH patients harboring mutations inDNMT3A. Disclosures Mazan: Selvita S.A.: Employment. Vassiliou:Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding.

An amendment to this paper has been published and can be accessed via a link at the top of the paper., info:eu-repo/semantics/published

Aims: To study the clinicopathological and prognostic value of cyclin D1 overexpression in patients with breast carcinoma. Methods and results: Immunohistochemistry was performed on paraffin-embedded tissue specimens from 290 invasive breast carcinomas to detect the proteins cyclin D1, oestrogen receptor (ER), progesterone receptor (PR), p53, c-erbB2, and topoisomerase IIa (topoIIa). Cyclin D1 staining was quantified using a computerized image analysis method. Cyclin D1 overexpression characterized smaller, ER-positive and PR-positive tumours (P = 0.017, P < 0.0001, and P < 0.0001, respectively), of a lower histological and nuclear grade (P = 0.011 and P < 0.0001, respectively), and with reduced expression of topoIIa (P = 0.001) and p53 (P < 0.001). Cyclin D1 was found to have an independent favourable impact on the overall survival of both the unselected cohort of patients (P = 0.011) and of patients with ER-negative and lymph node-positive tumours (P = 0.034 and P = 0.015, respectively). In triple-negative tumours, cyclin D1 overexpression was found to have independent favourable impacts on both overall and relapse-free survival (P = 0.002 for both). Conclusions: This is the first immunohistochemical study to dissociate the advantageous prognostic effect of cyclin D1 overexpression from its association with ER expression, and to provide evidence that cyclin D1 overexpression may be a marker of prolonged survival in patient subgroups with aggressive phenotypes.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer with a cumulative risk of ~1 in 2,000 children by the age of 15 years and an increasing incidence over the last 30 years.Whilst advances in the use of combination chemotherapy have significantly improved outcomes, current treatments remain toxic and can have long-term health consequences. T-cell acute lymphoblastic leukemia (T-ALL) represents between 15 to 25% of all ALL and affects both children and adults, and carries a worse prognosis. Activating mutations in the NOTCH1gene are found in more than 60% of cases and are being targeted therapeutically with compounds such as γ-secretase inhibitors (GSIs) and Notch inhibiting antibodies (mAbs). However, none of these approaches has entered mainstream therapy as yet. To address this, we performed a genome wide CRISPR-Cas9 screen in T-ALL cell lines driven by NOTCH1 overexpression (Jurkat, CEM-CCRF), or mutation (PEER) and in a line not dependent on NOTCH1 (Loucy) (Figure 1A). To discover NOTCH1-specific genetic vulnerabilities, we compared essential genes for NOTCH1-dependent T-ALL cell lines with those for Loucy and for 6 human myeloid leukemia cells (MV4;11, MOLM13, OCI-AML2, OCI-AML3, K562 and HL60)1. This identified 69 NOTCH-specific essential genes, including well known players in NOTCH signaling such as NOTCH1, RBPJ, BCL11B, GATA3 and CTPB1(Figure 1B). In addition to these genes, we also identified a separate group of genes associated to cellular pathways involved in the reduction of reactive oxygen species (ROS). One of these was NADK, the gene for nicotinamide adenine dinucleotide kinase, which we investigate further. NADK drives the conversion of NAD+to NADP+, which in its reduced form (NADPH) is used in the reduction of ROS levels through the production of reduced Glutathione. Exposure of Loucy (NOTCH1-WT) to the oxidant H2O2showed that it was not able to reduce intracellular ROS levels as efficiently as NOTCH1-driven lines, suggesting increased NADK activity in the latter. In keeping with this, intracellular NADP+/NADPH levels that were significantly higher in NOTCH1-driven cell lines than in Loucy (Figure 1C). In addition, pharmaceutical or genetic inhibition of NADK led to a significant increase in ROS level in NOTCH1-driven Jurkat cells, while Loucy cells were not affected (Figure 1D). This increase in ROS levels was associated with a significant decrease in cell proliferation (Figure 1E). It was previously reported that, in both human and mouse T-ALL cells, the intracellular domain of Notch1 (NiCD) suppresses production of ROS levels and imparts a higher resistance to H2O2,in so doing facilitating the proliferation of these cells2. We hypothesize that NADK is the mediator of this effect of NOTCH1 and this makes it a therapeutic vulnerability in NOTCH1-driven T-ALL and potentially in other NOTCH1-driven cancers. We currently testing this hypothesis in vivo using patient-derived T-ALL xenografts into immunocompromised mice and investigating the molecular events involved in the NOTCH1-NADK interaction. Our findings propose NADK as a novel therapeutic vulnerability in NOTCH1-driven T-ALL and demonstrate the power genome-wide CRISPR screens in finding novel genetic and therapeutic targets for this disease. 1. Tzelepis, K.et al.A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia. Cell Rep17, 1193-1205 (2016). 2. Giambra, V.et al.NOTCH1 promotes T cell leukemia-initiating activity by RUNX-mediated regulation of PKC-theta and reactive oxygen species. Nat Med18, 1693-8 (2012). Disclosures Vassiliou: KYMAB: Consultancy, Equity Ownership; Celgene: Research Funding.

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. Aberrant mRNA splicing plays a key role in cancer development and genes coding for several of the major components of the spliceosome are targeted by somatic mutations in numerous cancers including myelodysplastic syndromes and AML. Recently, myeloid neoplasms bearing spliceosome gene mutations were shown to be preferentially susceptible to pharmacological disruption of the spliceosome. Here we report that targeting the spliceosome can also be an effective therapeutic strategy in other types of AML. Recently, we generated a comprehensive catalogue of genetic vulnerabilities in AML using CRISPR-Cas9 genome-wide recessive screens and reported several novel intuitive and non-intuitive therapeutic candidates. Amongst these we identify SRPK1, the gene coding for a serine-threonine kinase that phosphorylates the major spliceosome protein SRSF1. Here, we demonstrate that targeted genetic disruption of SRPK1 in AML driven by MLL-fusion genes, led to differentiation and apoptosis. Additionally, mice transplanted with human AML cell lines carrying the MLL-AF9 fusion gene, namely MOLM-13 and THP-1, presented a significant prolongation of survival when SRPK1 was genetically disrupted by CRISPR-Cas9 editing. Similar effects were seen with pharmacological inhibition of SRPK1 in vitro and in vivo. At the molecular level we show that genetic or pharmacological inhibition of SRPK1 was associated with profound changes in the splicing of multiple genes involved in the MLL leukemogenic program in association with significant changes in enzymatic modifications of core histone tails. We proceeded to perform a genome-wide CRISPR drop-out screen for sensitizers of MOLM13 cells to pharmacological inhibition of SRPK1 and identified, amongst other genes, BRD4 as a sensitizer. We go on to show that the BRD inhibitor iBET-151 synergizes with SRPK1 inhibition to kill MOLM-13 both in vitro and in vivo. Preliminary data indicates that SRPK1 inhibition has overlapping molecular effects to BRD inhibition. We are currently investigating the molecular bases of this observation. Our work identifies SRPK1 as a novel therapeutic target in AML that can be used alone or in conjunctions with drugs targeting epigenetic modifications to improve their anti-leukemic effects. Citation Format: Konstantinos Tzelepis, Etienne De Braekeleer, Michael Seiler, Isaia Barbieri, Sam Robson, Yu Hsuen Yang, Malgorzata Gozdecka, Monika Dudek, Grace Collord, Oliver M. Dovey, Emmanouil Metzakopian, Dimitrios Garyfallos, Jonathan L. Cooper, Silvia Buonamici, Hannes Ponstingl, Michael R. Stratton, Allan Bradley, Brian J. Huntly, Cristina Pina, Tony Kouzarides, Kosuke Yusa, George S. Vassiliou. Modulation of splicing by inhibiting the kinase SRPK1 as a novel therapeutic strategy in myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2017-1158

Our purpose was to investigate the expression pattern of BRCA1 protein in sporadic breast carcinomas, as well as the clinicopathological and prognostic value of its subcellular localizations. Immunohistochemistry was performed on paraffin embedded tissue specimens from 111 sporadic, invasive breast carcinomas to detect the expression of the proteins BRCA1, ER, PR, erbB2, p53 and Ki67. BRCA1 protein was detected in the nuclei and the cytoplasm of the tumor cells. Nuclear BRCA1 immunoreactivity showed no relation with the classic clinicopathological markers and the expression of cerbB2, p53 and Ki67. Reduced expression of nuclear BRCA1 protein was found to exert an independent favorable impact on both the overall and relapse-free (RF) survival of the patients (p = 0.019 and p = 0.043, respectively). Cytoplasmic BRCA1 was associated with none of the classic histomorphological indices, except from the lymph node metastasis, with which its relation was found to be inverse (p = 0.05), prolonging the RF survival of the patients (p = 0.05). Our findings suggest that BRCA1 protein depicts different prognostic significance, according to its subcellular distribution. Nuclear detection of the protein was associated with a worse prognosis, while the cytoplasmic one was related to fewer recurrences as a result of fewer lymph node metastases.

To study the clinicopathological and prognostic value of cyclin D1 overexpression in patients with breast carcinoma. Immunohistochemistry was performed on paraffin-embedded tissue specimens from 290 invasive breast carcinomas to detect the proteins cyclin D1, oestrogen receptor (ER), progesterone receptor (PR), p53, c-erbB2, and topoisomerase IIα (topoIIα). Cyclin D1 staining was quantified using a computerized image analysis method. Cyclin D1 overexpression characterized smaller, ER-positive and PR-positive tumours (P = 0.017, P 0.0001, and P 0.0001, respectively), of a lower histological and nuclear grade (P = 0.011 and P 0.0001, respectively), and with reduced expression of topoIIα (P = 0.001) and p53 (P 0.001). Cyclin D1 was found to have an independent favourable impact on the overall survival of both the unselected cohort of patients (P = 0.011) and of patients with ER-negative and lymph node-positive tumours (P = 0.034 and P = 0.015, respectively). In triple-negative tumours, cyclin D1 overexpression was found to have independent favourable impacts on both overall and relapse-free survival (P = 0.002 for both). This is the first immunohistochemical study to dissociate the advantageous prognostic effect of cyclin D1 overexpression from its association with ER expression, and to provide evidence that cyclin D1 overexpression may be a marker of prolonged survival in patient subgroups with aggressive phenotypes.

Despite progress in understanding its genomics and molecular pathogenesis, the therapeutic landscape of acute myeloid leukaemia (AML) has changed little in the last 40 years. Whilst our improved molecular understanding of AML permits some optimism that progress may be forthcoming, an alternative approach for the identification of therapeutic targets is the agnostic interrogation of AML genomes for genetic vulnerabilities. In this study we apply a new and technically robust CRISPR-Cas9 platform to perform genome-wide screens for genetic vulnerabilities in human cancers. To do this, we develop and validate a CRISPR-based functional genomics toolkit composed of: i) lentiviral gRNA expression vectors harbouring an improved sgRNA scaffold, ii) Cas9 activity reporters for choosing cell line clones with high Cas9 nuclease activity and iii) an improved human genome-wide CRISPR library composed of 90,709 gRNAs targeting 18,010 genes. We first describe the timescale over which cells lacking individual essential genes are depleted from a pool of isogenic cells, thus providing the first such genome-wide framework for mammalian cells. As well as being of fundamental interest, such a temporal framework can be used to decide the length of time required for performing genetic screens and to select therapeutic targets. We then proceeded to perform drop-out screens with 30-day latencies in 5 AML cell lines (MV4-11, MOLM-13, OCI-AML2, OCI-AML3 and HL-60) and also in the non-AML lines HT-29 (colorectal adenocarcinoma) and HT-1080 (fibrosarcoma). Drop-out genes were identified using the MAGeCK algorithm as those showing significant depletion across their ≥5 cognate sgRNAs. From each cell line, more than 1,000 genes dropped out at FDR Next, we looked for genes that are specifically essential to AML cells by extracting genes depleted in at least 1 of the 5 AML cell lines, but not in HT-29 or HT-1080. This analysis identified approximately 150-200 essential genes for each cell line yielding a total of 510 AML-specific genes. Of these, 59 genes including RUNX1, CEBPA, CEBPB, MEN1, DOT1L and SMARCB1 were essential to 3 or more AML cell lines. GO analysis of these 59 genes showed particular enrichment in processes pertaining to chromatin modification and organisation, transcriptional regulation and nucleotide metabolism. We proceed to validate a number of novel drop-out genes using CRISPR-Cas9 with new sgRNAs and where possible with existing clinical/pre-clinical inhibitors. Furthermore, we identify oncogene-specific cell vulnerabilities, even for leukaemias driven by closely related oncogenes such as the MLL-AF4 (MV4-11) and MLL-AF9 (MOLM-13) fusion genes, which differed in their dependency on several genes including KAT2A and SRPK1. To validate these findings in primary cells, we generate a novel Rosa26-Ef1a-Cas9 mouse model and cross this with mice carrying Flt3-ITD. We then transformed Lin- haemopoietic cells from RosaCas9/+/Flt3ITD/+ mice using MLL-AF4- or MLL-AF9 -expressing retroviruses and validate the findings of our screens using sgRNAs against murine Kat2a and Srpk1. Taken together, these data dissecting the individual vulnerabilities of highly similar initiating mutations demonstrate the power of our screen to identify specific vulnerabilities for individual oncogenes and suggest that similar screens may also help to guide programmes of personalised medicine for patients based on the complement of somatic mutations within their cancer, which in some cases could be achieved through re-purposing of existing therapeutics. Disclosures McDermott: 14M Genomics: Other: co-founder, stock-holder and consultant.

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with a dismal clinical outcome and represents an unmet medical need. However, therapeutic progress will not be made without a greater understanding of the varied molecular pathogenesis of this heterogeneous disease. Recently, the majority of cases of the largest subgroup of AML, AML with a normal karyotype (AML-NK), were found to harbour recurrent loss-of-function mutations in epigenetic regulators. Germline mutations in the BCL6 Co-Repressor (BCOR) gene, situated at Xq27-28, are associated with two overlapping X-linked dominant disorders, the Oculo-facio-cardio-dental (OFCD) syndrome and syndromic micropthalmia. In addition, somatic mutations in BCOR were described within the last few years in adults and children with AML, particularly in cases arising in the context of aplastic anaemia and in patients with antecedent myelodysplastic syndromes. BCOR encodes a ubiquitously expressed protein which is involved in BCL6-mediated transcriptional repression. In contrast to other BCL6 partners, NCOR and SMRT, it was recently demonstrated to occupy the majority of its DNA binding sites independently from BCL6, reflecting its multiple interaction partners. BCOR also forms a Polycomb repressor complex 1 (PRC1)-like complex with PCGF1, KDM2B, RING1, SKP1, RYBP, and RNF2, and can bind to both class I and II histone deacetylases. Its spectrum of protein-binding partners and studies of mutant forms suggest that BCOR is an epigenetic modulator. Therefore, we predict that loss of BCOR in AML results in loss of its complex repressive function and leads to an activation of an important leukaemogenic transcriptional programme. To test this hypothesis, we generated a mouse model of leukaemia using CRISPR-Cas9 genome editing. Using a common endonuclease assay, we validated 6 different gRNA constructs against Bcor targeting the region of known human mutations described in AML. The three most efficient constructs were subsequently used to create a pool of lentiviral vectors. In order to increase the efficiency of targeting the haematopoietic progenitor cells, we harvested lineage depleted bone marrow cells from mice expressing the Cas9 cDNA from the Rosa locus (Rosa-Cas9). In parallel, we performed the same experiments with Rosa-Cas9 mice harbouring a Flt3 internal tandem duplication (Flt3-ITD), a mutation that can co-occur with BCOR mutations in individual AML patients. First, we transduced bone marrow lineage negative cells with lentiviruses carrying the three BCOR sgRNAs at 60-70% efficiency and performed a serial re-plating assay to investigate the clonogenic potential of targeted cells. We observed a significant increase in the colony numbers in both Cas9 only and Cas9 Flt3-ITD targeted cells in comparison to the empty lentiviral vector controls from the second re-plating onwards. Using a standard endonuclease assay we have confirmed the presence of Cas9-induced mutations within the target Bcor exons, indicative of clonal selection for Bcor targeted cells and an increased self-renewal potential of hematopoietic precursors that lack BCOR. Transduced cells were also injected into lethally irradiated recipient mice in the presence of helper cells to investigate development of leukaemia in vivo. We have been able to confirm the presence of the Bcor mutated cells in the blood of tested mice 12 weeks post injection and these mice continue to be monitored for evidence of leukemia. We are currently conducting integrated genomic analysis with ChIP-Seq, RNA-Seq and methylation analysis to link the BCOR mutations and their altered cellular phenotype to aberrant epigenetic control and pre/leukaemic gene expression programmes. Finally, we are also going to assess co-operativity of Flt3-ITD and BCOR mutations in AML leukaemogenesis. We anticipate that our work will inform the biology of AML-NK associated with BCOR, identify potential therapeutic targets and provide platforms for the assessment of novel therapeutics. 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.

Data-driven computational analysis and simulation identified drug repurposing opportunities for COVID-19., The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates the rapid development of new therapies against coronavirus disease 2019 (COVID-19) infection. Here, we present the identification of 200 approved drugs, appropriate for repurposing against COVID-19. We constructed a SARS-CoV-2–induced protein network, based on disease signatures defined by COVID-19 multiomics datasets, and cross-examined these pathways against approved drugs. This analysis identified 200 drugs predicted to target SARS-CoV-2–induced pathways, 40 of which are already in COVID-19 clinical trials, testifying to the validity of the approach. Using artificial neural network analysis, we classified these 200 drugs into nine distinct pathways, within two overarching mechanisms of action (MoAs): viral replication (126) and immune response (74). Two drugs (proguanil and sulfasalazine) implicated in viral replication were shown to inhibit replication in cell assays. This unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials.