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Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Waldenstrom macroglobulinemia (WM) and its precursor IgM gammopathy are distinct disorders characterized by clonal mature IgM-expressing B-cell outgrowth in the bone marrow. Here, we show by high-dimensional single-cell immunogenomic profiling of patient samples that these disorders originate in the setting of global B-cell compartment alterations, characterized by expansion of genomically aberrant extrafollicular B cells of the nonmalignant clonotype. Alterations in the immune microenvironment preceding malignant clonal expansion include myeloid inflammation and naïve B- and T-cell depletion. Host response to these early lesions involves clone-specific T-cell immunity that may include MYD88 mutation–specific responses. Hematopoietic progenitors carry the oncogenic MYD88 mutations characteristic of the malignant WM clone. These data support a model for WM pathogenesis wherein oncogenic alterations and signaling in progenitors, myeloid inflammation, and global alterations in extrafollicular B cells create the milieu promoting extranodal pattern of growth in differentiated malignant cells.Significance:These data provide evidence that growth of the malignant clone in WM is preceded by expansion of extrafollicular B cells, myeloid inflammation, and immune dysfunction in the preneoplastic phase. These changes may be related in part to MYD88 oncogenic signaling in pre–B progenitor cells and suggest a novel model for WM pathogenesis.This article is highlighted in the In This Issue feature, p. 549

supplementary figs 1-25 and supplementary tables 1-3

Supplementary Data from PDZ Proteins SCRIB and DLG1 Regulate Myeloma Cell Surface CD86 Expression, Growth, and Survival

Purpose:Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome.Experimental Design:CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for 1–6 months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA sequencing (RNA-seq) and assay for transpose accessible chromatin sequencing (ATAC-seq). Chromatin accessible regions were used to identify regulatory RNA expression in more than 700 samples from newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass trial (NCT01454297).Results:Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. More than 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with immunoglobulin heavy chain translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma.Conclusions:These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.

Enrichment of transcription factor consensus binding motifs in regulatory elements predictive of gene expression associated with poor overall survival.

Correlation of regulatory element transcription and gene expression in CoMMpass specimens.

ATAC-seq data for Emory patients.

Correlation of chromatin accessibility and gene expression for the 5% most variably expressed genes.

Regulatory elements predictive of gene expression for genes prognostic of overall survival in CoMMpass.

Regulatory element transcription in newly diagnosed myelomas from CoMMpass.

Gene set enrichment analysis of transcribed regulatory elements in CoMMpass.

Upregulation of NADPH oxidases (NOXs) in cancer cells leads to chronic increase in intracellular reactive oxygen species (ROS) and adaptation to a high ROS level for cell survival and, thereby, low sensitivity to radiotherapy. To overcome resistance to radiotherapy, we have developed a bioactive and CD44 targeted hyaluronic acid nanoparticle encapsulated with an NOX inhibitor, GKT831 (HANP/GKT831). We found that HANP/GKT831 had stronger inhibitory effects on ROS generation and cell proliferation than that of GKT831 alone in cancer cells. Systemic delivery of HANP/GKT831 led to the targeted accumulation in breast cancer patient derived xenograft (PDX) tumors in nude mice. Importantly, the combination of systemic delivery of HANP/GKT831 with a low dose of local radiotherapy significantly enhanced tumor growth inhibition in breast cancer PDX models. Our results showed that HANP/GKT831 primed tumor cells to radiation-induced DNA damage and cell death by downregulation of DNA repair function and oncogenic signal pathways.

The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response–dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11 , and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress–induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.

Background/Objectives: Despite intensive multimodal therapy, greater than 50% of children with high-risk neuroblastoma (HR NB) relapse with incurable disease. Next generation sequencing of primary HR NB tumors identified an increase in activating mutations in the RAS/RAF/MAPK pathway. Moreover, gene set enrichment analyses showed a significant decrease in expression of genes suppressed by the Yes-Associated Protein (YAP) at relapse, suggesting increased YAP transcriptional repression. YAP binds with TEAD family transcription factors to regulate gene expression. We have shown that YAP promotes chemotherapy and MEK inhibitor resistance in RAS-mutated NB tumors in vivo by suppressing the expression of Harakiri (HRK), a BH3-only pro-apoptotic protein activated in response to tumor environmental stress such as serum starvation. Our overall objective is to elucidate how YAP represses HRK and tumor suppressor genes globally, and to enhance MEK inhibitor potency by combining MEK inhibition with agents that inhibit YAP or induce HRK to restore the tumor environmental stress response and apoptosis in RAS-mutated NB. Design/Methods: We used publicly available databases to identify TEAD binding sites on the HRK gene locus in NB. To assess the global state of methylation, we treated NB cells, SK-N-AS (NRASQ61K mutation, MYCN non-amplified) and NLF (NF1 deletion, MYCN amplified), with demethylating agent azacitidine and evaluated HRK expression. To identify the specific TEAD (1-4) binding partner to YAP, we performed siRNA and co-immunoprecipitation studies. We further tested novel YAP-TEAD small molecule inhibitors with varying TEAD1-4 inhibition specificity in SK-N-AS and NLF cells in vitro. Results: We observed that TEAD binds near cis-regulatory regions on the HRK gene locus in NB. We found that HRK expression is restored when SK-N-AS and NLF cells are treated with azacitidine despite YAP expression increasing. We also identified TEAD2 as the specific binding partner to YAP in NB and found that TEAD2 is necessary for HRK regulation. Novel YAP-TEAD small molecule inhibitors affect NB cell viability under serum-deprived conditions in vitro, especially the inhibitor with highest specificity against TEAD2, and affect YAP-TEAD downstream targets. Conclusions: YAP-TEAD2 binding is essential for HRK regulation in RAS-mutated NB and thus is a logical therapeutic target to restore therapy response. Further studies are ongoing to test YAP-TEAD small molecule inhibitors in combination with MEK inhibitors and in vivo. Citation Format: Jenny Shim, Andrew Ho, Hunter C. Jonus, Adeiye A. Pilgrim, Benjamin G. Barwick, Tracy T. Tang, Lawrence H. Boise, Kelly C. Goldsmith. YAP-TEAD2 binding mediates therapy resistance in RAS-driven neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3552.

The molecular mechanisms underlying chemoresistance in some newly diagnosed multiple myeloma (MM) patients receiving standard therapies (lenalidomide, bortezomib, and dexamethasone) are poorly understood. Identifying clinically relevant gene networks associated with death due to MM may uncover novel mechanisms, drug targets, and prognostic biomarkers to improve the treatment of the disease. This study used data from the MMRF CoMMpass RNA-seq dataset (N = 270) for weighted gene co-expression network analysis (WGCNA), which identified 21 modules of co-expressed genes. Genes differentially expressed in patients with poor outcomes were assessed using two independent sample t-tests (dead and alive MM patients). The clinical performance of biomarker candidates was evaluated using overall survival via a log-rank Kaplan–Meier and ROC test. Four distinct modules (M10, M13, M15, and M20) were significantly correlated with MM vital status and differentially expressed between the dead (poor outcomes) and the alive MM patients within two years. The biological functions of modules positively correlated with death (M10, M13, and M20) were G-protein coupled receptor protein, cell–cell adhesion, cell cycle regulation genes, and cellular membrane fusion genes. In contrast, a negatively correlated module to MM mortality (M15) was the regulation of B-cell activation and lymphocyte differentiation. MM biomarkers CTAG2, MAGEA6, CCND2, NEK2, and E2F2 were co-expressed in positively correlated modules to MM vital status, which was associated with MM’s lower overall survival.

Improvements in tumor immunotherapies depend on better understanding of the anti-tumor T cell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8(+) T cells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1(+) stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8(+) T cells. Murine tumor models revealed that tumor-specific CD8(+) T cells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8(+) T cell activation in response to cancer is different from that of canonical CD8(+) T cell activation to acute viruses, and it proposes two stages of tumor-specific CD8(+) T cell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.

PTEN (phosphatase and tensin homolog) is a known tumor suppressor gene (TSG) in solid tumors and hematologic malignancies, including multiple myeloma (MM), yet several aspects of its biology and clinical implications remain incompletely understood in MM. For instance, PTEN mutations/deletions are infrequent in newly diagnosed MM in the CoMMpass study (6 and 9 cases with heterozygous mutations or loss of one PTEN gene copy, respectively in 957 patients, IA16; no evidence of bi-allelic loss), and there is no enrichment for PTEN transcript suppression or biallelic PTEN loss at relapse. Moreover, while MM cell lines are considered to reflect advanced relapsed/refractory MM, nearly all are PTEN-proficient (with notable exceptions e.g. biallelic loss in OPM2). These considerations raise a question whether PTEN loss is indeed functionally associated with a competitive advantage for MM cells compared to their PTEN-proficient counterparts. In genome-scale CRISPR-Cas9 gene editing (knockout, KO) screens we observed that 16/19 MM cell lines exhibited CERES or CHRONOS scores >0.4, consistent with the proposed role of PTEN as a negative regulator of survival and proliferation: the 3 outlier cell lines included OPM2 (biallelic PTEN deletion) and 2 other lines (SKMM2 and AMO1) with no obvious genetic events suppressing PTEN function or causing constitutive activation of PI3K/Akt/mTOR signaling. We performed subgenome-scale CRISPR KO screens with focused libraries of single guide RNAs (sgRNAs) for several known (including PTEN) or candidate TSGs and control sgRNAs from MM and other tumors in vitro and confirmed that in vitro enrichment of PTEN KO in several PTEN-proficient (but not -deficient lines), consistent with the genome-scale CRISPR study results. These observations were further confirmed with in vitro competition assays where isogenic Cas9+ cells transduced with sgRNAs against PTEN or olfactory receptor (OR) genes (as DNA-cutting negative controls) were mixed, cultured for 4-12 weeks and DNA sequenced for the distribution of their sgRNAs, and determined a significant relative enrichment of PTEN KOs compared to their OR KO counterparts. Importantly, when cells harboring focused sub-genome scale sgRNA library were implanted in vivo in a bicalcium phosphate scaffold-based bone marrow (BM)-like model with a "humanized" mesenchymal bone stromal cell compartment, we observed that CRISPR KO of PTEN represented the top enriched gene perturbation in the PTEN-proficient MM cell lines MM.1S and KMS11 (not the AMO1 line) in vivo, consistent with the in vitro results. Collectively, these observations indicate that while PTEN deficiency is infrequent in newly diagnosed MM patient samples and in preclinical cell line models that reflect advanced disease, PTEN KO is associated with major "fitness" advantage for MM cells in preclinical in vitro and in vivo xenograft models, i.e. systems in which tumor cell "fitness" does not account for potential immunogenicity of human tumor cells. We hypothesized that PTEN KO remain highly immunogenic which curtails their "fitness" advantage: indeed, we observed in a series of CRISPR KO screens in PTEN-proficient cell lines from MM, other hematologic malignancies or solid tumors, PTEN loss was not associated with increased resistance to allogeneic donor-derived NK cells (in-house studies) or T-cell treatment (e.g. 41T, RENCA cells; GSE149933). In fact, for some MM cell lines (e.g. MM1S, KMS11), PTEN KO led to more pronounced sensitivity to NK cells (compared to control OR KOs) in genome-scale or focused CRISPR screens, and in validation experiments with individual sgRNAs for PTEN (vs. OR gene sgRNAs). While our functional genomic studies document that PTEN loss confers a generalizable "fitness advantage" for MM lines in vitro and in immunocompromised in vivo models, the limited, if any, association of advanced MM with PTEN loss raises intriguing questions about the potentially complex role of PTEN as a TSG. While alternative hypotheses are plausible, our results point to the immune responses of PTEN-deficient tumor cells as an additional factor that can reconcile our integrated observations in molecular annotated clinical samples and preclinical functional genomic studies. Disclosures Tsherniak: Cedilla Therapeutics: Consultancy; Foghorn Therapeutics: Consultancy; The Center for Protein Degradation: Consultancy; SAB: Membership on an entity's Board of Directors or advisory committees. Licht: Epizyme: Research Funding. Boise: Abbvie: Consultancy; AstraZeneca: Consultancy, Research Funding. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; EMD Serono: Research Funding; Abbvie: Research Funding; Nurix: Research Funding; Janssen/Johnson & Johnson: Research Funding; BMS: Research Funding; Ionis Pharmaceuticals: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; TEVA: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding.

Multiple myeloma (MM) is an incurable form of plasma cell cancer in which primary and secondary chromosomal translocations routinely juxtapose oncogenes to plasma cell-specific super-enhancers. Coincidentally, drugs which target super-enhancers have had success clinically. For example, immunomodulatory imide drugs (IMiDs) degrade super-enhancer-binding pioneer factors IKAROS and AIOLOS, while glucocorticoids (Dexamethasone) and proteasome inhibitors (Bortezomib) have the ability to transrepress or block the processing of super-enhancer-forming NF-κB proteins, respectively. Currently, alternative enhancer-targeting drugs are also in clinical development, like p300 inhibitors which target the acetyl-binding bromodomains and/or histone acetyl transferase activity of the chromatin-regulating coactivator homologs CBP and EP300. Despite showing therapeutic promise, our understanding of how these drugs function, alone or together, remains incomplete. Case in point, we find that IMiD-induced degradation of its target proteins IKAROS and AIOLOS does not guarantee a therapeutic response in vitro, and patients successfully treated with IMiDs eventually relapse; meanwhile, coactivator-targeting therapies like p300 inhibitors are often too toxic in vivo, and lack a therapeutic window. To improve the outcomes of MM patients we need to understand the heterogeneous genetics and transcription-factor milieus of the myeloma enhancer landscape, as well as how to increase the precision of enhancer-disrupting drugs. To accomplish this, our lab utilizes more than 60 human myeloma cell lines that have been extensively characterized at the genetic, proteomic, and drug-therapeutic-response levels. Additionally, we have generated a highly-predictive immunocompetent mouse model (Vk*MYC hCRBN+) that develops human-like MM and is sensitive to both IMiDs and a new class of therapeutics termed "degronimids" (normal mice do not respond to IMiDs or degronimids). Our central hypothesis is that combining a broad coactivator-targeting drug (e.g., the p300 inhibitor GNE-781), with a MM-specific transcription factor-targeting drug (e.g., IMiDs) restricts toxicities to myeloma cells and thus improves the therapeutic window. Currently, we are testing a variety of coactivator-targeting compounds alongside traditional IMiD therapies and other preclinical transcription factor-targeting drugs both in vivo and in vitro. We show that Vk*MYC hCRBN+ mice are exquisitely sensitive to GNE-781, requiring one fourth of the dose needed to treat other cancers and therefore avoiding the neutropenia and thrombocytopenia seen at higher doses. Second, we show that although IMiDs and GNE-781 induce an effective but transient response in vivo as single agents, the combination of the two drugs proved curative, with a progressive deepening of the anti-tumor response occurring even after therapy is discontinued. Ongoing experiments aim to determine how this drug combination, and other coactivator + transcription factor-targeting combinations, permanently disrupt myeloma-specific super-enhancers. Disclosures Neri: BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy. Boise: AstraZeneca: Honoraria, Research Funding; AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees. Chesi: Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma.

INTRODUCTION Proteasome inhibitors (PI) such as bortezomib (Velcade), carfilzomib (Kyprolis) and ixazomib (Ninlaro)) have been shown to be efficacious in multiple myeloma (MM) therapy. However, despite being an effective first line therapy, resistance to PI usually develops, leading to relapse and refractory disease. Previous studies have implicated a role of OXPHOS, glycolysis, antioxidants and serine metabolism in PI resistance. Mitochondrial metabolism plays a central role in malignant progression not only by generating ATP but also by providing precursors for synthesis of several biomolecules such as proteins, nucleotides, fatty acids and antioxidants that can influence the efficacy of MM therapies. We previously determined reduced mitochondrial electron transport chain (ETC) activity promotes sensitivity to the BCL-2 antagonist, venetoclax. However, the relationship between the metabolic state of the mitochondria and proteasome inhibitor (PI) sensitivity is not fully understood. Unexpectedly, we found ETC inhibition or reduced ETC activity to promote resistance to PIs. Here, we investigate the mechanistic basis for divergent effects of mitochondrial stress on sensitivity to PIs and venetoclax. METHODS We have used RNA-Seq and carbon isotope tracing using labeled U 13C-glucose or U 13C-glutamine, flow cytometry and western blot analysis in MM cell lines treated with mitochondrial Complex I inhibitor, IACS-010759 (IACS) +/- Bortezomib; and immunostaining of MM patient samples and interrogation of Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 15). RESULTS We find that mitochondrial ETC (complex I-V) inhibition antagonizes bortezomib (BTZ) and carfilzomib (CFZ) induced cell death in MM in contrast to promoting sensitivity to venetoclax. Additionally, cell lines exhibiting intrinsically reduced ETC activity were more resistant to PI in comparison to cell lines with higher ETC activity. Evaluation of CoMMpass MM trial (NCT0145429, IA15) and serial samples from 50 patients before PI treatment and after relapse, show pathways related to OXPHOS and TCA cycle to be downregulated in poor survival patients, corroborating our in vitro observations on reduced ETC activity promoting resistance to PI. To elucidate the mechanistic basis of ETC-inhibition induced PI resistance we performed RNA-Seq and U 13C-glucose and glutamine tracing in L363 cells treated with the ETC Complex I inhibitor IACS +/- BTZ. RNA-Seq analysis and further confirmation by western blot analysis reveals integrated stress response (ISR) upregulation, ATF4 induction, and suppression of protein translation and global protein ubiquitination levels, likely responsible for resistance to proteasome inhibition in cells co-treated with IACS and BTZ compared to BTZ alone. Stable isotope tracing reveals an upregulation of reductive carboxylation; while RNA-Seq data and flow cytometry demonstrate increase in the cystine/glutamate transporter SLC7A11. The ensuing metabolic rewiring in mitochondrially suppressed MM induces several metabolic vulnerabilities including sensitivity to the SLC7A11 inhibitor, erastin. We also show that knockdown of ATF4 re-sensitizes ETC-inhibited cells to BTZ while ablating sensitivity to venetoclax (previously reported). Furthermore, examination of patient samples demonstrates inter-cellular heterogeneity in ATF4 expression. Our results thus, support the role ATF4 as key determinants of PI and BCL-2 antagonist efficacy. CONCLUSION We show mitochondrial ETC inhibition induces ISR mediated resistance to PI. These ETC-inhibited cells are however sensitive to BCL-2 antagonists and afford additional metabolic vulnerabilities that can be capitalized upon to target metabolic heterogeneity in MM. Our study underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic heterogeneity-mediated resistance. Disclosures Lonial: Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Jaye: Stemline Therapeutics: Honoraria. Nooka: Janssen Oncology: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Sanofi: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Adaptive technologies: Consultancy; Karyopharm Therapeutics: Consultancy.

For many genes that are recurrently mutated in patient-derived samples or cell lines from multiple myeloma (MM), the functional roles of these genes have remained incompletely understood. We have sought to address the functional implications of these genes through extensive CRISPR-Cas9-based functional genomics studies for loss-of-function (LOF, e.g. CRISPR-based gene editing) and gain of function (GOF, e.g. CRISPR-based gene activation) in MM cell lines in vitro and in vivo. We focused on 96 genes mutated in >2% of newly diagnosed MM patients in the CoMMpass (IA17) study (after excluding non-expressed genes [ In these LOF and GOF functional studies, there were several examples of recurrently mutated genes which performed in vitro and in vivo in a manner consistent with prior knowledge on these genes as essential for MM survival/growth (e.g. NRAS or KRAS in MM lines harboring mutations for those genes, FGFR3 in t(4;14) lines) or, conversely, as tumor suppressor genes (e.g. TENT5C/FAM46C, TRAF3, TP53). Beyond these genes, which served as "positive controls", other genes with notable functional roles identified in these studies include recurrently essential genes such as SETD2, the E3 ligases HUWE1 and HERC2, or the RNA binding protein genes RBMX and ELAVL1. Interestingly, however, the large majority of genes recurrently mutated in MM do not exhibit a recurrent role as either essential genes/drivers of MM cell growth or tumor suppressor genes in these in vitro or in vivo functional genomics studies. For instance, genes such as LTB, ATM, DUSP2, the phosphatases PTPRD, PTPRF, PTPRM do not exhibit recurrent functional role as major positive or negative regulators of MM cell growth in these studies, regardless of the mutational or copy number status of these genes in the cell lines tested. The fact that these observations were concordant in both in vitro and in vivo CRISPR studies suggests that the majority of genes recurrently mutated in MM patients do not seem to impact, either when lost or gained, the MM cell survival/proliferation in not only cell autonomous in vitro conditions, but also in the context of interaction with the local microenvironment of a "humanized" BM-like milieu. Given the immunocompromised nature of these in vivo systems, we also examined whether these recurrently mutated genes could influence the ability of MM cells to escape immune surveillance. However, in our in vitro genome-scale LOF or GOF CRISPR screens in three MM cell lines exposed to allogeneic donor-derived NK cells, perturbations of genes recurrently mutated in MM patient samples also did not emerge as "hits" associated with NK cell resistance. The fact that most recurrently mutated genes in MM did not elicit a clear phenotype in either loss- or gain-of-function studies with endpoints focusing on "cell fitness" (survival/proliferation) or in the context of exposure to immune effector cells such as NK cells, does not preclude alternative functional roles that may conceivably involve e.g. responses to other types of immune effector cells; cooperative interactions with other genes; roles in DNA repair (e.g. a plausible scenario in the case of ATM); impact on MM self-renewal; or potential role in early stages of myelomagenesis, but not in its late/advanced forms (which are generally considered to be represented by cell lines). More broadly, this study highlights the significance of identifying candidate drivers of MM cell biology by integrating conventional mutational analyses with functional characterization of the respective genes of interest, ideally through assessment of diverse endpoints related to both cell-autonomous and non-autonomous tumor cell behavior. Disclosures Tsherniak: Cedilla Therapeutics: Consultancy; Foghorn Therapeutics: Consultancy; The Center for Protein Degradation: Consultancy; SAB: Membership on an entity's Board of Directors or advisory committees. Licht: Epizyme: Research Funding. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Mitsiades: Nurix: Research Funding; EMD Serono: Research Funding; Novartis: Research Funding; BMS: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; Arch Oncology: Research Funding; Sanofi: Research Funding; H3 Biomedicine: Research Funding; FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees.

Background: Moderate mitochondrial stress induced by multiple mediators but most notably ROS can lead to activation of persistent mito-protective mechanisms termed "Mitohormesis". As a result of massive protein synthesis, malignant plasma cells (PCs) from MM patients (pts) undergo substantial ER stress but in addition high rates of Ig synthesis contributes to overproduction of ROS. We hypothesized that MM cells exploit mitohormesis to maintain ROS in the hometic zone, thereby increasing mitochondrial fitness to avoid apoptosis. We therefore set out to determine if the processes of mitohormesis are activated in MM and whether unmitigated mitochondrial stress can be exploited as a therapeutic strategy in MM. Results: Protective stress mechanisms of mitohormesis include the activation of the mitochondrial UPR (UPR MT),a mitochondrial-to-nuclear signaling pathway mediated by CHOP and ATF5 that upregulates mitochondrial import proteins, chaperones and proteases to maintain mitochondrial proteastasis. We first demonstrated that UPR MT activation occurs with progression from precursor to overt MM. Using a UPR MTgene signature derived from published gene-sets we observed upregulation of UPR MT genes in single-cell RNA sequencing (scRNA-seq) data generated from PCs derived from Vκ*MYC mice (a transgenic mouse model of MM) spanning the spectrum of the disease. UPR MT gene signature scores in PCs from mice increased with disease progression with the highest levels found in late-MM> int-MM> early MM>wild type mice. Similarly, analysis of publicly available gene expression datasets (GSE6477) that includes normal donors, MGUS and newly diagnosed MM (NDMM) revealed higher expression of UPR MT genes in the majority of NDMM, weak expression in MGUS and absence in normal PCs. To assess the impact of UPR MT expression on pt outcomes we calculated a UPR MT index score derived from the median expression of 12 mtUPR classifier genes across the MMRF CoMMpass dataset of NDMM pts. Stratifying pts by UPR MT expression score we found that pts in the top quartile had a significantly shorter PFS and OS compared to pts with the lowest quartile weighted score. Next, we postulated that perturbation of the mitochondrial import protein, Translocase of the Inner Membrane 23 (TIM23) would exaggerate mitochondrial stress as mitochondrial import efficiency is a key regulator of the UPR MT. First, we demonstrated that TIM23 complex genes are enriched in pts from the CoMMpass dataset with poor risk (1q gain and PR gene signature) and that shorter PFS and OS is associated with a higher weighted score of TIM23 complex genes. We then demonstrated that genetic (shRNA) knockdown or pharmacologic inhibition of TIM23 with MB-10, a small molecule inhibitor of TIM23 induced apoptosis of MM cell lines and primary pt PCs. Further non-transformed cell lines, CD138 - non-MM cells and normal donor hematopoietic progenitor cells were less susceptible to the effects of MB-10. Consistent with activation of the UPR MT, treatment of MM cells resulted in increased cytosolic ATF4, CHOP and a shift of ATF5 to the nuclear fraction. Activation of the CHOP-dependent branch of the UPR MT resulted in in upregulation of mitochondrial-targeted proteins, cpn10 and ClpP. Interestingly, MB-10 also induced XBP1 splicing demonstrating that inhibition of TIM23 complex can simultaneously activate the IRE1/XBP1 branch of integrated stress response (ISR), This led us to hypothesize that targeting TIM23 as an alternative means of activating the ISR could overcome acquired resistance to proteosome inhibitors (PIs). Indeed, PI-resistant and parental isogenic cell lines were equally susceptible to MB-10 as measured by IC50 values of cell growth. Finally, we demonstrated that doxycycline inducible knockdown of TIM23 in a mouse xenograft model induced tumor regression with significantly small tumor volumes at the end of 17 days of doxycycline treatment compared to tumors expressing an inducible control vector. Conclusions: These data demonstrate that mitohormesis and UPR MT activation is associated with MM progression and worse clinical outcomes. Further we show that disrupting mitochondrial protein import results in unmitigated mitochondrial stress that switches the UPR MT from an adaptive cytoprotective to cytotoxic proapoptotic response. Thus, targeting mitochondrial import proteins such as TIM23 may represent novel therapeutic targets for MM. Disclosures Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties. Trudel: Janssen: Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Roche: Consultancy; Sanofi: Honoraria; Pfizer: Honoraria, Research Funding; Genentech: Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding.