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RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules., Nanopore direct RNA Sequencing data contain information about the presence of RNA modifications, but their detection poses substantial challenges. Here the authors introduce Nanocompore, a new methodology for modification detection from Nanopore data.

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.

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.

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.

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).

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

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.

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.

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.