ALBERT

Description: BACKGROUND: Patients with relapsed and/or refractory non-Hodgkin's lymphoma (NHL), especially those with aggressive lymphomas, have overall poor prognosis. Novel targets and therapies are under investigation. Molibresib (GSK525762) is a potent and specific inhibitor of the bromodomain and extraterminal domain (BET) family of proteins, the inhibition of which prevents transcriptional complex assembly and the subsequent expression of oncogenic drivers. Molibresib inhibits growth in NHL cell lines, both in vitro and in vivo. Study BET116183 was designed to evaluate the safety, tolerability, and preliminary efficacy of molibresib in relapsed and refractory hematologic malignancies. Here we report the results from the NHL dose escalation cohort. METHODS: Eligible subjects were adults with relapsed or refractory NHL. An accelerated dose titration was employed with one subject per dose level until the occurrence of a ≥Grade 2 drug-related toxicity; thereafter, subjects were enrolled in a standard 3+3 design. A Neuenschwander continual reassessment method (N-CRM) model was used to provide guidance for the next dose level. Dose escalation continued until the maximum tolerated dose (MTD) was identified. All data, including safety, tolerability, pharmacokinetics (PK), and efficacy, were used to identify the recommended part 2 dose (RP2D). RESULTS: From 14 May 2014 to the data cutoff date of 24 June 2018, 27 NHL subjects were enrolled and received at least one dose of study drug. Of these, 19 (70%) had B-cell lymphomas (diffuse large B-cell lymphoma [DLBCL], mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma , and Burkitt's lymphoma); eight subjects (30%) had T-cell lymphomas (cutaneous T-cell lymphoma [CTCL], anaplastic T-cell lymphoma [ATCL], peripheral T-cell lymphoma, and adult T-cell leukemia/lymphoma). The median age was 64 years (range 24 to 76); 20 subjects (76%) were male and 7 subjects (24%) were female. The median number of prior treatments was 3 (range 1 to 〉 4). From the starting dose of 10 mg molibresib orally once daily (QD), the dose was escalated to 80 mg QD. The median time on study was 1.4 months (range 0.2 to 20 months). Two dose-limiting toxicities (DLTs) were identified in subjects treated at 60 mg QD, though one was subsequently determined not to be a DLT. One subject experienced Grade 4 thrombocytopenia related to study drug. A second subject experienced a Grade 2 mechanical fall; this event was later revised to unrelated to study drug. Across all dose levels, all subjects experienced an adverse event (AE); 25 subjects (93%) experienced at least one AE that was deemed to be related to molibresib treatment. The most common related AEs across all dose levels were thrombocytopenia (n = 21 [78%]), fatigue (n = 6 [22%]), nausea (n = 6 [22%]), diarrhea (n = 4 [15%]), and rash (n = 4 [15%]). Blood bilirubin was increased in 3 subjects (11%), and prothrombin time and activated partial thromboplastin time were prolonged in 2 subjects each (7%). Common Grade 3 and Grade 4 related events included thrombocytopenia (n = 19 [70%]), as well as anemia, asthenia, and increased blood bilirubin (n = 2 [7%] each). No Grade 5 related AEs were reported. Among all subjects, 11 (41%) required dose reduction for toxicity: 7 subjects at the 60 mg dose level (39% treated at that dose) and 4 at the 80 mg dose level (57% treated at that dose). PK analyses showed dose-proportionality after single and repeat dosing, with large variability between subjects. One subject with DLBCL achieved a complete remission that was durable through week 54 on study. Four additional subjects (one DLBCL and 3 CTCL) achieved partial remission, for an objective response rate (ORR) of 5/27 (18.5%). Five more subjects had stable disease as best response. Of six CTCL/ATCL subjects enrolled, three subjects had partial response for an ORR of 50% in this sub-population. CONCLUSIONS: This is the first study evaluating the safety and efficacy of the BET inhibitor molibresib in NHL subjects. Overall, thrombocytopenia and other AEs were monitorable, manageable and reversible. The RP2D was identified as 60 mg QD. Single-agent activity was observed across multiple NHL subtypes at both 60 mg and 80 mg doses; most notable was a 50% response rate in subjects with CTCL. Because of the promising data, Part 2 of the BET116183 study is currently open and enrolling subjects with CTCL to better define the clinical activity of BET bromodomain inhibition in this histology. Disclosures Dickinson: GSK: Consultancy. Kamdar:Genentech: Consultancy; Seattle Genetics: Speakers Bureau. Mateos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Alegre:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Kim:Roche: Research Funding; Mundipharma: Research Funding; J&J: Research Funding; Novartis: Research Funding; Kyowa-Kirin: Research Funding; Celltrion: Research Funding; Takeda: Research Funding. Martín:Janssen: Honoraria, Other: Travel expenses; Celgene: Consultancy, Honoraria, Other: Travel expenses; Roche: Consultancy, Honoraria, Other: Travel expenses; Servier: Honoraria, Other: Travel expenses. Horner:GSK: Employment. Winnberg:GSK: Employment. Mathew:GSK: Employment. Botbyl:GSK: Employment. Karpinich:GSK: Employment. Kremer:GSK: Employment. Dhar:GSK: Employment. Karadimitris:GSK: Research Funding; Gilead: Honoraria; Celgene: Research Funding.

Description: Ectopic expression of FGFR3 associated with t(4;14), frequently containing the activating mutation K650E (TDII), has been implicated in the pathogenesis of human multiple myeloma. To investigate the role of autophosphorylated tyrosine residues in FGFR3 signal transduction and transformation, we characterized a series of FGFR3 TDII mutants with single or multiple Y→F substitutions. Phenylalanine substitution of a non-activation loop tyrosine residue Y760, essential for PLCγ binding and activation, significantly attenuated FGFR3 TDII-mediated PLCγ activation, as well as transformation in Ba/F3 cells and a murine bone marrow transplant (BMT) leukemia model. In contrast, single substitution of other non-activation loop tyrosine residues Y577, Y724 or Y770 had minimal to moderate effects on TDII-dependent transformation. Substitution of all the four non-activation loop tyrosine residues significantly attenuated, but did not abolish FGFR3 TDII transforming activity. However, substitution of both activation loop tyrosine residues Y647/Y648 resulted in complete abrogation of FGFR3 TDII kinase activity and transforming activity in vitro and in vivo. Similar observations were obtained in the context of constitutively activated fusion TEL-FGFR3 associated with peripheral T-cell lymphomas (PTCL) with chromosomal translocation t(4;12)(p16;p13). Moreover, two independent EμSR-FGFR3 TDII transgenic mouse lines developed pro-B cell lymphoma, and PLCγ was highly activated in primary lymphoma cells as assessed by tyrosine phosphorylation, along with elevated activation of STAT3 and expression of Bcl-XL in the tumor tissues. These data indicate that engagement of multiple signaling pathways, including PLCγ-dependent and PLCγ-independent signaling pathways, are required for full hematopoietic transformation by constitutively activated FGFR3 mutants.

Description: Reccurent translocation t(4;14) associated ectopic expression of FGFR3, sometimes containing the activation mutation K650E (TDII), has been identified in 25% of human multiple myeloma (MM) patients and cell lines. However, current empirically-derived cytotoxic chemotherapy does not effectively treat this disease. One potential therapeutic strategy of treating MM is to inhibit the tyrosine kinase activity of FGFR3. In this report, we evaluated the efficacy of PKC412 (N-benzoyl-staurosporine), a small molecule tyrosine kinase inhibitor, for the treatment of FGFR3 mutants induced diseases. PKC412 effectively inhibits the tyrosine kinase activity and activation of downstream effector pathways of FGFR3 TDII or the constitutively activated TEL-FGFR3 fusion that was reported in a subtype of human peripheral T-cell lymphoma (PTCL), as well as proliferation of hematopoietic Ba/F3 cells transformed by the FGFR3 mutants. Furthermore, PKC412 drastically inhibits proliferation of four different multiple myeloma-derived primary cell lines that are associated with t(4;14) and expression of dysregulated FGFR3. Moreover, oral-gavage treatment with PKC412 resulted in statistically significant prolongation of survival in the murine bone marrow transplant (BMT) models of FGFR3 TDII-induced pre-B cell lymphoma or TEL-FGFR3 fusion-induced myeloproliferative disease, which suggests suitable pharmacokinetic and toxicity profiles of PKC412 for clinical use. Together, our data establish the small molecule inhibitor PKC412 as a molecularly targeted therapy for multiple myeloma and other human malignancies expressing activated FGFR3.

Description: The de novo DNA Methyltransferase 3A (DNMT3A) gene is one of the most commonly mutated gene in acute myeloid leukaemia with a normal karyotype (AML-NK). Approximately two thirds of mutations are heterozygous and affect codon R882, located within the methytransferase domain. Also, we and others have shown that these mutations occur in a significant proportion of haematologically normal people and behave as leukaemia-initiating changes. DNMT3A mediates the transfer of a methyl group from s-adenosylmethionine (SAM) to the 5' carbon of cytosine residues at CpG dinucleotides. The DNMT3A protein domains bind to unmethylated H3K4, a repressive histone mark, as well as the H3K36Me3 mark associated with active transcription. DNA methylation is linked to histone modifications and DNMT3A binds to class I and II histone deacetylases (HDAC) and histone methyltransferases (HMT), such as SETDB1 and G9a, as well components of Polycomb repressive complex 2 (PRC2). DNMT3AR882H has been reported to exert a dominant-negative effect on wild type (WT) DNMT3A, with a significant reduction of its enzymatic activity. In AML samples, the R882 mutation correlates with global hypomethylation, particularly at CpG islands, shores and promoters, although some promoter hypermethylation has also been reported. However, the molecular mechanism, through which DNMT3AR882H alters methylation to drives leukaemia, remains unknown. In order to understand the molecular and functional consequences of the DNMT3AR882H mutation in leukaemogenesis we developed a mouse model (Dnmt3afloxR882H/+), whereby targeting of the Dnmt3a allele does not inactivate the native locus. This allows conditional expression of the mutant protein within haematopoietic stem and progenitor cells that have developed in the presence of normal levels of Dnmt3a. Dnmt3afloxR882H/+ mice were crossed with mice carrying an interferon-inducible Cre recombinase allele (Mx1-Cre). Upon Cre induction with pIpC the endogenous Dnmt3a exon 23 (last exon), was efficiently replaced by the human exon 23 carrying the R882H mutation. This was confirmed at both the DNA and the RNA level. In order to investigate the molecular effects of Dnmt3aR882H, we analysed pre-leukaemic haematopoietic cells (in mice 5-6 weeks post Cre-induction). We observed that Dnmt3aR882H/+ mutant bone marrow (BM) cells had markedly enhanced self-renewal potential as manifested by increased serial replating in cytokine supplemented methylcellulose media in comparison to WT mice. We next examined the relative haemopoietic reconstitution efficiency of Dnmt3aR882H/+ BM cells in a competitive setting. Dnmt3aR882H/+ BM (CD45.2) cells were mixed with WT competitor BM (CD45.1) cells at a ratio of 1:2 and transplanted into lethally irradiated recipient mice (CD45.1/45.2). The analysis of blood chimerism in recipients indicated an increased contribution from Dnmt3aR882H/+ in comparison to WT cells, indicating the enhanced ability of Dnmt3aR882H/+ BM cells to engraft and reconstitute recipient mice. Currently, we are addressing the role of Dnmt3aR882H in the spontaneous leukaemia development alone as well as in rational combinations with other mutations present in human AML. Additionally, to elucidate the molecular mechanism underlying the striking haematopoietic phenotypes ofDnmt3aR882H/+ we have performed analyses of DNA methylation and hydroxymethylation patterns in the haematopoietic progenitor compartment (lineage negative cell population) of Dnmt3aR882H/+ and WT mice, using reduced representation oxidative bisulfite sequencing (RRoxBS) in the pre-leukaemic setting. Moreover, due to cross-talk between Dnmt3a mediated DNA methylation and other chromatin states, we have analysed Dnmt3aR882H-associated changes in histone tail modifications, using ChIP-Seq. As the DNA and histone modifications associated with Dnmt3aR882H are likely to lead to alter global gene expression, we also performed RNAseq analysis in Dnmt3aR882H/+ and WT mice. Currently, we are conducting an integrated analysis, coupling DNA and histone modifications to aberrant gene expression and Dnmt3aR882H-dependent phenotypes. We predict that this work will provide a detailed understanding of the molecular and cellular effects of DNMT3A-R882H and in so doing reveal rational therapeutic approaches for patients with this common and aggressive subtype of AML. Disclosures No relevant conflicts of interest to declare.

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

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

Description: Introduction: The increasing delineation of acute myeloid leukemia (AML) has identified a number of genetic mutations which may be amenable to targeted therapies. However, such mutations typically only occur in a minority of patients, and this relative paucity presents challenges in drug development. Even for more common mutations such as FLT3 ITD, randomised trials can take many years to complete, and there is the issue of how to deal with patients who are tested but not eligible. Earlier phase trials therefore tend to be single arm studies, and often recruit in the relapsed/refractory population, where eligibility is known up front, and it is possible to obtain an early read out for efficacy. Such is the case for the recent evaluations of enasidenib and ivosidenib in IDH1/2 mutated patients. However, with single-arm studies there a need to contextualise results. We therefore looked at outcomes from the United Kingdom NCRI trials of AML for patients with IDH1/IDH2 mutations who were relapsed or refractory to therapy. Methods: A database search identified patients within the UK NCRI AML trials with an IDH1/IDH2 mutation, who had received intensive induction and who were either: in second relapse, relapsed post-transplant, refractory to two courses of induction, or who relapsed within 1 year of remission. Outcomes were measured from the point of eligibility: patients who were multiply eligible were included only once, at their first point of eligibility. The primary outcome was overall survival, with achievement of complete remission, with or without peripheral count recovery, as secondary outcome. Cox regression analysis was used to identify prognostic factors within the cohort of patients. Cytogenetics are evaluated using the MRC classification. Results: A total of 757 patients were identified with IDH1/2 mutation (IDH1 alone n=247; IDH2 alone n=504, both n=6). Of these 211 patients satisfied the relapsed/refractory criteria (IDH1 alone n=81; IDH2 alone n=128; both IDH1/2 n=2; refractory n=28; relapsed post SCT n=34; relapsed within 1 year with no SCT n=138; second relapse n=11 - Table). Median age was 54 years (range 22-77); 51% were male; and 95% of patients had intermediate risk cytogenetics. Remissions were achieved in 43/211 patients (20%; refractory 50%; relapsed post SCT 15%; relapsed within 1 year 17%; second relapse 9% - Table). Patients with IDH1 mutations had a remission rate of 23%; for IDH2 mutated patients, the rate was 18%. Median survival was 4.4 months for IDH1 mutated patients, and 6.6 months for IDH2 mutations; 2 year survival was 17%, 21% respectively. Split by age, median survival was 4.0 and 9.4 months respectively (2-year survival 19%; 27%) for patients aged

Description: Acute leukemias are classified into myeloid and lymphoid subtypes according to their phenotypic characteristics. Leukemic cells, however, sometimes co-express myeloid and lymphoid phenotypes, or switch their phenotype from myeloid to lymphoid lineages and vice versa. The conventional classification of leukemias is based mainly upon the concept that leukemic phenotype reflects a progenitor stage at which leukemic transformation occurs, or that it represents a stage at which transformed hematopoietic stem cells (HSCs) become incapable of further differentiation. Here we propose another possibility that phenotype of leukemias could be determined by instructive signals from the leukemic transformation mechanism itself. We found that TEL/PDGFβR (T/P), a tyrosine kinase fusion isolated from chronic myelomonocytic leukemia, can instruct myeloid lineage commitment and conversion at stem and progenitor stages of hematopoiesis. The T/P gene was transduced into purified progenitors or HSCs by using a retrovirus carrying a green fluorescent protein reporter. HSCs transduced with T/P (T/P+ HSCs) spontaneously formed GM colonies without cytokines. Furthermore, T/P+ HSCs were incapable of differentiation into B cells on OP9 stromal layer in the presence of IL-7. To test the effect of T/P on lymphoid commitment more precisely, we transduced T/P into purified common lymphoid progenitors (CLPs) that normally differentiate only into T, B and NK lineages. In a limiting dilution assay, 1 in 7 control-GFP transduced CLPs generated B cell progeny in vitro, while only 1 in 500 T/P+ CLPs differentiated into B cells. Instead, surprisingly, the majority of T/P+ CLPs and even T/P+ thymic proT cells quickly differentiated into granulocytes and monocytes in vitro. We then transplanted T/P+ CLPs into lethally irradiated congenic mice. T/P+ CLPs again differentiated into Gr-1+ granulocytes and monocytes in vivo. Gene expression analyses showed that T/P+ CLPs upregulated GM-related molecules including C/EBPα and GM-CSFRα immediately after T/P transduction, while transduction of either C/EBPα or GM-CSFRα also reprogrammed CLPs into the myeloid lineage as we reported previously. Thus, T/P signaling can activate these critical GM-related molecules in lymphoid progenitors to convert them into the myeloid lineage. These data collectively suggest that at least some types of oncogenic tyrosine kinase fusions can specify leukemic phenotypes, through activating critical signals for lineage commitment.

Description: Abstract 55 Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which confer a poor prognosis and are often refractory to conventional therapies. Recent efforts have begun to unravel the molecular pathogenesis of these malignancies. Several groups have demonstrated that MLL-fusions associate with two macromolecular chromatin complexes; the polymerase associated factor (PAFc) complex, which interacts with the N-terminal domain of MLL, a portion of the protein that is retained in all the described fusions, or the super elongation complex (SEC), via interaction with the C-terminal fusion partner. These complexes play an integral role in regulating transcriptional elongation and this function appears to be aberrantly co-opted by the MLL-fusions to initiate and perpetuate transcriptional programmes that culminate in leukaemia. In this study we used a systematic global proteomic survey incorporating quantitative mass spectrometry to demonstrate that MLL-fusions, as part of SEC and PAFc complexes, are associated with the BET family of acetyl lysine recognition chromatin “adaptor” proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. Targeting the BET proteins to alter aberrant transcriptional elongation has recently been demonstrated to be possible using small molecule inhibitors that selectively bind the tandem bromodomain at the amino-terminus of the ubiquitously expressed BET proteins (BRD2/BRD3/BRD4). We developed a novel class of potent small molecule inhibitors to the BET family, which is chemically distinct to previously published BET-inhibitors. We then used this new compound (I-BET151) to demonstrate its profound and selective efficacy against human MLL-fusion leukaemic cell lines in liquid culture as well as clonogenic assays in methylcellulose. We also establish that primary murine progenitors retrovirally transformed with MLL-ENL and MLL-AF9 are equally susceptible to treatment with I-BET151. We show that the main phenotypic consequence of BET inhibition in MLL fusion leukaemia is a dramatic early induction of cell cycle arrest and apoptosis. Global gene-expression profiling, following I-BET151 treatment in two different human MLL-fusion leukaemia cell lines (expressing MLL-AF4 and MLL-AF9), highlights a common differentially expressed gene signature that accounts for this phenotype. Importantly, chromatin immunoprecipitation analyses at direct MLL target genes including BCL2, C-MYC and CDK6, indicate that I-BET151 selectively inhibits the recruitment of BET family members BRD3/BRD4, and SEC and PAFc components. These events result in the inefficient phosphorylation and release of paused POL-II from the TSS of these genes providing mechanistic insight into the mode of action of I-BET151 in MLL-fusion leukaemia. We subsequently established the therapeutic efficacy of I-BET151 in vivo by demonstrating dramatic disease control in murine models of MLL-AF4 and MLL-AF9 leukaemia. Finally, we also demonstrate that I-BET151 accelerates apoptosis in primary leukaemic cells from a large number of patients with various MLL-fusion leukaemias, by affecting a similar transcription programme to that identified in the human leukaemic cell lines. Importantly, we also demonstrate that I-BET151 significantly reduces the clonogenic potential of isolated primary leukaemic stem cells, suggesting that disease eradication may be possible. These data highlight a new paradigm for drug discovery targeting the protein-protein interactions of chromatin-associated proteins. We demonstrate that small molecules that perturb the interaction of BRD3/4 with chromatin have therapeutic potential in MLL fusion leukaemias and moreover, we provide the molecular mechanism to account for this therapeutic efficacy. Finally, our results emphasize an emerging role for targeting aberrant transcriptional elongation in oncogenesis. Disclosures: Prinjha: GSK: Employment. Chung:GSK: Employment. Lugo:GSK: Employment. Beinke:GSK: Employment. Soden:GSK: Employment. Mirguet:GSK: Employment. Jeffrey:GSK: Employment. Lee:GSK: Employment. Kouzarides:GSK: Consultancy.

Description: 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