Neoantigen peptides arising from genetic alterations may serve as targets for personalized cancer vaccines and as positive predictors of response to immune checkpoint therapy. Mutations in genes regulating RNA splicing are common in hematological malignancies leading to dysregulated splicing and intron retention (IR). In this study, we investigated IR as a potential source of tumor neoantigens in multiple myeloma (MM) patients and the relationship of IR-induced neoantigens (IR-neoAg) with clinical outcomes. MM-specific IR events were identified in RNA-sequencing data from the Multiple Myeloma Research Foundation CoMMpass study after removing IR events that also occurred in normal plasma cells. We quantified the IR-neoAg load by assessing IR-induced novel peptides that were predicted to bind to major histocompatibility complex (MHC) molecules. We found that high IR-neoAg load was associated with poor overall survival in both newly diagnosed and relapsed MM patients. Further analyses revealed that poor outcome in MM patients with high IR-neoAg load was associated with high expression levels of T-cell co-inhibitory molecules and elevated interferon signaling activity. We also found that MM cells exhibiting high IR levels had lower MHC-II protein abundance and treatment of MM cells with a spliceosome inhibitor resulted in increased MHC-I protein abundance. Our findings suggest that IR-neoAg may represent a novel biomarker of MM patient clinical outcome and further that targeting RNA splicing may serve as a potential therapeutic strategy to prevent MM immune escape and promote response to checkpoint blockade.

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Multiple myeloma (MM) is an incurable malignancy of mature plasma cells. Despite major advances in the therapeutic armamentarium of MM, only 50% of patients survive more than 5 years after diagnosis, with significantly lower rates (21%) for high-risk patients. Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA (B-cell maturation antigen) shows high response rates in relapsed/refractory patients. However, most patients have disease remission that lasts less than 18 months, prompting the search for additional and synergistic therapeutic approaches. We unbiasedly mapped the cell surface proteome of MM by integrating Mass-Spectrometry (MS) and RNA-seq analyses from 7 MM cell lines and 904 primary MM patient samples bearing high-risk cytogenetics. To identify cell surface proteins, we ran a pool of 4,761 proteins and 16,000+ transcripts through five repositories. An integrated scoring database was developed by scoring each ID based on the number of databases (0-5) it was identified in, with 0 if the molecule was not found in any and 5 if the protein was found in all five. We identified 402 proteins with a surface score of 3 or higher in MM cell lines and patient samples by transcriptomics and proteomics. We prioritized the 326 candidates that were more highly expressed in patients. Based on functional enrichment analyses, we found the proteins formed three main networks with immune mechanisms representing the largest cluster (227 out of 326 cell surface proteins) followed by transporters and adhesion proteins.Based on a pipeline we previously established (1), we further selected 97 candidates minimally expressed in normal tissues. This list included current therapeutic targets such as BCMA, SLAMF7, ITGB7 and LY9. Validation in primary patient samples by western blot and flow-cytometric analyses, enabled the identification of 10 top candidates (CCR1, CD320, FCRL3, IL12RB1, ITGA4, LAX1, LILRB4, LRRC8D, SEMA4A, SLAMF6) that resulted most frequently and highly expressed. We found that LAX1, LILRB4 and SEMA4A significantly impact myeloma patient overall survival based on Kaplan-Meier analysis in the MM Research Foundation (MMRF) cohort (2). CCR1, IL12RB1, LILRB4 and SEMA4A were upregulated by the treatment with Bortezomib or Venetoclax that conversely, decreased BCMA expression in MM U266 cells. By stratifying the patient population, we found that the SEMA4A and LAX1 were up-regulated in patients with t(4;14) compared to patients with no cytogenetic abnormality; LILRB4 in patients with t(14;16) and CCR1 patients with t(14;16) and t(14;20). By calculating co-expression levels CCR1-LILRB4 and CCR1-FCRL3 resulted co-expressed in 100% of patients. For safety purposes (3), we excluded candidates with high (>55%) protein abundance in highly-purified normal hematopoietic stem cells and activated T-cells, narrowing down the list to 6 top candidates (CCR1, FCRL3, IL12RB1, LILRB4, LRRC8D, SEMA4A). To define the function of this group of promising cell surface targets, we used a CRISPR/Cas9 inducible system in KMS11 MM cells. We found that knock-out of CCR1, LRRC8D and SEMA4A individually reduces the MM cell growth by ~60%, 50% and 50% respectively, and almost completely abrogates MM cell migration through porous chambers by >80%. By co-culturing irradiated KO and control MM cells with healthy donor T-cells we also found that lack of CCR1 increased T-cell proliferation by 50% compared to controls and enhanced killing of MM cells, suggesting that CCR1 may suppress T-cell mediated immune responses in addition to play a role in MM cell survival and migration. This study suggests the contribution of an altered MM surfaceome to disease development and may lead to potential novel immunotherapeutic approaches for high-risk MM. References 1. Perna F et al., Cancer Cell 2017 3. Dong C et al., in press Oncogene 2021 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Multiple myeloma (MM) is a devastating plasma cell malignancy characterized by the expansion of aberrant monoclonal plasma cells in the bone marrow, leading to severe clinical manifestations and poor prognosis, particularly in relapsed/refractory cases. Identifying novel therapeutic targets is crucial to improve treatment outcomes in these patients. In this study, we investigated the role of the protein arginine methyltransferase 1 (PRMT1) in MM pathogenesis and explored its potential as a therapeutic target. We observed that PRMT1, responsible for most asymmetric di-methylation in cells, exhibited the highest expression among PRMT family members in MM cell lines and primary MM cells. Importantly, PRMT1 expression was significantly elevated in relapsed/refractory patients compared to newly diagnosed patients. High expression of PRMT1 expression was strongly associated with poor prognosis. We found that genetic or enzymatic inhibition of PRMT1 impaired MM cell growth, induced cell cycle arrest, and triggered cell death. Treatment with MS023, a potent PRMT type I inhibitor, demonstrated a robust inhibitory effect on the viability of primary cells isolated from newly diagnosed and proteasome inhibitor-relapsed/refractory patients in a dose-dependent manner. Suppression of PRMT1 downregulated genes related to cell division and upregulated genes associated with apoptosis pathway. We also found that genes related to immune response and lymphocyte activation were significantly upregulated in PRMT1-suppressed cells. Notably, the activation status of T cells was strikingly enhanced upon co-culturing with PRMT1-KO MM cells. In vivo studies using a xenograft model revealed that targeting PRMT1 by either CRISPR/Cas9-mediated knockout or MS023 treatment significantly attenuated MM tumor growth and prolonged the survival of tumor-bearing mice. Histological analysis further confirmed increased apoptotic cell death in MS023-treated tumors. Collectively, our findings establish PRMT1 as an indispensable and novel therapeutic vulnerability in MM. The elevated expression of PRMT1 in relapsed/refractory patients underscores its potential as a target for overcoming treatment resistance. Moreover, our results highlight the efficacy of MS023 as a promising therapeutic agent against MM, offering new avenues for therapeutic approaches in relapsed/refractory MM.

Loss of function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of AML patients with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early pre-leukemic event, which when combined with other genetic lesions result in full blown leukemia. Here, we show that loss of Dnmt3a in HSC/Ps results in myeloproliferation, which is associated with hyperactivation of the PI3Kinase pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-seq analysis on drug treated Dnmt3a -/-HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment and extracellular matrix compared to controls. Remarkably, drug treated leukemic mice showed a reversal in the enhanced fetal liver HSC like gene signature observed in vehicle treated Dnmt3a -/-LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a new target for treating DNMT3A mutation driven myeloid malignancies.

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