Patient Multiple Myeloma Cells Research Articles

Discovery of Prolyl-tRNA Synthetase As a Novel Target in Multiple Myeloma

Discovery of Prolyl-tRNA Synthetase As a Novel Target in Multiple Myeloma

Novel Cytokine-Mediated Mechanism of Action Identified By Quantitative Seroproteomics in Multiple Myeloma Patients Treated with Tagraxofusp, a Novel CD123-Directed Targeted Therapy

Introduction Plasmacytoid dendritic cells (pDCs) express CD123/IL-3Rα and promote tumor growth and immunosuppression in multiple myeloma (MM) (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia, 2018, 32:843-846). Tagraxofusp is a novel targeted therapy directed against CD123, and is FDA-approved for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm [BPDCN]). Tagraxofusp can also trigger anti-MM activity by reducing the viability of immunologically defective and tumor-promoting pDCs in MM. Furthermore, tagraxofusp synergistically enhances the anti-MM activity of anti-MM agents bortezomib and pomalidomide. Our preclinical findings led to a recently completed phase 1/2 clinical trial of tagraxofusp with pomalidomide/dexamethasone in relapsed/refractory MM patients (NCT02661022). Results demonstrated preliminary safety and efficacy, with 5 of 9 heavily pretreated patients achieving durable partial response (PR) (ASH 2019). Here, we report the early results of our translational correlative studies using bone marrow (BM), peripheral blood (PB), and serum from the study cohort.Methods Tagraxofusp is a bioengineered targeted therapy directed to CD123 comprised of human IL-3 fused to a truncated diphtheria toxin (DT) payload (Stemline Therapeutics, NY). pDCs and patient MM cells were purified from BM/PB samples after informed consent, and quantified using FACS, as described (Ray et al, Leukemia, 2018). A novel high throughput seroproteomics platform SOMAscan was used to analyze 1,310 protein analytes in serum samples from MM patients (n = 9). SOMAscan data were subjected to meta-analysis to generate heatmaps, followed by hierarchical cluster analysis. SOMAscan results were validated with ELISA using supernatants from MM patient pDCs cultured with or without tagraxofusp.Results Analysis of BM/PB samples from MM patients receiving tagraxofusp therapy showed a distinct reduction in the frequency of viable pDCs [average 2% at screening vs 0.75% post-tagraxofusp; n = 6; p = 0.036]. Of note, pDCs isolated from tagraxofusp-treated patients showed decreased ability to trigger MM cell growth. SOMAscan analysis of patient serum before and after tagraxofusp therapy showed alterations in the levels of 100 proteins [Median Fold Change in expression: 0.39 to 4.5; n = 6; 3 each; p < 0.05]. Importantly, tagraxofusp treatment reduced pDC-related soluble proteins including IFN-α (fold change: 0.8, treated vs untreated; p < 0.05). Pathway analysis further show that treatment affected immune signaling. For example, tagraxofusp decreased the levels of immunosuppressive proteins, soluble CD40L and IL1R2 (0.071-fold and 0.088 fold vs untreated; p = 0.02 and p = 0.013, respectively), promoting immune response. Moreover, analysis of end of treatment samples showed decreased soluble C-reactive protein, affecting the complement cascade after treatment (0.53-fold, p = 0.0173) via the downregulation of several C-C motif soluble chemokines (p < 0.05). Our earlier study showed that pDC-MM interactions triggered secretion of IL-3, which in turn promotes both pDC survival and MM cell growth. Importantly, tagraxofusp in this trial decreased serum IL-3 levels (fold change 0.75, treated vs untreated; p < 0.05).Conclusions In the present study, we validate the target specificity of tagraxofusp against MM pDCs in relapsed and refractory MM patients enrolled in a phase 1/2 clinical trial. A future clinical trial of tagraxofusp in combination with bortezomib and pomalidomide will examine the utility of tagraxofusp to improve outcome in patients with relapsed refractory MM. DisclosuresMo: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eli Lilly: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy. Olguin: Stemline Therapeutics, New York, NY: Current Employment. Chen: Stemline Therapeutics, New York, NY: Current Employment. Brooks: Stemline Therapeutics: Current Employment. Mughal: Stemline: Current Employment, Current holder of stock options in a privately-held company; Oxford University Press, Informa: Other: financial benefit and/or patents . Richardson: Janssen: Consultancy; Celgene/BMS: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Oncopeptides: Consultancy, Research Funding; AstraZeneca: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Protocol Intelligence: Consultancy; Regeneron: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: Oncopeptides: Consultancy; C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics: 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; Bristol Myers Squibb: 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; 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; 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.

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

Aberrant CDK7 Activity Drives the Cell Cycle and Transcriptional Dysregulation to Support Multiple Myeloma Growth: An Attractive Molecular Vulnerability

Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models.Gene expression analysis after CDK7 inhibition in several MM cell lines revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs.To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented.In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. DisclosuresShirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: 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; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: 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; 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. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

Identification of Novel Targets Based on Splicing Alterations for Undruggable RAS/CDK Signaling Cascade in Multiple Myeloma

Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets.Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC<0.5) MM cytotoxicity triggered by this combination treatment, which triggered dose-dependent manner G0/G1 phase growth arrest. We assessed early death cascade in MM cells after Ei+Ci treatment, and demonstrated significant priming to selective peptides BIM, BAD, and MS1 or HRK, suggesting dependency on BCL2 and MCL1 or on BCL-XL proteins. Our studies showed that Ei+Ci treatment induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling including c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of Ei and Ci. Patient MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that this combination can overcome the protective effects of the MM BM milieu. Moreover, our in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. We evaluated the effect of Ei+Ci treatment on target gene expression in BM cells isolated from flushed femurs of treated animals with Ei, Ci or Ei+Ci, and observed downregulation of Erk1/2-CDK4/6-dependent gene signature. Therefore, we suggest that these inhibitors selectively target Erk1/2, CDK4/6 and their downstream substrates both in vitro/vivo.We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p<2e-11) upregulation of genes with progression from MGUS to sMM, and, also to overt MM . SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 is linked with poor outcomes. SNRPB proteins are also overexpressed in MM cells. Our studies evaluating SNRPB effects on RNA splicing showed both upregulation of transcripts with full intron retention and transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. We evaluated RAVER1 and SNRPB expression in BM cells from animals treated with Ei and Ci alone or in combination. We observed significant downregulation of RAVER1/SNRPB (p=0.001) in BM samples obtained from animals treated with Ei+Ci. We observed decreased intron retention events in genes in treated samples, consistent with our in vitro analyses in MM cell lines and patient samples. Thus, RAVER1/SNRPB overexpression contributes to the aberrant transcriptome splicing associated with RAS/CDK cascade in MM.Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. DisclosuresLetai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Anderson: 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; Gilead: 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; Sanofi-Aventis: 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.

Identifying Long Noncoding RNA Dependencies Using CRISPR Interference (CRISPRi)-Based Platform in Multiple Myeloma

Identifying Long Noncoding RNA Dependencies Using CRISPR Interference (CRISPRi)-Based Platform in Multiple Myeloma

CD44 Expression in Different Plasma Cell Diseases

CD44 expression in different plasma cell diseasesIntroductionMyeloma is a genetically complex disease which develops via a multistep process whereby plasma cells are driven towards malignancy through the accumulation of genetic “hits” over time. This multistep process permits myeloma to have four recognisable clinical stages including monoclonal gammopathy of undetermined significance (MGUS) , smouldering multiple myeloma (SMM) , multiple myeloma (MM) and plasma cell leukemia (PCL). CD44 as a complex adhesion molecule, is highly expressed in a variety of solid tumors and involved in metastasis and chemotherapy resistance . Previous studies had found that CD44 was also highly expressed in MM, and associated with advanced clinical stage, extramedullary myeloma (EM) and poor survival. However the relation of CD44 expression in different plasma cell disease remain unknown.Patients and MethodsNewly diagnosed MGUS (n=12), SMM (n=11), MM (n=133) and PCL (n = 8) patients who were hospitalized in our hospital from December 2017 to December 2020 were enrolled. The diagnosis was based on the criteria of the International Myeloma Working Group. CD44 was detected by flow cytometry as follows: 2 ml of heparin anticoagulated bone marrow was collected from the patients at the time of diagnosis, 1×10 6 cells were detected, and a gate was set for identifying abnormal plasma cells characterized by CD138 and CD38. CD44 positive was defined as more than 20% of the plasma cells expressed CD44.ResultsFrom December 2017 to December 2020, a total of 164 patients diagnosed in our hospital were included in the study, including 12 MGUS patients, 11 SMM patients, 133 MM patients and 8 PCL patients. The expression proportion (7.9%±11.9%, 11.2%±15.5%, 40.2%±37.7%, 81.9%±25.6%, P < 0.001) and expression intensity (the mean fluorescence intensity was 1 742.8±1 023.1, 2 915.7±923.0, 6 692.6±11 275.5, 25 359.6±22 604.5, P=0.001) of CD44 on the monoclonal plasma cells of MGUS, SMM, MM and PCL patients gradually increased. In 133 MM patients, 73 cases were for CD44 positive. CD44 positive patients were more likely to show ＞3 focal bone lesion (75.3% VS 50.0%, P=0.002), and international staging system（ISS）III stage (68.5% VS 51.7%, P=0.048) compared those with CD44 negative. CD44 expression rate was significantly higher in MM patients with extramedullary disease (n=26) than those without (n=107) (56.7%VS 36.2%, P=0.031)Conclusion: Our study showed that from MGUS, SMM, MM to PCL, the CD44 expression on abnormal plasma cells was gradually increased. CD44 overexpression was associated with osteolytic lesions, advanced ISS staging and extramedullary myeloma. The results indicated that CD44 was related to the invasion degree of plasma cell disease, and could be used as a marker for differential diagnosis between different plasma cell disease. DisclosuresNo relevant conflicts of interest to declare.

Super-Enhancer-Driven PPP1R15B As an Oncogenic and Potential Therapeutic Target in Multiple Myeloma

Background: Multiple myeloma (MM) is a hematological malignancy arising from immunoglobulin-secreting plasma cells. Despite recent progress in stem-cell transplantation, high-dose chemotherapy and novel therapies, MM remains incurable and most patients experience relapse. In addition of genetic alterations, growing evidence has suggested that alterations in epigenetic landscape contribute to pathogenesis of MM. Super enhancers (SE) are large clusters of putative enhancers with aberrantly strong binding of mediators and transcription-regulating proteins. MM cells are highly dependent on unfolded protein response (UPR) signaling pathways due to high level of endoplasmic reticulum (ER) stress. Phosphorylation of eIF2α can attenuate protein translation. The PPP1R15B (denoted as R15B hereafter) gene encodes a regulatory subunit of an eIF2α-specific phosphatase complex. In this study, we identified SE-driven oncogenes specific in MM with a particular focus on a candidate SE-associated gene R15B, whose functional roles in MM remain largely elusive.Methods: We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples and normal CD138+ plasma cells, memory B cells (controls). ROSE analysis was used to systematically annotate SEs and their associated genes between tumors and controls. A combination of public data mining, RNA interference (RNAi), overexpression and CRISPR/Cas9 technologies followed by functional assays were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line NCI-H929 with R15B knockdown and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on highly integrative chromatin immunoprecipitation (HiChIP) followed by sequencing.Results: We have identified R15B as one of the SE-associated genes specific to MM patient samples and MM cell lines. The expression of R15B was 6- to 50-fold higher in a panel of 8 MM cell lines than normal controls. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. Knockdown and knockout of R15B significantly reduced cell viability, clonogenicity and induced G2/M phase cell cycle arrest. Gene Ontology (GO) enrichment and pathway analysis of downregulated genes in response to R15B knockdown indicates that these genes are significantly associated with G2/M checkpoint hallmark, oxidative phosphorylation, unfolded protein response and E2F1-related pathways. Loss of R15B may induce apoptosis via activation of UPR pro-apoptotic executors PUMA and NOXA. ChIP-qPCR assays indicates that transcription factor C/EBP-β is strongly enriched at R15B SE region. Furthermore, we examined the therapeutic effects of Salubrinal, a selective inhibitor of eIF2α phosphorylation, on MM cells. Salubrinal inhibited MM cell proliferation in a dose-dependent manner, and MM cell lines with higher R15B expression were more sensitive to Salubrinal than those with lower R15B level.Conclusions: Our integrative approaches identified R15B as a novel SE-driven oncogene, and may form a positive feedback loop with C/EBP-β. Salubrinal selectively inhibits proliferation of MM cells with high R15B expression. Hence, we propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation. DisclosuresChng: Novartis: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Honoraria; Amgen: Honoraria, Research Funding.

Delineating CDK9 Regulated Molecular Events for the Development of Rationally Derived Multiple Myeloma Treatment Strategies

Background: Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and the development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome.Methods: Following expression profiling, CRISPR loss-of-function screens and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses.Results: Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, SMM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines verified their dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, but not other genes (e.g. Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 induced a reduction of mRNA/ protein levels of Mcl-1, cMyc, RNA Pol II, but not of other potential targets (e.g. Bcl-2) in a dose- and time-dependent manner. Moreover, Thal-sns-032 reduced Mdm2 and thereby increased p53 protein levels. Consequently, Thal-sns-032 inhibited tumor cell proliferation and survival both in tumor cell- and tumor cell:BMSC co- cultures. Rationally, derived combination strategies of Thal-sns-032 for example with venetoclax, but also other investigational and established MM therapies induced synergistic anti-MM effects within the tumor microenvironment.Conclusion: In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derive MM combination treatment strategies. DisclosuresVallet: Pfizer: Honoraria; MSD: Honoraria; Roche Pharmaceuticals: Consultancy. Podar: Celgene: Consultancy, Honoraria; Amgen Inc.: Consultancy, Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

Bispecific Vγ9Vδ2-T and Type 1 NKT Cell Engager Lava-051 As First-in-Class Clinical Candidate to Target CD1d Expressing CLL, MM and AML

TAK1-inhibitors are cytotoxic for multiple myeloma cells alone and in combination with melphalan.

Multiple myeloma (MM) is an incurable cancer caused by malignant transformation of plasma cells. Transforming growth factor-β activated kinase 1 (MAP3K7, TAK1) is a major regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. Both NF-κB and MAPK control expression of genes with vital roles for drug resistance in MM. TAK1 is an attractive drug target as it switches these survival pathways to cell death. Our analysis showed that patients with high MAP3K7 expression in the tumor had shorter overall and progression free survival. The TAK1-inhibitors NG25 and 5Z-7-oxozeaenol (5Z-7) were cytotoxic to MM cell lines and patient cells. NG25 reduced expression of MYC and E2F controlled genes, involved in tumor cell growth, cell cycle progression and drug resilience. TAK1 can be activated by genotoxic stress. NG25 and 5Z-7 induced both synergistic and additive cytotoxicity in combination with the alkylating agent melphalan. Melphalan activated TAK1, NF-κB, and the MAPKs p38 and c-Jun N-terminal kinase (JNK), as well as a transcriptional UV-response. This was blocked by NG25, and instead apoptosis was activated. MM induce elevated bone-degradation resulting in myeloma bone disease (MBD), which is the main cause of disability and morbidity in MM patients. NG25 and 5Z-7 reduced differentiation and viability of human bone degrading osteoclasts, suggesting that TAK1-inhibition can have a double beneficial effect for patients. In sum, TAK1 is a promising drug target for MM treatment.

P-006: Expanded natural killer cells with daratumumab, lenalidomide and dexamethasone combination potentiates antimyeloma activity in a myeloma xenograft model

<h3>Background</h3> Advances in the treatment of multiple myeloma (MM) significantly improved overall survival of patients. Nonetheless, MM still remains an incurable disease. This emphasizes urgent need for novel combination therapies for the treatment of MM. Recently, natural killer (NK) cells are becoming an attractive and safe tool in the field of immunotherapy. <h3>Methods</h3> Here, we investigated how daratumumab, lenalidomide, and dexamethasone (DRd) treatment potentiate antitumor effects of NK cells in a MM xenograft mouse model. NK cells were expanded and activated using K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells of MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. Tumor bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNK), Rd, Rd + eNK, DRd, and DRd + eNK. DRd treatment strongly enhanced the cytotoxicity of eNK by upregulating NK cell's activation ligands and effector function. <h3>Results</h3> DRd combination with eNK significantly prolonged mouse survival and reduced MM progression and serum M-protein level. Moreover, DRd treatment significantly increased eNK persistence and homing into MM sites. <h3>Conclusions</h3> Our findings suggest that DRd treatment augments the anti-myeloma effects of ex vivo expanded and activated NK cells by modulating immune responses in MM-bearing mice.

P-003: Improving NK cell function in Multiple Myeloma with NKTR-255, a novel polymer-conjugated human IL-15

<h3>Background</h3> Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of NK cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal for new immunotherapeutic approaches, increasing tumor cell recognition, avoiding tumor escape and potentially enhancing the effect of other drugs. <h3>Methods</h3> Here we investigate the potential of NKTR-255, a novel polymer-conjugated human IL-15 to engage the IL-15 pathway and overcome the inhibitory status observed in NK cells from MM patients. For this purpose, we have analyzed ex vivo and in vivo effects of NKTR-255 on phenotypic features, effector functions and cytotoxicity of NK cells against MM cells. <h3>Results</h3> We observed that incubation with NKTR-255 was able to tilt the balance towards an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of MM patients, with increased expression of activating receptors (NKG2D, NKp46, NKp30, DNAM-1, CD69, TRAIL) on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. For a more accurate assessment of the effect of NKTR-255 on NK cell activity in an autologous setting in the presence of the bone marrow (BM) milieu, we cultured whole BM samples from non-treated newly-diagnosed MM patients with increasing doses of NKTR-255 for 5 days. NK cells experienced a dose-dependent induction of proliferation and activation (as shown by increased expression of CD69 and NKG2D), which translated in a reduced viability of CD138+ MM cells in the presence of NKTR-255. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared to placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we also tested in vitro and in vivo efficacy of a combination of NKTR-255 with daratumumab. We observed a more efficient antibody-dependent cellular cytotoxicity against MM cells in vitro and decreased tumor growth in vivo, where NKTR-255 rescued CD38+ NK cell levels from depletion by daratumumab. <h3>Conclusions</h3> Taken together, these results support the restoration and expansion of NK cell activity in MM with NKTR-255, providing rationale for its clinical use as a novel immunotherapeutic approach for MM patients alone or in combination with monoclonal antibodies or other immunomodulatory drugs.

P-081: A novel circulating miRNA involved in T cell senescence of multiple myeloma patients

<h3>Background</h3> Many circulating miRNAs were reported as biomarkers of multiple myeloma (MM),but the functions of these circulating miRNAs are not clarified recently. The aim of this work is to identify novel biomarkers of circulating miRNAs in diagnosis and prognosis for MM and reveal their function in the pathogenesis of MM. <h3>Methods</h3> MiRNAs profiling in serum(n=15) and MM cells(CD138+, n=10) were analyzed by RNA-sequencing of MM patients. The common dysregulated miRNAs(P<0.05) were identified via Veen Diagram analysis in serum and MM cells between MM patients and healthy donors, then validated by RT-PCR. ROC analysis and Kaplan-Meier(K-M) analysis were performed to evaluate the differently expressed circulating miRNAs in diagnosis and prognosis in MM patients, respectively. Exosomes of serum were isolated and characterized by dynamic light scattering, transmission microscopy, and western blot analysis. <h3>Results</h3> There were stable and specific miRNA expression profiles in serum and tumor cells of MM patients. The miRNA profiling of MM serum showed that there were 74 differential circulating miRNAs(P<0.05), among which 56 miRNAs(75.7%) were down-regulated and 18 miRNAs were up-regulated(24.3%). MiRNA profiling of MM cells showed that there were 376 miRNAs differentially expressed(P<0.05), of which 87(23.1%) were down-regulated and 289(76.9%) were up-regulated. Veen analysis found 36 common dysregulated miRNAs both in serum and tumor cells of MM. There were 22.3% of differential miRNAs located on chromosome 1 and 17. Five circulating miRNAs(miR-27b-3p, miR-145-3p, miR-628-3p, miR-342-5p and miR-30e-3p) were further confirmed differentially expressed in MM patients(n=201) by RT-PCR. K-M analysis showed that MM patients with low level of miR-27b, miR-145, and miR-628 had shortened PFS and OS. Strikingly, comparing to the decreased level of miR-27b in MM patients serum, miR-27b was enriched in the exosome of serum. MM patients with low level of serum miR-27b displayed lower CD3+T cells and CD3+CD4+T cells in peripheral blood. Our further study found increased proportation of senescent T cells in MM patients with low level of serum miR-27b, especially in CD8+T cells. In order to confirm T cell senescent phenotype, PBMCs were co-cultured with MM patients serum exosomes which mimic the in vivo situation. Flow cytometry analysis showed the CD28 expression was notably decreased on the CD3+ T cells in the co-culture group. In addition, RT-PCR and flow cytometry analysis showed that overexpression of miR-27b significantly down-regulated the level of CD28 in jurkat cells, a T cell line. These findings suggested an immune suppressive microenvironment in MM patients with lower level of serum miR-27b. <h3>Conclusion</h3> Circulating miRNA works as a usful biomarker in diagnosis and prognosis of MM. Strigently, serum miR-27b is a novel circulating miRNA involved in T cell senescence of multiple myeloma patients which indicated the poor outcome of patients with MM.

P-086: Quantitative seroproteomics analysis of Multiple Myeloma patients treated with Tagraxofusp, a novel CD123-directed targeted therapy, identifies novel cytokine-mediated mechanism of action

<h3>Background</h3> We previously described a tumor-promoting and immunosuppressive role of plasmacytoid dendritic cells (pDCs; CD123/IL-3R+) in multiple myeloma (MM) pathogenesis (Chauhan et al. Cancer Cell, 2009; Ray et al, Leukemia, 2018). Tagraxofusp, an FDA-approved (for patients with blastic plasmacytoid dendritic cell neoplasm [BPDCN]) novel targeted therapy directed against CD123, can trigger anti-MM activity by decreasing the viability of MM-promoting pDCs. These observations led to a recently completed phase 1/2 clinical trial of tagraxofusp and pomalidomide/dexamethasone in relapsed/refractory MM patients (NCT02661022). The treatment regimen demonstrated preliminary safety and efficacy, with 5 of 9 heavily pretreated patients achieving durable partial response (PR) (ASH 2019). Here, we report the early results of our translational correlative studies using bone marrow (BM), peripheral blood (PB), and serum from the study cohort. <h3>Methods</h3> Tagraxofusp is a bioengineered targeted therapy directed to CD123 developed by fusing human IL-3 to a truncated diphtheria toxin (DT) payload (Stemline Therapeutics, NY). pDCs and patient MM cells were purified from BM/PB samples after informed consent, and quantified using FACS, as described (Ray et al, Leukemia, 2018). A novel high throughput seroproteomics platform SOMAscan was used to analyze 1,310 protein analytes in serum samples from MM patients (n = 9). SOMAscan data were subjected to meta-analysis to generate heatmaps, followed by hierarchical cluster analysis. SOMAscan results were validated with ELISA using supernatants from MM patient pDCs cultured with or without tagraxofusp. <h3>Results</h3> Analysis of BM/PB samples from MM patients receiving tagraxofusp therapy showed a distinct reduction in the frequency of viable pDCs [average 2% at screening vs 0.75% post-tagraxofusp; n=6; p=0.036]. Of note, pDCs isolated from tagraxofusp-treated patients showed decreased ability to trigger MM cell growth. SOMAscan analysis of patient serum before and after tagraxofusp therapy showed alterations in the levels of 100 proteins [Median Fold Change in expression: 0.39 to 4.5; n=6; 3 each; p < 0.05]. Importantly, tagraxofusp treatment reduced pDC-related soluble proteins including IFN-γ fold change: 0.8, treated vs untreated; p < 0.05). Our earlier study showed that pDC-MM interactions triggered secretion of IL-3, which in turn promotes both pDC survival and MM cell growth. Importantly, tagraxofusp decreased serum IL-3 fold change 0.75, treated vs untreated; p < 0.05), consistent with tagraxofusp attenuating the survival of tumor-promoting pDCs. <h3>Conclusions</h3> Our correlative science studies validate the target specificity of tagraxofusp against MM pDCs in relapsed and refractory MM patients enrolled in a phase 1/2 clinical trial. Our study favors further evaluation for this novel therapeutic to improve the clinical outcome of patients with MM. Further combination studies are planned.

OAB-021: BCMA-specific ADC MEDI2228 and Daratumumab induce synergistic myeloma cytotoxicity via enhanced IFN-driven innate immune responses and expression of CD38 and NKG2D ligands

<h3>Background</h3> Efforts are required to improve potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma (MM). <h3>Methods</h3> We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate MEDI2228 which may augment efficacy of these immunotherapies. RNA sequencing followed by gene set enrichment analysis showed that MEDI2228 significantly enriched IFN I-signaling and induced type I interferon (IFN I)-stimulated genes (ISGs) in MM cell lines. The most MEDI2228-enhanced IFN-driven genes include chemokines/cytokines and receptors (i.e., CXCL9/10, CCL4L1/2, CCL22, CCL1/3/4/5, CCL3L1/3, TNFSF9/10, CSF2 (GM-CSF), CCR7), RSAD2, CASP1, ISGs (i.e., XAF1, TRIMs, IFITs, ISG15, GBP2/3, OAS1/2/L, RNASEL, MX1/2, FAS), RUNX3, GZMB, IKBKE, IRF1/6/7/9, STAT1/2/4/6, MB21D1(CGAS), TMEM173 (STING), IFIT1/2/3/5, and SOCS1/2/3. <h3>Results</h3> Regardless of genetic heterogeneity and resistance to current anti-MM therapies, MEDI2228 induced dose- and time-dependent DNA damage-ATM/ATR-CHK1/2 pathways, activation of cGAS-STING-TBK1-IRF3 and STAT1-IRF1-signaling cascades, as well as increased CD38 expression. It overcame CD38 downregulation triggered by IL6 and bone marrow stromal cell culture supernatant (BMSC-sup), via activation of STAT1-IRF1 without phosphorylation of STAT3 in immunomodulatory drugs (IMiDs)- and bortezomib-resistant MM cell lines. In contrast, MEDI2228 did not change CD38 expression and survival in BCMA-negative NK effector cells. Significantly, MEDI2228 with anti-CD38 monoclonal antibody Daratumumab (Dara) synergistically induced NK-mediated lysis of MM cell lines and autologous resistant patient MM cells, even in the presence of BMSC-sup and BMSCs. In parallel, MEDI2228 increased membrane expression of NKG2D ligands (NKG2DLs), i.e., MICA/B and ULBPs, in all MM cells tested, including Dara-resistant patient cells, correlating with enhanced MM cell susceptibility to NK cell killing. Since MEDI2228, but not its MMAF-ADC homolog M3 even used at >1-log higher concentrations enhanced surface expression of CD38 and NKG2DLs on MM cells, the potent DDR-mediated immunomodulation triggered by MEDI2228 vs M3 is critical in rendering MM cells more susceptible to Dara-induced NK cell killing. Importantly, M2 still activated STAT1/IRF1 signaling to induce CD38 and MICA/B expression in MM tumors grown in mice. All MM1S tumor-bearing NSG mice reconsituted with human NK cells became tumor-free following a single low dose M2 with Dara treatment, with 100% host survival. <h3>Conclusions</h3> Taken together, our data showed that MEDI2228 restored MM sensitivity to CD38 targeting by Dara without depleting NK cells and potentiated immunogenic cell death of MM cells. These results therefore provide the mechanistic rationale for clinical evaluation of combination CD38- and BCMA-directed immunotherapies to further improve patient outcome in MM.

P-225: A first-in-human study of FOR46 in patients with triple refractory Multiple Myeloma

<h3>Background</h3> Cluster of differentiation 46 (CD46) is highly expressed in multiple myeloma, especially in patients (pts) with gain of chromosome 1q and in the refractory setting. In vitro studies show upregulation and enhanced cytotoxicity upon treatment with pomalidomide, dexamethasone or lenalidomide. FOR46, an anti-CD46 antibody-drug conjugate with vc-MMAE, recognizes a tumor selective epitope of CD46 and is taken up by malignant cells by macropinocytosis, which is a relatively tumor selective uptake mechanism. <h3>Methods</h3> Pts with MM were enrolled who had progressed on proteasome inhibitors, immunomodulatory drugs and CD38-targeted therapies. An accelerated titration followed by 3+3 dose escalation design was used. FOR46, at protocol specified doses, was infused intravenously over 30-60 minutes on Day 1 of 21-day cycles. Following excess toxicity (neutropenia and fatigue) in a patient (pt) with high body mass index (BMI), dosing was changed from actual weight (AW) to adjusted body weight (ABW). G-CSF secondary prophylaxis was required for pts experiencing grade (gr) ≥ 3 neutropenia during a previous treatment cycle. The initial protocol had 2.4 mg/kg AW as the highest dose. When the MTD was not defined using ABW dosing, escalation was held pending protocol amendment to allow a higher dose and expansion to 10 patients at 2.4 mg/kg ABW began. Safety was evaluated using CTCAE v5.0 and efficacy was evaluated per IMWG criteria. Dexamethasone was only allowed for infusion reaction prophylaxis. CD46 antigen density was determined on patient MM cells via flow cytometry. <h3>Results</h3> Fifteen pts were enrolled at 6 pre-defined dose levels from 0.1 to 2.4 mg/kg with 1 patient each at the 0.1, 0.3 and 0.6 mg/kg dose levels, 3 at 1.2 and 1.8 mg/kg and 6 at 2.4 mg/kg. Median age was 68 (range 33 – 79) with 4 females. Gain 1q was present in 9 pts, absent in 5 pts and unknown in 1. The median number of prior lines of therapy was 6 (range 3-17). The only dose-limiting toxicity (DLT) was gr 4 neutropenia in 1 high BMI patient dosed by AW. This was the only DLT among 6 pts at 2.4 mg/kg dosed by a mix of AW (n=3) and ABW (n=3). One of 3 at 2.4 mg/kg ABW had non-dose limiting gr 4 neutropenia. The most common related adverse event was gr 4 neutropenia in 3 pts (20%). One patient (6.7%) had gr 4 thrombocytopenia and 1 each (6.7%) had gr 3 AST elevation, neutropenia, anemia, nausea, and peripheral neuropathy (PN). Of the 6 response-evaluable pts in the 1.8 and 2.4 mg/kg cohorts, 3 had partial responses (PRs) lasting 21, 30, and 15 weeks, respectively. Of the PRs, 1 pt did not have gain of 1q21. In addition to the fifteen pts in dose escalation cohorts, 10 have been enrolled in an expansion cohort at 2.4 mg/kg by ABW. <h3>Conclusion</h3> FOR46 demonstrates an acceptable toxicity profile using ABW dosing. There is encouraging evidence of efficacy in triple refractory MM. A protocol amendment allowing additional dose escalation to 2.7 mg/kg by ABW has been approved. NCT03650491

P-093: Ubiquitin receptor PSMD4/Rpn10 as therapeutic target in Multiple Myeloma

<h3>Background</h3> Our prior studies showed that targeting Ubiquitin Receptor (UbR) Rpn13 upstream of the 20S proteasome signaling cascade overcomes proteasome-inhibitor (PI)-resistance (Yan et al. Leukemia 2016; Yan et al, 2021 BCJ–please complete reference details). Besides Rpn13, 19S-associated UbR PSMD4/Rpn10 also plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. In the present study, we show that inhibition of PSMD4 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. <h3>Methods</h3> Cell viability, and apoptosis assays were performed using WST/ CellTiter-Glo assay, and Annexin V staining, respectively. PSMD4 knockout 293 cell line was generated using CRISPR/Cas9. Doxcycycline (Dox)-inducible PSMD4-knockdown (KD) MM cell line was generated using short hairpin RNA (shRNA). A xenograft human MM model was used to characterize the role of PSMD4 on tumor progression. Statistical significance was assessed with Student's t test. <h3>Results</h3> 1) MM patient gene expression profiling database showed that PSMD4 expression inversely correlates with overall survival (n=175; p=0.00064). 2) RT-PCR, immunoblotting, and immunohistochemistry of MM patient BM showed higher PSMD4 levels in patient MM cells and MM cell lines versus normal cells. 3) Transient transfection of MM cells including, bortezomib-resistant MM cells with PSMD4-siRNA decreased their viability; conversely, transfection with PSMD4-WT rescued cells from growth-inhibitory activity of PSMD4-siRNA. Western blotting confirmed knockdown of PSMD4 by PSMD4-siRNA versus scrambled (scr)-siRNA, and restoration of PSMD4 levels in cells transfected with PSMD4-WT versus PSMD4-siRNA. 4) CRISPR/Cas9-PSMD4-KO showed reduced cell growth. 5) Both PSMD4-KO and PSMD4-KD cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation; 6) PSMD4 blockade triggered apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling in MM cells; and finally, 7) Transplantation of Dox-inducible PSMD4-KD MM.1S cells in mice correlated with significantly reduced tumor growth. <h3>Conclusion</h3> Our preclinical data validates targeting UbR PSMD4/Rpn10 to enhance cytotoxicity and overcome PI resistance in MM, as well as provides the basis for the development and clinical evaluation of PSMD4 inhibitors to improve patient outcome.

P-082: Delineating CDK9- regulated molecular events for the development of rationally derived multiple myeloma treatment strategies

<h3>Background</h3> Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome. <h3>Methods</h3> Following expression profiling, CRISPR loss-of-function screen and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses. <h3>Results</h3> Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, smoldering MM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines confirmed their growth dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, but not other genes (e.g. Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 induced a reduction of mRNA/ protein levels of Mcl-1, cMyc, RNA Pol II, and IRF4 but not of other potential targets (e.g. Bcl-2) in a dose- and time-dependent manner. Moreover, Thal-sns-032 reduced Mdm2 and thereby increased p53 protein levels. Consequently, Thal-sns-032 inhibited tumor cell proliferation and survival both in tumor cell- and tumor cell:BMSC co-cultures. Rationally derived combination strategies of Thal-sns-032 for example with venetoclax, but also other investigational and established MM therapies induced synergistic anti-MM effects within the tumor microenvironment. <h3>Conclusion</h3> In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derived MM combination treatment strategies.

P-098: Super-enhancer-driven PPP1R15B as an oncogenic and potential therapeutic target in Multiple Myeloma

<h3>Background</h3> Growing evidence suggests that alterations in epigenetic landscape contribute to pathogenesis of multiple myeloma (MM). MM cells are highly dependent on unfolded protein response signaling pathways due to high level of endoplasmic reticulum stress. Phosphorylation of eIF2a can attenuate protein translation. The PPP1R15B(denoted as R15B hereafter) gene encodes a regulatory subunit of eIF2a-specific phosphatase complex. In this study,we identified super enhancer (SE)-driven oncogenes specific in MM with a particular focus on a candidate gene R15B, whose functional roles in MM remain largely elusive. <h3>Methods</h3> We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples, normal CD138+ plasma cells and memory B cells. ROSE analysis was used to annotate SEs and their associated genes. A combination of public data mining, RNAi, overexpression and CRISPR/Cas9 technologies were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line H929 with R15B KD and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on HiChIP. <h3>Results</h3> We have identified R15B as one of the SE-associated genes specific to MM patient samples and cell lines. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. R15B KD or KO significantly reduced cell viability, clonogenicity and induced G2/M arrest. ChIP-qPCR assays showed that C/EBP-β is strongly enriched at R15B SE region. We also found that salubrinal, a selective inhibitor of eIF2a phosphorylation, inhibited MM cell proliferation in a dose-dependent manner. <h3>Conclusions</h3> Our integrative approaches identified R15B as a novel SE-driven oncogene. We propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation.