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Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

In Brazil, until the 1980s, the context of blood as transfusion therapy was marked by paid donations. Thus, self-interest has surpassed solidarity as a motivator to donate. Recruiting donors involves advising the population due to the difficulties related to the myths around donation. With the COVID-19 pandemic, recruiting convalescent plasma (CP) donors has been a hard. This is an observational, prospective and non-interventionist study carried out in a hemotherapy unit of the Unified Health System, in central-western Brazil. Data collection was carried out from 06/19/2020 to 07/31/2020. The subjects were contacted by the Recruitment and Collection (CR) sector, through an active search, using lists of patients previously diagnosed with COVID-19. The study was also published on social and traditional networks, which resulted in self-reference. Convalescent COVID-19 patients tested, of both genders, aged between 18 and 60 years, weight over 60 kg, without symptoms for more than 14 days, and nulliparous donors were invited to the study. Those who met the criteria were scheduled for clinical and serological screening. The subjects eligible for donation, with IgG reagent, signed the Free and Informed Consent Form. Individuals with positive RT-PCR and / or non-reactive IgG were excluded. During the study period, RC made 308 and received 1,797 calls (2,105 contacts), generating 242 (11.5%) screening appointments, 173 (8.2%) of which resulted from self-referral and 69 (3.2%) from active search. Of these, 131 (6.2%) subjects attended the appointment. After clinical screening, 37 (28.25%) subjects were ineligible, 37 (28.25%) after serological tests and 57 (43.5%) were eligible for donation. The ineligibility causes in clinical and serological screening are described in table 1. Many countries face difficulties in meeting the demand for blood and its components during the pandemic (Barone & DeSimone. Transfusion, 2020), especially in those where blood commercialization is prohibited, as in Brazil. The purpose of recruiting donors is to make blood donation habitual to Brazilians, as it occurs in developed countries. Figure 2 shows self-referral rates after dissemination in traditional media. The ads focused on the donor's ability to save lives by encouraging altruism (Ronse, et al. 2018).On the other hand, despite attracting more people, most were not eligible for donation, demonstrating a great capacity to raise awarenessamong the population, but it was necessary to improve criterias and demonstrate them clearly for the likely donor. Of the 26 donors, 22 (84.6%) are older than 29. For these, awareness-raising occurred mainly through the television media 9(34.6%) and 5(19.3%) through personal contact. In the youngest 4(15.4%), the stimulus was social networks (Sümnig, et al. Transfusion, 2018). Marketing was important for recruitment. As blood donation is not usual for most brazilians, it is essential to plan, develop, evaluate strategies, enabling new forms of collection. Another difficulty encountered was the logistics for this donation type. As the donor is convalescent, the recruitment, screening, and collection was restricted to a physical space, isolated from conventional donos (Bloch, et al. J Clin Invest. 2020). In conclusion, the COVID-19 pandemic has become a public health challenge worldwide. Many recovered patients could donate CP. However, it is necessary to define the ideal requirements for donor selection to ensure the therapeutic viability and efficacy of PC transfusion. Blood collection teams need to strengthen strategies to inform the population about blood donation needs. The information available in the traditional and digital media about the donation process can increase the donation rate and guarantee a safe blood component. Strategies such as a greater number of insertions in social networks with well-defined criteria for donating plasma from a convalescent donor, clarification of exclusion criteria in the means of greater reach, creation of easily accessible channels to the donor (registrations, central doubts),in addition to stratifying by age group and proposing different dissemination strategies and thanksgiving for the donation, forming a network of donations. The combined efforts of these actions will contribute with expert advice and experience, technical guidance and additional support to potentially save more lives. Disclosures No relevant conflicts of interest to declare.

Multiple Myeloma (MM) is a malignancy driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, somatic mutations and copy number abnormalities all contribute to myelomagenesis, however true drivers of the disease have not been well defined. To identify new targetable dependencies in MM, we generated high-quality active enhancer landscape using large cohort of primary patient myeloma cells (n=70), MM cell lines, and normal plasma cells. We integrated this data with an in-house curated atlas of 600+ active enhancer profile across a wide range of tumor types and normal tissues. Combining these data with gene expression and genetic dependency (CRISPR KO) enabled a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among these, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well as symptomatic MM compared to normal plasma cells. To functionally validate the role of BOB-1 in MM, we performed loss-of-function studies using shRNA, siRNAs and antisense GapMers specific for BOB-1 and observed a significant impact on MM cell viability and cell cycle arrest. Transcriptomic analysis upon BOB-1 depletion by RNA-sequencing revealed a small set of genes commonly modulated in all 3 MM cell lines tested including the plasma cell differentiation related transcription factor XBP1 and heme oxygenase (HMOX1). Importantly, we observed ribosome biogenesis, RNA polymerase 1A transcription and mRNA translation and elongation processes to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Bob1 KD resulted in a rapid and robust decrease in the level of transcription of rDNA by RNA polymerase I as determined by qRT-PCR quantification of pre-rRNA (47S). In addition, ChiP assay revealed decreased binding of RNA polymerase 1A to the 18S ribosomal DNA promoter region in BOB-1 depleted cells compared to control. These data indicate that BOB1 downregulation results in the suppression of RNA-polymerase I activity in MM cells. RNA Pol I-dependent transcription governs abundance of rRNA and directly regulates cellular translational and proliferative capacity. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. We observed that in MM cells compared to control cells, BOB-1 KD decreased, while its overexpression significantly enhanced de novo protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular light chains (kappa or lambda) production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result of decreased protein production, BOB-1 depletion was associated with induction of resistance to proteasome inhibition suggesting that high expression of BOB-1 may be one the factors driving the exquisite sensitivity of MM cells to proteasome inhibitor. Interestingly, mass spectrometry analysis revealed BOB1 in a protein complex with mTOR, Raptor and mLST8 proteins which are members of mTORC1 complex which is also involved in ribosomal function and may suggest the mechanism of action of Bob-1 at molecular level. In conclusion, here we report BOB1 as a specific proximal dependency in MM cells with potential role in modulating the protein load/capacity balance and ribosomal biogenesis essential for MM cell protein production function and therefore their sensitivity to proteasome inhibition. Disclosures Shirasaki: FIMECS: Consultancy. Mitsiades: BMS: Research Funding; Nurix: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Abbvie: Research Funding; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Karyopharm: Research Funding; Sanofi: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria. Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria. Munshi: Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Pfizer: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Novartis: Consultancy.

Multiple myeloma (MM) is marked by several genetic abnormalities, including chromosome translocation t(11;14). Overexpression of anti-apoptotic BCL-2 in t(11;14) MM promotes disease progression, prompting clinical use of the BH3 mimetic and BCL-2 inhibitor venetoclax in combination with proteasome inhibitor therapy. Despite high initial response rates and prolonged progression-free survival, patients commonly relapse. To delineate mechanisms contributing to acquired drug resistance we modeled responses to venetoclax in two highly sensitive MM cell lines (KMS27 and KMS-12PE). Colonies generated from a surviving cell were cultured in high-dose venetoclax to generate monoclonal drug-tolerant expanded persister (DTEP) clones. To determine whether venetoclax resistance in DTEP clones is mediated by transcriptional adaptation via genomic or epigenomic regulation and transcriptional reprogramming, we conducted whole-genome sequencing (WGS) and RNA-seq of the clones. WGS analysis did not show significant differences between parental and resistant clones, but transcriptomic analysis showed shared and unique transcriptome signatures in DTEP clones. Gene set enrichment analysis of the common significantly modulated genes in resistant clones revealed that PKA-ERK-CREB and K-Ras pathway genes were significantly upregulated, whereas apoptotic genes were downregulated in resistant clones compared to parental cells. Importantly, ectopically expressed ERK in venetoclax-sensitive cells conferred a resistant phenotype that was rescued using two specific ERK inhibitors in DTEP clones. These data confirm a key role for ERK activation in acquired venetoclax resistance. Resistant clones were further characterized by reduced mitochondrial priming assessed by dynamic BH3 profiling, with altered expression of anti-apoptotic regulators including MCL-1, BCL-xL, and BCL-W and the replaced BCL-2: BIM complex by both MCL-1 and BCL-xL. Because these data suggested a functional substitution between anti-apoptotic BCL-2 family members in cells with acquired resistance to venetoclax, we next evaluated if MCL-1 or BCL-xL are codependent in MM cells that are insensitive or resistant to venetoclax. Simultaneous inhibition of MCL-1 (via S63845) or BCL-xL (via A155463) and BCL-2 (via venetoclax) increased BIM release and enhanced cell death in resistant clones (vs single agents), with combination index values < 0.3 in all doses. Upregulation of BCL-xL or MCL-1 in MM cells also mediated primary venetoclax resistance independent of genetic hallmarks (e.g. t [11;14]-translocated cells). Thus, simultaneous inhibition of MCL-1 or BCL-xL and BCL-2 triggered synergistic cytotoxicity in MM cell lines intrinsically resistant to venetoclax. These data suggest that combined inhibition of BCL-2 and BCL-xL may overcome venetoclax resistance. However, the dependence of BCL-xL in mature platelets had triggered thrombocytopenia for patients under therapy using BCL-xL inhibitor. To further explore the potential clinical application of targeting BCL-xL, we employed novel BCl-2/BCL-xL dual inhibitor, BH3 mimetic pelcitoclax (APG-1252). Using pro-drug strategy for design, pelcitoclax has limited cell permeability during circulation, and was converted to a more potent metabolite APG-1252-M1 in tumors/tissues. APG-1252-M1 was thus used for in vitro cell based assays. We discovered that APG-1252-M1 induced cytotoxicity in MM cell lines intrinsically resistant to venetoclax (regardless of genetic background or BCL-2:BCL-xL ratio) and also significantly reduced MM cell viability in clones with acquired venetoclax resistance, overcoming ERK activation and decreasing BIM sequestration by BCL-xL. In vivo study using pelcitoclax is ongoing and will be presented at the meeting. In conclusion, we report that venetoclax resistance in MM evolves from outgrowth of persister clones displaying activation of the ERK pathway and a shift in mitochondrial dependency towards BCL-xL, which can potentially be effectively targeted via the novel BCL-2/BCL-xL inhibitor pelcitoclax (APG-1252), which is currently in clinical investigation for solid tumors (NCT03080311). Disclosures Deng: Ascentage Pharma Group: Current Employment. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Novartis: Consultancy; Janssen: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Amgen: Consultancy; Abbvie: Consultancy; Legend: Consultancy; Pfizer: Consultancy.

Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-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; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

To identify therapeutically actionable genetic dependencies we have pursued various approaches to derive a deeper understanding of the oncogenic hallmarks of myelomagenesis. We have studied the long noncoding RNA (lncRNA) landscape in multiple myeloma (MM) and identified a large number of differentially expressed lncRNAs in MM versus normal plasma cells. These lncRNAs presumably drive the tumorigenesis and MM cell growth, and in turn be susceptible to therapeutic intervention. To this end, we have developed and utilized a CRISPR interference (CRISPRi)-based platform for decoding and targeting the lncRNA dependencies (LongDEPs) in MM. In this study, we have used RNA-seq of patient-derived CD138+ MM cells (n=360) and MM cell lines (n=70) to generate a priority list of 913 expressed intergenic lncRNAs. Then, to systematically interrogate the role of these lncRNAs in MM cell growth, we have performed a CRISPRi viability screen transducing 3 MM cell lines engineered to express a dCAS9-KRAB fusion protein, with a pooled library consisting of 7 sgRNAs against each of the 913 transcription start sites (TSS) and 576 negative control sgRNAs. Relative representation of sgRNAs was assessed by deep sequencing after 3 weeks and analyzed using the MAGeCK robust rank aggregation (RRA) algorithm. The most enriched or depleted sgRNAs were further tested in a secondary CRISPRi viability screen. Focusing on depleted sgRNAs, we have identified >30 unique LongDEPs; which were further validated via an antisense oligonucleotide (ASO)-based loss-of-function study in a panel of MM cell lines (n=11). A comparative transcritpomic analysis comparing data from 360 newly-diagnosed and clinically-annotated MM patients and 16 healthy donors showed significant upregulation of these LongDEPs in MM patient cells. Of note, specific longDEPs were found selectively upregulated in genetically-defined patient subsets, including high-risk MM carrying t(4;14), 1q gain or del17p. Moreover, at least 18 LongDEPs were identified as independent risk-predictors of clinical outcome in newly-diagnosed MM patients. The lncRNA RROL was identified as a leading LongDEP, with a dependency score on a par with positive controls such as IRF4 or MYC. This lncRNA is specifically overexpressed in MM patients after disease relapse, and its higher expression in newly diagnosed MM patients could predict a worse clinical outcome. We have validated the essential role of RROL in support of the proliferation and survival of MM cells both in vitro and in vivo in NOD SCID mice, using ASO-based loss-of-function studies. To explain this effect, we have characterized its role in the control of the pro-survival de novo lipogenesis (DNL) pathway via an unbiased lipid profiling and by measuring the incorporation of C 14-radiolabeled glucose into the lipid pool. Mechanistically, we have shown that RROL promotes the DNL pathway via transcriptional regulation of rate-limiting enzymes including ACC1. Using in vitro (RNA protein pull down) and in cellulo (RNA yeast-3-hibrid) assays, we have identified the transcription factor c-MYC as a relevant protein interactor of RROL. This interaction occurs at the chromatin level and is required for i) MYC occupancy at DNL gene loci (e.g. ACC1), as shown by both ChIP-qPCR and single molecule dual RNA FISH coupled with immunofluorescence; ii) MYC interaction with a number of transcriptional co-activators, including WDR82, as assessed in vitro in 3 MM cell lines using co-immunoprecipitation followed by Mass spectrometry (Co-IP/MS) and in cellulo using the proximity-dependent biotin identification assay (BioID) in Flp-In T-REx cells expressing a FLAG-BirA*-MYC fusion protein. Overall, our data indicate that RROL provides the chromatin scaffold to assemble a transcriptionally activated ribonucleoprotein complex - minimally composed by RROL, MYC and WDR82 - at gene regulatory loci of DNL rate-limiting enzymes. To develop therapeutic inhibitors of LongDEPs, starting with RROL, we have tested >70 ASOs following a multi-step screening approach. The anti-MM activity of 2 leading compounds was demonstrated in vitro and in vivo in 2 clinically relevant animal models, including a BLI-based orthotopic model. In conclusion, our work establish LongDEPs as an additional source of genetic dependencies in MM paving the way for their biologic, clinical and therapeutic characterization in this disease context. Disclosures Young: Dewpoint: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Membership on an entity's Board of Directors or advisory committees; Omega Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Gryaznov: MAIA Therapeutics: Current Employment. Anderson: Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: 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; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Novartis: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Legend: Consultancy; Abbvie: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy.

Integrated genomic analysis including whole genome and RNA sequencing in primary multiple myeloma (MM) cells have reported a universal loss of BCL7A gene in MM patients compared to normal plasma cells (PC). Our Genetic modulation in in-vitro and in-vivo MM models have also validated the loss of BCL7A as an oncogenic event responsible for acquisition of a more proliferative phenotype in MM cells. We performed a comparative mass spectrometric analysis confirming BCL7A as a member of the canonical m-SWI/SNF chromatin remodeling complex. We therefore performed the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to assess genome-wide changes in DNA accessibility upon BCL7A gain or loss. We found that loss of BCL7A in wild-type KMS12BM and NCI-H929 cells resulted in enrichment of important transcription factor motifs in the transcriptionally active sites of chromatin. We identified 36 de novo accessible and 1079 de novo inaccessible regions across the genome after BCL7A shRNA KD (knock-down). These genomic regions with altered accessibility were associated with genes involved in protein binding (FDR < 0.001), GTPase activation (FDR = 0.016), and GTPase regulation (FDR = 0.030). Candidate transcription factors for the genomic regions with altered accessibility were identified by querying a database of human ChiP-seq experiments (ReMap 2020). IRF4 was identified to be enriched in regions of accessible chromatin upon BCL7A loss. IRF4 is an oncoprotein transcription factor and is a direct target of myc, generating a feedback loop in MM cells. IRF4 dependency is central to myeloma cell proliferation. Most importantly, we found that in addition to functions within the m-SWI/SNF complex, BCL7A forms a protein complex with IRF4. Altogether these data suggest a role for BCL7A in driving IRF4 oncogenic activities in MM. To understand how BCL7A-dependent changes may influence IRF4 activity, we performed CHiP assay using IRF4 antibody in BCL7A KO (knock-out) and AB (add-back) KMS12BM and NCI-H929 cells. We observed increased binding of IRF4 to the promoter of its target genes like PRDM1, CDK6, STAG2, PIM2, SQLE, Myc, CANX, IRF4, SCD, ELL2, CASP3 in BCL7A KO cells, while the binding of these target genes was significantly decreased in AB cells. While, CHiP assay using IRF4 antibody in control and ectopically expressed BCL7A in AMO1 and KMS11 cells showed significantly low binding of IRF4 to the promoter of most of its target genes in BCL7A overexpressed cells compared to control. Integrated transcriptomic analysis following BCL7A KD and overexpression revealed the existence of a set of genes transcriptionally regulated by IRF4 to be significantly upregulated following BCL7A depletion and downregulated following ectopic expression of BCL7A. To investigate whether these genes are involved in the phenotypic and functional effects observed in MM after BCL7A depletion, we performed LOF studies (si-RNA screen) in scrambled and BCL7A KD MM cells. Among others, we observed that MM cells are highly sensitive to the inhibition of EEF1B2, RPS3A, SOX2, DCC and NDUFA1 only in the context of BCL7A loss, implicating a role as critical effector molecules downstream of the IRF4-BCL7A transcriptional network. In conclusion, we observe that BCL7A binds to IRF4, functionally restricting its activity. The universally observed down regulation of BCL7A in MM provides the necessary molecular change to allow IRF4 to exert its required transcriptional activity to induce MM cell growth. Our results now provide the basis to understand the mechanism of development of IRF4 dependency in MM. Disclosures Anderson: Pfizer: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: 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; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Janssen: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Pfizer: Consultancy.

Venetoclax is the first BCL-2 inhibitor approved for the treatment of patients with chronic lymphocytic leukemia (CLL) alone or in combination with anti-CD20 monoclonal antibodies (MoAbs). Initial studies have shown high efficacy of venetoclax monotherapy or in combination with rituximab with an overall response rate of 79-92% in patients with relapsed/refractory (R/R) CLL. To investigate the use of venetoclax combinations in a real-world population, we designed this observational retrospective and prospective study aimed at defining the outcome of patients with R/R CLL treated with venetoclax-based regimens outside clinical trials in Italy. Efficacy (progression-free survival -PFS-, overall response rate -ORR-, complete response -CR-, measurable residual disease -MRD-) and safety (most frequent adverse events -AE-, grade 3-4 AE, laboratory and clinical tumor lysis syndrome -TLS-) data were collected within the study through a web-based platform after patients signed written informed consent. As of July 8 th, 2021 124 subjects (85 males, 39 females) were enrolled into the study (Table 1). Median age at venetoclax initiation was 70 years (44-91), median CIRS score 4 (0-14), median creatinine clearance 70 ml/min (22-123). A relevant proportion of patients presented adverse prognostic features, including TP53 aberrations [32% TP53 mutation and/or del(17p)], unmutated IGHV (77%). At the time of venetoclax initiation, patients had received a median of 2 previous therapies (1-8), with 57% previously exposed to BTK and/or PI3K inhibitors. 67/117 (57%) patients received venetoclax alone, while 50 were treated with a combination of venetoclax + rituximab (VenR), information on treatment plan was missing in 7 patients. Patients on venetoclax monotherapy compared to those receiving VenR showed a higher percentage of elevated LDH at baseline (64% vs 38%), were more heavily pretreated (median lines of therapy 3 vs 1), and had more frequently received treatment with ibrutinib (66% vs 27%) and/or idelalisib + rituximab (29% vs 4%). After a median follow-up of 13.7 months (0-41.9), the estimated 12m-PFS was 82% (CI 74-90%) (Figure 1), and the estimated 12m-overall survival (OS) was 83% (CI 76-91%). The best ORR in the whole cohort was 85% (CI 95% 76-91%) with a CR rate of 40%; best response was reached after a median of 3.9 months of treatment (range 0.6-30.5). The best ORR was not different in patients receiving VenR vs venetoclax alone (83% vs 88% respectively, p = n.s.), while the CR rate was significantly higher in those receiving VenR vs venetoclax alone (57% vs 30%, p=0.011). The ORR and CR rate in patients previously exposed to BTK and/or PI3K inhibitors were significantly lower (77% and 29% respectively), and the 12m-PFS was shorter in patients previously treated with ibrutinib compared to ibrutinib-naïve (75% vs 89%, p=0.005). Twenty subjects discontinued venetoclax (median time to discontinuation 4.1 months, range 0.4-10.8), 8 due to disease progression, 3 Richter's transformation, 7 AEs, 1 was lost to follow-up and 1 underwent planned allogeneic stem cell transplantation. Venetoclax-based regimens were well tolerated, with the most frequent AE of any grade related to venetoclax being neutropenia (79.6%), grade 3-4 in 61.9% (Table 2). In the whole cohort one grade 3 clinical TLS occurred and resolved without sequelae, and only 2/124 patients experienced laboratory TLS during ramp-up phase (both grade 1). No treatment-related death was reported. In conclusion, this analysis presents the results of one of the largest cohort of patients with R/R CLL treated with venetoclax-based regimens outside clinical trials and confirms the favourable efficacy and safety profile of the drug. In this patient population highly enriched for unfavorable disease features (1 out of 3 carrying TP53 aberrations, almost 80% with unmutated IGHV) venetoclax-based regimens were able to obtain a response in a high proportion of cases, with a very short time to best response (less than 4 months). As expected, response duration was shorter in patients previously exposed to BTKi/PI3Ki. When we compared the outcome of patients treated with venetoclax monotherapy vs VenR, the addition of anti-CD20 MoAb allowed to reach deeper responses by significantly increasing the CR rate. As the study and its data collection are still ongoing, the updated analysis of an expanded cohort with longer follow-up will be presented at the meeting. Figure 1 Figure 1. Disclosures Scarfo: Janssen: Honoraria, Other: Travel grants; Astra Zeneca: Honoraria; Abbvie: Honoraria. Quaglia: Roche: Other: Travel grants; Janssen: Honoraria, Other: Travel grants, Speakers Bureau; Sandoz: Consultancy; AstraZeneca: Honoraria. Marasca: AbbVie: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; Janssen: Honoraria, Other: Travel grants. Sanna: Astra Zeneca: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Murru: Abbvie: Consultancy, Honoraria, Other: travel and accommodation; Janssen: Consultancy, Honoraria. Laurenti: Roche: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria; BeiGene: Honoraria. Gaidano: Incyte: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Reda: Beigene: Consultancy; Astra Zeneca: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Molica: Astrazeneca: Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Sportoletti: AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Galimberti: AbbVie, Janssen: Honoraria, Other: Travel grants; Incyte: Speakers Bureau. Mauro: AbbVie: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Cuneo: Janssen: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau. Ghia: AstraZeneca: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; ArQule/MSD: Consultancy, Honoraria; Sunesis: Research Funding.