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Acute myeloid leukemia (AML) remains an incurable blood cancer largely due to rapid emergence of resistance to conventional treatments. Thus, new therapeutic modalities are greatly needed to halt AML development. Here, using genetic and xenograft mouse models, we reveal that inhibition of the ubiquitin ligase RNF5 in human AML cell lines and in MLL-AF9-driven AML severely decreased the leukemogenic potential of those cells and prolonged survival of model leukemic mice. These findings suggest the possibility that targeting a single gene, namely RNF5, could effectively inhibit different AML subtypes. We initially focused on RNF5 as its expression is upregulated in AML patient cohorts as well as in AML-derived cell lines compared with normal hematopoietic cells. Furthermore, high RNF5 expression in AML patient specimens correlated with poor prognosis, relapse and short overall patient survival. By contrast, specimens from AML patients who responded to therapy exhibited low RNF5 levels. In vitro, RNF5 loss impaired the clonogenic potential of MLL-AF9-transduced bone marrow cells and markedly attenuated growth and survival of AML but not CML or T-ALL cell lines, in which RNF5 is also highly expressed. High-throughput screen and bioinformatics analysis identified RNF5 and ER-associated degradation (ERAD) components, as augmenting AML cell sensitivity to histone deacetylase (HDAC) inhibition. Indeed, inhibition of RNF5 sensitized AML cells to HDAC inhibitors. Correspondingly, a favorable prognosis was observed in AML patients exhibiting low expression of RNF5 and HDAC. Collectivity, our studies identify a potential new therapeutic modality based on targeting RNF5 to inhibit AML and suggest that RNF5 expression could serve as a prognostic marker and means to stratify patients for treatment with HDAC inhibitors. Disclosures Ofran: AbbVie: Membership on an entity's Board of Directors or advisory committees. Vuori:Bionano Genomics: Membership on an entity's Board of Directors or advisory committees.

The aberrant and constitutive activation of the HOXA cluster genes and the their-co-factor MEIS1 (HOX/MEIS) is a recurrent feature in several types of myeloid and lymphoid leukemias. Aberrant HOX/MEIS expression has been shown to drive limitless leukemia stem cell self-renewal and is therefore an attractive target for therapy in acute myeloid leukemia (AML). However, since HOX/MEIS genes encode DNA-binding transcription factors, small molecules targeting these proteins directly are lacking. Furthermore, targeting the HOX/MEIS network is complicated by the fact that these genes are coordinately regulated and have redundant functions in sustaining leukemic self-renewal. One way of therapeutically targeting aberrant HOX/MEIS transcription is the identification and pharmacologic inhibition of upstream chromatin regulators that coordinately modulate their expression. In order to identify such chromatin regulators, we made use of an endogenous GFP reporter knocked-in to the MEIS1 locus in the high HOX/MEIS-expressing U937 human AML cell line. Using this system, we first performed a high-throughput flow-cytometry-based small-molecule inhibitor screen with a library of 261 compounds targeting epigenetic regulators. In our screen, the most potent hits that reproducibly showed >50% MEIS1-GFP inhibition were small molecules that targeted DOT1L, the histone methyltransferase. DOT1L inhibitors have already been well-characterized as HOX/MEIS regulators and most epigenetic regulators are not targeted by existing compound libraries. Therefore, we decided to use a genetic screening approach to more extensively interrogate the landscape of epigenetic regulators of HOX/MEIS expression in AML. For this, we designed a custom computational pipeline and built a CRISPR library of 10,000 sgRNAs targeting functionally conserved protein domains of all catalogued chromatin modulatory proteins (> 600 proteins - 5 sgRNAs per conserved domain). This list of epigenetic regulators included histone modifying enzymes, chromatin readers, nucleosome remodelers, adaptor proteins and proteins involved in DNA and RNA modifications, as well as other transcriptional regulators. Using this comprehensive, domain-focused CRISPR library, we conducted a phenotypic enrichment screen. Specifically, we used flow cytometry to purify the top 20% GFP-MEIS1 (high) and bottom 20% GFP-MEIS1 (low) expressing cells and identified sgRNAs that were enriched particularly in the GFP-MEIS1 -low vs -high fraction using next generation sequencing. Given the extent and complexity of the CRISPR library, our approach uncovered members of six distinct chromatin modifying complexes as MEIS1 regulators (MAGeCKFlute pipeline, 2 SD > mean) and we could validate > 10 of these hits as bonafide regulators of MEIS1 as well as HOXA genes. We also demonstrated their essentiality for the proliferation of HOX-driven AML cells using arrayed sgRNA competition assays. These validated hits included several known as well as novel chromatin readers and writers amenable to small-molecule targeting. We focused our attention on the KAT7/JADE3 complex and the casein kinase 2 (CK2) family that we validated as potent and selective regulators of HOX/MEIS expression in AML cells. Our studies demonstrated that genetic depletion of components of the KAT7 complex or of the CK2 family could reverse HOX/MEIS activation in human AML cells, leading to a progressive loss of proliferative potential. Importantly, the use of the clinical-grade CK2 inhibitor CX4945 (Silmitasertib) caused a concentration-dependent down-regulation of HOX/MEIS expression in models of HOX-driven AML, leading to significant anti-leukemia effects. Our study provides a framework for the multiplexed identification of actionable dependencies targeting therapeutically recalcitrant oncogenic networks in cancer. Specifically for AML, since Silmitasertib is in Phase 2 trials for treatment of other cancers, our studies may solve the long-standing problem of targeting leukemia stem cells in AML potentially overcoming therapy refractoriness in this devastating disease. Disclosures No relevant conflicts of interest to declare.

Introduction: MAPK pathway mutations are present in more than 50% of patients (pts) with relapsed/refractory multiple myeloma (RRMM; Xu et al. Oncogenesis 2017; Kortum et al. Blood 2016). While MEK inhibitors have demonstrated limited single-agent activity in RRMM, combination with BCL-2 inhibition with or without PD-L1 inhibition may improve efficacy, by shifting the apoptotic balance towards cell death and increasing CD8+ T cell recognition and enhancing the anti-tumor immune response. We conducted a Phase Ib/II study of cobimetinib (C) as a single agent and in novel combinations with venetoclax (V) with or without atezolizumab (A) in pts with RRMM (NCT03312530). Methods: Pts with RRMM who had received 3-5 prior therapies, including a proteasome inhibitor and an immunomodulatory drug, were enrolled. Safety run-in (SRI) cohorts, SRI 1 and SRI 2, evaluated the safety of C+V and C+V+A, respectively. Pts were then randomized 1:2:2 to C (Arm A), C+V (Arm B), or C+V+A (Arm C). Pts received C 60 mg PO daily on Day (D) 1-21 (Arm A), or C 40 mg PO daily on D1-21 and V 800 mg PO daily on D1-28 (SRI 1, Arm B) and A 840 mg IV on D1 and D15 (SRI 2, Arm C) on 28-day cycles. Responses were assessed using IMWG 2016 criteria. Pharmacokinetic (PK) analyses were performed. Exploratory biomarker assessments included cytogenetics, mutational analysis, and PD-L1 and BCL-2 family expression (RNAseq). Results: As of May 6 2020, 49 pts were enrolled (SRI 1, n=6; SRI 2, n=6; Arm A, n=6; Arm B, n=16; Arm C, n=15). Median age was 65 years (range 44-79), 63% were male, 94% had ECOG PS 0-1, and 47% had ISS II/III disease. Median prior lines of therapy was 4 (range 3-5), with prior ASCT in 43% and prior daratumumab in 41% of pts. 24% of pts had high-risk cytogenetics (del(17p), t(4;14), t(14;16)), 20% were t(11;14) positive, 51% had RAS mutations (KRAS, NRAS, BRAF, NF-1), and 31% had high PD-L1 expression. The most common adverse events (AEs) of any grade (Gr; C/C+V/C+V+A) were diarrhea (33%/82%/91%), nausea (17%/50%/67%), anemia (17%/46%/57%), neutropenia (0/32%/57%), thrombocytopenia (0/27%/33%), blood creatine phosphokinase increase (17%/32%/24%), and rash (50%/14%/33%). The most common Gr 3-4 AEs were neutropenia (0/14%/38%), anemia (0/23%/24%), thrombocytopenia (0/18%/24%), and pneumonia (0/14%/14%). The most common serious AE was pneumonia (0/23%/14%). Two AEs of tumor lysis syndrome (TLS) were reported, with 1 meeting Howard criteria for laboratory TLS (t(11;14) pt on C+V, no dose interruption). Treatment discontinuation due to AEs was noted in 17%, 18%, and 14% of C, C+V, and C+V+A pts, respectively. At a median follow-up of 13.4 months (mo), there were 27 (55%) deaths overall (4 [67%]/14 [64%]/9 [43%]). The leading cause of death was progressive disease (PD; 2 [50%]/11 [79%]/7 [78%]). Two treatment-related deaths were observed (C+V: pneumonia; C+V+A: general physical health deterioration in the setting of PD). Overall survival (OS) for C, C+V, and C+V+A was 12.9, 12.4, and 23.3 mo respectively, and comparable with historical OS in 3L+ pts (Usmani et al. Oncologist 2016; Kumar et al. Leukemia 2017). No clinically relevant drug-drug interactions were identified between C, V, and A based on preliminary PK analysis. The overall response rate (ORR; ≥partial response) was 0% (0/6) for C, 27% (6/22) for C+V, and 29% (6/21) for C+V+A (Figure A). Compared with the non-t(11;14) pts, responses to C+V and C+V+A were higher among t(11;14) pts, with an ORR of 50% (2/4) and 100% (5/5), respectively. Median duration of response was 11.5 mo for C+V (range 2-18) versus 5.1 mo for C+V+A pts (range 2-15), with individual pt characteristics likely contributing to the observed difference. Prolonged disease stability and durable responses were noted in a subset of pts, irrespective of t(11;14) and/or RAS mutation status (Figure B/C). At data cut-off, 4 pts remain on treatment, 3 of whom are t(11;14) positive and 1 who is t(11;14) negative and RAS wild-type but BCL2/BCL2L1 (BCL-XL) high (log2≥2.3). Conclusions: C, C+V and C+V+A demonstrated manageable safety and tolerability in heavily pretreated pts with RRMM. Preliminary efficacy results show moderate activity of the combinations in all-comers and higher activity in t(11;14) pts, supporting a biomarker-driven approach in the development of V in MM. Further biomarker analyses may provide insight into the pt subgroups that may benefit from combined MAPK and BCL-2 inhibition, and the contribution of PD-L1 inhibition. Disclosures Schjesvold: Novartis, Amgen, Celgene, Takeda, Janssen, Oncopeptides, MSD, Sanofi: Consultancy; Celgene, Amgen, Janssen, Oncopeptides: Research Funding; Amgen, Celgene, Takeda, Janssen,Novartis, SkyliteDX, Oncopeptides, Sanofi: Honoraria. Ribrag:Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; arGEN-X-BVBA: Research Funding; BAY1000394 studies on MCL: Patents & Royalties; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Honoraria; AZD: Honoraria, Other; Pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; argenX: Current equity holder in publicly-traded company, Research Funding; Institut Gustave Roussy: Current Employment; Nanostring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Rodriguez-Otero:GlaxoSmithKline: Consultancy, Current Employment, Current equity holder in publicly-traded company, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Kite: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Abbvie: Consultancy, Honoraria; Medscape: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria. Robak:Abbvie, Pharmacyclics, Gilead, GlaxoSmithKline, Novartis, Janssen, F. Hoffmann-La Roche, Acerta, AstraZeneca, BioGene, UCB: Research Funding; F. Hoffmann-La Roche, Abbvie, Pharmacyclics, Novartis, Janssen, Acerta, AstraZeneca, BioGene, UCB, Sandoz: Honoraria. Hansson:Amgen, Celgene, Takeda, Janssen Cilag: Consultancy. Hajek:Celgene, Novartis, Amgen, Takeda, Janssen, BMS: Research Funding; Takeda, Amgen, Oncopeptides, Sanofi, Janssen, Novartis, Celgene: Honoraria; BMS, Takeda, Amgen, Oncopeptides, Sanofi, Janssen: Membership on an entity's Board of Directors or advisory committees. Amor:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinez-López:Janssen, BMS, Sanofi, Novartis, Incyte, F. Hoffmann-La Roche and Amgen: Honoraria, Other: Advisory boards; Hosea and Altum: Membership on an entity's Board of Directors or advisory committees; Janssen, Novartis, BMS, Incyte: Consultancy. Onishi:Genentech, Inc.: Current Employment, Current equity holder in publicly-traded company; F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Gallo:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Raval:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Deshpande:Syneos Health: Current Employment. Malhi:Genentech, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Hong:F. Hoffmann-La Roche: Current equity holder in publicly-traded company; Genentech, Inc.: Current Employment. Raab:Amgen: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Venetoclax (ABT-199/GDC-0199) is a highly selective, potent, oral B-cell lymphoma-2 (BCL-2) inhibitor. Cobimetinib (GDC-0973/RG7420) is an orally bioavailable small molecule inhibitor of mitogen-activated protein kinase kinase 1 (MAP2K1 or MEK1). Atezolizumab (RG7446/MPDL-3280A) is a monoclonal antibody directed against programmed death-1 ligand-1 (PD-L1).

Introduction: Sickle cell disease (SCD) poses a considerable health burden in lower middle income country (LMIC) like India. One ICMR survey reported about 20% of children with SCD died by the age of two years and 30% of children with SCD amongst the tribal community in India died before they reached adulthood. Data on mortality rate in the adult population with SCD is sparse. Our centre data shows that despite the availability of Hydroxyurea and supportive care, almost one-third of hospitalized Sickle Cell Crisis patients develop life threatening complications. Manual Partial Exchange Transfusion is a cost-effective intervention to decrease mortality in Sickle Cell Crisis. But perhaps, it is an underutilized therapy. In India, the cost incurred for each session of exchange transfusion, if done by the RBC apheresis machine, is $420. Whereas Manual Exchange Transfusion costs only $70(including the cost of the central venous catheter) for the first session followed by $15 for each subsequent session. Aim: A single centre retrospective data analysis to evaluate the outcome of Sickle Cell Crisis patients undergoing Manual Partial Exchange Transfusion during hospitalization. Material and Method: A total of 553 SCD patients on regular follow up at the Haemato Oncology Care Centre (HOCC) from July 2012 to July 2020 were evaluated.169 patients were hospitalized for treatment of Sickle Cell Crisis at the Bhailal Amin General Hospital and Sterling Hospital Vadodara. The data was retrieved after the IRB/Apex committee approvals for retrospective analysis. The indication for exchange transfusion was Acute Chest Syndrome 19(35%), Hepatic cell crisis 11(20%), Vaso Occlusive Crisis not responding to the conservative line of therapy in 48 hours of treatment 11(20%), Cerebrovascular Accident (CVA) 5 (9%), Avascular Necrosis of Femur with excruciating pain 3 (6%), Complicated Dengue with Multi-organ failure 3 (6 %), Priapism 1 (2%) and Splenic Sequestration 1 (2%). Central venous access was secured with a central venous catheter or dialysis catheter. Simple packed cell volume was transfused to 11(20%) patients and 5(9%) patients received platelet transfusion. The cut-off values for HB and Platelet count were 8 gm% and 50000/cumm, respectively at the time of the Manual Partial Exchange Transfusion procedure. Blood volume withdrawn was 5 to 10 ml /kg, followed by an equal volume of packed cell volume transfusion at every session. The procedure was repeated every 12 hr or 24 hr depending on the clinical condition of the patient. HbS value was reassessed post 4 sessions with repeat testing done after 2 to 4 sessions if the observed HbS value was more than 30% or more than 10% (for CVA) after 4 sessions. The endpoint was clinical recovery with HbS less than 30% or no clinical recovery despite the achievement of HbS less than 10%. In CVA the endpoint was HBS less than 10%. Results: Manual Partial Exchange Transfusion was carried out in 54/169 (32 %) hospitalized patients. The median age was 25yrs (range 5 yrs to 69 yrs), with male predominance [Males 42(77%) and females 12(23%)].Pre procedure HbF value was 20% in 18(33%). A total of 50 out of 54 (93%) patients recovered completely. 28 (52%) patients were hemodynamically stable with normal SPO2 on room air at the time of Manual Partial Exchange Transfusion with an average of 7 days of hospitalization. All of these patients recovered completely with less than 5 sessions of Manual Partial Exchange Transfusion. 26(49%) patients were critically ill and had an average of 12 days of hospitalization. They were on Ventilator and Inotrope support at the time of Manual Partial Exchange Transfusion. 14/26(54%) critically ill patients recovered completely with an average of 6 sessions of Manual Partial Exchange Transfusion. 12/26(46%) critically ill patients succumbed even though post exchange HBS value decreased to less than 10%. The overall mortality rate of SCD patients in this analysis was 12/553 (2.1%), significantly lower than what was historically reported as 30% in the ICMR survey. Underlying Dengue viral infection associated with Multi-organ dysfunction and Fulminant hepatic failure were risk factors for mortality observed in our study. Conclusion: Manual Partial Exchange Transfusion is highly effective in reducing mortality in Sickle Cell Crisis. It is feasible and cost-effective in small centres lacking apheresis machine facility. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually compared to many other cancers, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation via MDM2i and minimize toxicity, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. These screens identified BRD4 inhibition as a candidate synthetic lethal partner of MDM2i. BRD4 is a member of the Bromodomains and Extraterminal (BET) family of proteins, a transcriptional co-activator and already a candidate AML therapeutic target. Surprisingly, we found inhibition of BRD4 alone induces expression of some of p53 target genes. We unexpectedly reveal that BRD4 binds to p53 target genes and acts as a transcriptional repressor of these genes. Synergistic cell killing by the drug combination (MDM2i + BET inhibitor (BETi)) depends on synergistic activation of p53 target genes, such as PUMA and NOXA, due to simultaneous stabilization of p53 by MDM2i and relief of BRD4-mediated repression by BETi. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type TP53in vitro, against two mouse models of AML in vivo, and against primary human patient blasts in vitro. Furthermore, we used BET PROTACs to selectively and completely induce degradation of BRD4 in cells. Consistent with results from BETi, BET degraders and MDM2i synergize to suppress cell viability with superior potency. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, co-targeting wild-type TP53 and a transcriptional repressor function of BRD4 represent a novel synthetic lethal vulnerability in AML. Disclosures Latif: AbbVie: Consultancy, Honoraria; Takeda UK: Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Kite: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria. Higgins:Roche: Current Employment, Current equity holder in publicly-traded company, Other: Support of parent study and funding of editorial support. Copland:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Roche: Research Funding; Epizyme: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; Gilead: Speakers Bureau.