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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.

Supplemental Materials & Methods and Figures 1-6. Sup. Figure 1. The effect of antiestrogens against ER+ BrCa spheroid cultures is attenuated by BMSCs. Sup. Figure 2. BMSCs attenuate the antiestrogen response of BrCa xenografts in vivo. Sup. Figure 3. BMSCs attenuate hormone-induced gene expression in cancer spheroids with varying effect on HR expression. Sup. Figure 4. BMSCs paracrine effect induces HT resistance in PrCa/BrCa 3D spheroids via IL-6 secretion or in IL-6-independent manner. Sup. Figure 5. Transcriptional signatures of genes downregulated (log2FC< -1) or upregulated (log2FC > 1) in MCF7 spheroids cocultured (8 days) with HS5 (vs. monoculture) do not correlate with relapse-free survival of 342 ER-negative BrCa patients (stratified using the upper tertile of the respective signature as cutoff). Sup. Figure 6. BrCa spheroids in coculture with BMSC acquire increased dependence on growth factor receptor and downstream signaling.

Although hormonal therapy (HT) inhibits the growth of hormone receptor–positive (HR+) breast and prostate cancers, HT resistance frequently develops within the complex metastatic microenvironment of the host organ (often the bone), a setting poorly recapitulated in 2D culture systems. To address this limitation, we cultured HR+ breast cancer and prostate cancer spheroids and patient-derived organoids in 3D extracellular matrices (ECM) alone or together with bone marrow stromal cells (BMSC). In 3D monocultures, antiestrogens and antiandrogens induced anoikis by abrogating anchorage-independent growth of HR+ cancer cells but exhibited only modest effects against tumor cells residing in the ECM niche. In contrast, BMSC induced hormone-independent growth of breast cancer and prostate cancer spheroids and restored lumen filling in the presence of HR-targeting agents. Molecular and functional characterization of BMSC-induced hormone independence and HT resistance in anchorage-independent cells revealed distinct context-dependent mechanisms. Cocultures of ZR75-1 and LNCaP with BMSCs exhibited paracrine IL6-induced HT resistance via attenuation of HR protein expression, which was reversed by inhibition of IL6 or JAK signaling. Paracrine IL6/JAK/STAT3-mediated HT resistance was confirmed in patient-derived organoids cocultured with BMSCs. Distinctly, MCF7 and T47D spheroids retained ER protein expression in cocultures but acquired redundant compensatory signals enabling anchorage independence via ERK and PI3K bypass cascades activated in a non–IL6-dependent manner. Collectively, these data characterize the pleiotropic hormone-independent mechanisms underlying acquisition and restoration of anchorage-independent growth in HR+ tumors. Combined analysis of tumor and microenvironmental biomarkers in metastatic biopsies of HT-resistant patients can help refine treatment approaches.Significance:This study uncovers a previously underappreciated dependency of tumor cells on HR signaling for anchorage-independent growth and highlights how the metastatic microenvironment restores this malignant property of cancer cells during hormone therapy.

SUMMARYNatural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators, adhesion molecules, protein fucosylation genes, and transcriptional regulators. CRISPR screens with a single-cell transcriptomic readout revealed how these cancer cell genes influenced the gene expression landscape of both cell types, including regulation of activation states in both cancer and NK cells by IFNγ signaling. Our findings provide a resource for rational design of NK cell-based therapies in blood cancers.HIGHLIGHTSTranscriptomic states of interacting NK cells and cancer cells depend on cancer cell lineageMolecular correlates of increased sensitivity of myeloid compared to B-lymphoid cancers include activating receptor ligands NCR3LG1, PVR, and ULBP1New regulators of NK cell resistance from 12 genome-scale CRISPR screens include blood cancer-specific regulators SELPLG, SPN, and MYBSingle-cell transcriptomics CRISPR screens targeting 65 genome-wide screen hits identify MHC-I, IFNy, and NF-κB regulation as underlying mechanisms

Systemic breast cancer treatments often fail to achieve complete and sustained responses due to drug-persistent residual tumor foci, the “seed” for eventual relapse. Recent clinical studies have revealed that the chemo-persistent tumor cells undergo extensive transcriptional reprogramming in response to neo-adjuvant treatment; however, the impact of this acquired molecular signature on the ability of residual cancer cells to survive during therapy is not clear. To further study the molecular hallmarks of chemo-persistent cancer cells, we analyzed the transcriptional signatures of post-treatment residual tumors in a large number of breast cancer patients from several neoadjuvant clinical studies. We observed that the treatment-persistent tumor cells had a distinct cellular state which molecularly resembles that of the embryonic diapause, a dormant stage of transiently suspended development in undifferentiated epiblasts triggered by stress and induced by suppression of Myc activity and overall biosynthesis. Remarkably, the propensity for residual tumors with an embryonic diapause-like (EDL) molecular signature was significantly associated with worse outcome in breast cancer patients. To dissect this distinct cancer cell state, we developed novel in vitro models using 3D breast cancer organoids which responded to cytotoxic treatment by generating longitudinally-persistent residual organoid fractions that phenotypically and molecularly simulated the in vivo emergence of post-treatment residual tumors in preclinical and clinical settings. The treatment-persistent tumor fractions in cancer organoids and in the respective in vivo xenografts did not exhibit significant genetic changes compared to baseline tumor cells, but had reduced apoptotic priming and an EDL transcriptional reprograming similar to that of residual tumors in patients. Similarly to embryonic diapause, residual persistent fractions in cancer organoids, xenografts and patient tumors had markedly suppressed Myc transcriptional output and biosynthetic levels. Ectopic induction of MYC expression enhanced acute chemotherapeutic cytotoxicity in breast cancer organoids. Conversely, suppression of MYC or pharmacological inhibition of Myc transcriptional co-activators, BET bromodomains, abrogated chemotherapeutic cytotoxicity and induced in breast cancer cells an EDL molecular signature characterized by below-baseline redox stress levels which were maintained during drug exposure. High-throughput interrogation of residual persistent breast cancer organoids indicated broad refractoriness to specific anticancer drug classes thought to operate through induction of cellular stress (e.g. agents targeting DNA or DNA-repair). However, maintaining the residual cells in dormancy after completion of cytotoxic chemotherapy via inhibition of Brd4/Myc axis or pharmacologically interfering with the diapause-like transcriptional reprogramming of treatment-persistent cancer cells represent potential therapeutic strategies to target chemo-persistent tumor cells. Overall, our study shows that breast tumors dynamically co-opt the stress survival mechanism of embryonic diapause to persist during treatment, and reveals an unexpected role of Myc as regulator of cancer cell entry into transient drug-refractory dormancy. The diapause-like persister organoid cancer models provide ex vivo tractability for studying the otherwise elusive, dormant, drug-refractory residual tumors, with potential implications in personalized medicine and drug discovery. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Dhvanir Kansara, Juliette Bouyssou, Jennifer Roth, Michal Sheffer, Rinath Jeselsohn, Nathanael Gray, Ulrich Steidl, Boris Bartholdy, Myles Brown, Aedin Culhane, Constantine Mitsiades. Treatment persistence of residual breast tumors through an embryonic diapause-like cancer cell state with suppressed myc activity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS1-07.

Background: Natural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. Methods: We performed multiplexed single-cell RNA-seq (scRNA-seq) on co-cultures of NK cells and 26 cell lines representing diverse blood cancers. Using screens of pooled DNA-barcoded cell lines (PRISM), we quantified the sensitivity of over 60 blood cancer cell lines and integrated the results with CCLE multi-omics to uncover molecular correlates of tumor cell susceptibility to NK cells. We performed 12 genome-scale CRISPR loss-of-function and gain-of-function screens of cancer-cell intrinsic NK cell resistance mechanisms across 7 blood cancer cell lines. Finally, we investigated the mechanisms-of-action of 65 genome-scale screen hits using CRISPR screens with scRNA-seq readout in both tumor and NK cells. Results: At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype. NK cells transitioned either into an activated state characterized by 4-1BB, GITR, TIM-3, and TIGIT or a state marked by type I interferon signature. Tumor cells responded to NK cell attack by activating interferon gamma (IFNy) signaling, inducing MHC class I. The activation states correlated with sensitivity to NK cells, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. Molecular correlates of increased sensitivity included expression of activating receptor ligands NCR3LG1, PVR, and ULBP1, whereas TRAF3 mutations correlated with resistance. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators and adhesion molecules. The screens identified new ​​blood cancer-specific NK cell inhibitory regulators (SELPLG, SPN, and MYB ) and genes previously underappreciated in NK cell evasion, including protein fucosylation and transcriptional regulators (e.g. GFI1B). CRISPR screens with scRNA-seq readout identified MHC-I, IFNy, and NF-κB regulation as underlying mechanisms. Cancer cell knockout of positive regulators of NK cell response (CD58, NCR3LG1) induced an inactive NK cell state, providing experimental evidence how cancer cell-intrinsic genetic alterations can shape the molecular profile of attacking immune cells to promote immune evasion. Conclusions: By integrating diverse functional genomics screens and patient genomic profiles, we provide a comprehensive landscape of potential biomarkers and functionally validated genetic mechanisms which influence how NK cells recognize and kill malignant cells. The results offer a roadmap to facilitate development of NK-cell based immunotherapy for blood cancers and beyond. Citation Format: Olli Dufva, Sara Gandolfi, Jani Huuhtanen, Olga Dashevsky, Khalid Saeed, Jay Klievink, Petra Nygren, Jonas Bouhlal, Jenni Lahtela, Anna Näätänen, Bishwa Ghimire, Tiina Hannunen, Pekka Ellonen, Hanna Duàn, Jason Theodoropoulos, Essi Laajala, Jouni Härkönen, Petri Pölönen, Merja Heinäniemi, Shizuka Yamano, Ryosuke Shirasaki, David Barbie, Jennifer Roth, Rizwan Romee, Michal Sheffer, Harri Lähdesmäki, Dean A. Lee, Ricardo De Matos Simoes, Matti Kankainen, Constantine S. Mitsiades, Satu Mustjoki. Single-cell functional genomics of natural killer cell evasion by tumor cells [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 667.

Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced KrasG12D-driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR and blocks EGFR-induced RAS activation. Moreover, we show that inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. Our results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer.

Multiple myeloma (MM), the second most common hematologic malignancy in adults, is characterized by clonal proliferation of malignant plasma cells and tumor-induced complications, including MM-induced bone disease (MBD). Up to 90% of MM patients develop MBD during the course of their disease, which is a major cause of morbidity and mortality. MM therapy focuses primarily on agents that seek to eradicate the tumor cells and others which inhibit bone resorption. In contrast, no drugs are currently available to promote bone formation in MM patients and reverse MBD. Towards addressing this unmet therapeutic need, we applied a modified version of our “humanized” bone marrow (BM)-like scaffold (huBMsc)-based xenograft model, where MM cells and bone marrow mesenchymal stromal cells (BMMSCs) are co-implanted. In this system, we investigated the impact of MM cells on BMMSCs to identify markers that can be targeted with the intent to reverse MBD. After confirming that MM cells indeed inhibit bone formation in vivo, we performed transcriptional analysis of ex vivo expanded “unexposed” control BMMSCs vs. “MM-exposed” BMMSCs and found 332 differentially expressed genes. Next, we compared these data with transcriptional profiles of BMMSCs after in vitro differentiation towards osteoblasts, as well as publicly available data of transcriptional profiles of BMMSCs obtained from MM, ALL, and AML patients. Combined, this analysis identified 8, MM-specific, differentially expressed genes, including the small leucine-rich proteoglycans (SLRPs), ASPN, OGN and OMD, that are 1) suppressed in the presence of MM (xenograft model and patient-derived BMMSCs); 2) induced during in vitro osteogenic differentiation; and 3) not dysregulated in BMMSCs in other hematological malignancies. As reported previously, we observed that bone morphogenetic protein (BMP)6 can upregulate the expression of these SLRPs in BMMSCs, while inducing osteogenic differentiation. Moreover, incubation of MM cells with BMP6 in either mono- or co-cultures with stromal cell lines, resulted in reduced MM cell viability. In addition, pretreatment of BMMSCs with BMP6 completely abolished their support of MM proliferation. Interestingly, BMP6, in contrast to BMP2 and BMP4, did not induce, but inhibited receptor activator of nuclear kappa-B ligand (RANKL)-dependent osteoclast generation. Collectively, our analyses identify BMP6 as a master regulator that promotes osteogenic differentiation, inhibits RANKL-dependent osteoclastogenesis and can directly induce MM cell death. Therefore, by reshaping the BM microenvironment to become less tumor-supportive, BMP6 represents an attractive candidate for MM treatment. Citation Format: Jiaxian Wang, Thomas Baardemans, Ricardo de Matos Simoes, Olga Dashevsky, Huipin Yuan, Joost D. de Bruijn, Anton C. Martens, Constantine S. Mitsiades, Richard W. Groen. BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6374.

Background Natural killer (NK) cell-based therapy is a promising approach to improve treatment responses in multiple myeloma (MM), which remains incurable. However, genetic determinants and mechanisms of sensitivity or resistance to NK cells in MM are incompletely understood. Here, we investigated the transcriptional impact of genes identified as regulators of sensitivity to NK cells in genome-wide CRISPR screens and integrated the findings with patient-derived data. Methods To systematically dissect the mechanisms by which select genes identified in our previous genome-wide studies influence MM cell response to NK cells, we performed pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform. Based on genome-wide screen ranking and biological relevance, 31 genes were selected. Pools of MM1.S and LP1 expressing sgRNAs targeting select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection and assignment to cells, followed by differential gene-expression analysis and patient data correlation. Results The analysis of the transcriptomic responses in MM cells upon NK cell exposure revealed that engagement by NK cells resulted in activation of IFNy-JAK-STAT signaling and upregulation of HLA class I genes, including the inhibitory NKG2A ligand HLA-E, as well as HLA class II. We then evaluated the impact of each sgRNA perturbation on gene expression in each condition, compared to the untreated control. Silencing of IFNy signaling mediators (JAK1, JAK2, STAT1, IFNGR2) prevented the induction of the IFNy response upon NK cell exposure, providing a mechanism for the observed sensitization to NK cells via reduced expression of HLA I genes. Disruption of TRAF2, NFKBIA, or NFKBIB, which also sensitized MM1.S to NK cells, induced NF-kB signaling. The induced NF-kB signature included increased expression of the death receptor FAS, pivotal for susceptibility to NK cells in the MM1.S CRISPR screen. We then correlated the findings with patient data from the CoMMpass dataset. Mutations in the NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes also identified experimentally by CROP-seq. Also, NFKBIA mutations were linked to reduced HLA-E expression in both MM patients and CROP-seq, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. Interestingly, patients with NLRC5 mutations had lower HLA-E expression consistent with CROP-seq data, indicating that although rare, NLRC5 mutations may predispose MM cells to higher NK-sensitivity. Conclusions Our data shed light on mechanisms of response to NK cells in MM and identify potential therapeutic targets for combination treatment. Furthermore, patient data correlation highlights subgroups that might have an increased susceptibility to NK cell treatment, highlighting the potential of such studies in the identification of predictive biomarkers.

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generate models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts and in cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, abrogates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity, or interfering with the diapause-like adaptation by inhibiting cyclin-dependent kinase 9 (CDK9), represent potential therapeutic strategies against chemotherapy-persistent tumor cells. Our study demonstrates that cancer co-opts a mechanism similar to diapause with adaptive inactivation of Myc to persist during treatment.

Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced Kras

Background: The biology and treatment response of human multiple myeloma (MM) cells in vivo is influenced by interactions with mesenchymal bone marrow stromal cells (BMSCs). For several key BMSC-derived cytokines (including IL-6) only the human, not murine, form optimally interacts with their respective receptor(s) on human MM cells. To better simulate the treatment responses of human MM cells in the BM, "humanized" BM-like niches in vivo have been engineered with biocompatible ceramic scaffolds "functionalized" via osteogenic differentiation of human mesenchymal BMSCs and implanted subcutaneously in immunocompromised mice. Aim: To determine if the patterns of genetic dependencies elucidated through in vitro CRISPR-based functional genomic studies are recapitulated when human MM cells are grown in mice within BM-like scaffolds with "humanized" mesenchymal stromal compartment. Methods: Cas9+ MM cell lines KMS11 and XG7 were transduced with a library of 1372 single-guide RNAs (sgRNAs) targeting 184 genes of interest (4 sgRNAs/gene), including 89 genes preferentially essential for MM cell lines compared to other neoplasms in vitro; broad-spectrum oncogenic targets (e.g. KRAS, MYC); tumor suppressor genes (e.g. PTEN); genes with limited in vitro essentiality in MM cells, but significantly higher expression vs. non-MM lines (e.g. ZFP91, ZBP1); and 155 olfactory receptor (OR) genes (typically not expressed or biologically inactive in tumor cells) as "DNA cutting" control sgRNAs. MM cells transduced with this focused CRISPR knockout (KO) library were injected into "humanized" scaffolds implanted subcutaneously (s.c.) in NSG mice. Tumors were collected when mice reached criteria for euthanasia and next-generation sequencing quantified the changes in sgRNA distribution at the end vs. start of experiment. Results: A large majority of genes defined by in vitro CRISPR KO screens as MM-preferential dependencies were also essential for MM cells in BM-like scaffolds. Among 57 MM-preferential dependencies in vitro which were plausible dependencies for KMS11 cells (e.g. CERES scores -0.4) for KMS11 cells, 29 genes exhibited in vivo essentiality for both KMS11 and XG7 cells: several of these latter "in vivo dependencies" are recurrently essential for other MM lines in vitro (e.g. ZBTB38, UBE2J1, TCF3, DNAJB11), while also others have limited if any in vitro essentiality (e.g. ZBP1, PYGO2) across MM despite significantly higher transcript levels vs. other neoplasias. Notably, several genes with increased essentiality in the "humanized" BM scaffolds vs. in vitro also had stronger in vivo dependency in the BM scaffolds vs. when growth of the same MM cells as s.c. plasmacytomas (e.g. BCL2, PIM2, UBE2J1, SYVN1, ALG9, AMFR). Co-culture with BMSCs or IL-6 treatment induces several of these transcripts in MM cells suggesting that increased dependency of MM on these genes in the "humanized" BM model is due at least partly to its human cytokines. Conclusions: This study provides evidence for significant impact of the "humanized" BM-like niche on the patterns of genetic dependencies for human MM cells. Most genes preferentially essential for MM cells in vitro remain essential for their in vivo growth in the "humanized" BM model. Notably, several genes that do not meet criteria for dependency in vitro show such metrics in "humanized" BM scaffolds, but not sc plasmacytomas. These observations highlight important implications of the "humanized" BM-like in vivo model for current and future efforts to define and prioritize therapeutic targets for MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Janssen/Johnson & Johnson: Research Funding; Arch Oncology: Research Funding; TEVA: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; EMD Serono: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while >80% and >75% of these genes have detectable transcript levels (RPKM>1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Pharmacological inhibition of the canonical proteasome by inhibitors targeting its beta5 (PSMB5) subunit represent a cornerstone of the therapeutic management of plasma cell neoplasias. However, proteasome inhibitors have limited clinical applications in other hematologic malignancies. Notably, a broad spectrum of cell lines and patient-derived samples from lymphoma, leukemia and myeloma tend to express high levels of transcripts for and exhibit substantial activity of the immunoproteasome, an alternative proteasome particle which is enriched in hematopoietic/immune tissues. Established proteasome inhibitors used in clinical practice (e.g. bortezomib, carfilzomib, ixazomib) target both the canonical 20S proteasome and the immunoproteasome. We reasoned though that targeting of the immunoproteasome with more selective small molecule inhibitors that largely spare the canonical proteasome may provide insights into the specific functional role of the immunoproteasome in hematologic neoplasias, and importantly may also uncover underappreciated therapeutic opportunities related to targeting of hematologic neoplasias beyond myeloma, that might be more dependent on the immunoproteasome vs. the canonical proteasome; as well as potential benefits related to the therapeutic index of these agents. To address these questions, we characterized the activity of the previously disclosed selective immunoproteasome inhibitor M3258, which targets the LMP7 (beta5i subunit). The bortezomib-resistant cell line MM1VDR, which has a "gatekeeper" PSMB5 mutation preventing bortezomib binding to the canonical proteasome, remained sensitive to in vitro treatment with M3258, in further support of the functional specificity of this inhibitor. To obtain a comprehensive assessment of the activity of M3258 across hematologic malignancies, we studied a pool of 74 "DNA-barcoded" cell lines (PRISM system) from hematologic malignancies, including multiple myeloma (MM), leukemias and lymphomas, which were treated for 48hr with M3258. MM cell lines (9/16) were strongly represented among the sensitive lines which displayed 50% reduction in their cell viability in this analysis; and their responsiveness did not differ significantly from the M3258-responsive MM lines. Importantly, we observed no correlation between the responses of leukemia, lymphoma or MM lines to in vitro treatment with M3258 vs. clinically-relevant pulse of bortezomib. Furthermore, 7 of the 15 lymphoma/leukemia lines that were M3258-responsive had limited, if any, response to a clinically-relevant pulse of bortezomib. Taken together, these observations suggest that immunoproteasome inhibition with M3258 can be active against subsets of hematologic neoplasias with resistance/limited responsiveness to canonical proteasome inhibitors. To further examine the in vivo relevance of these observations, we studied the in vivo activity of M3258 against the MM.1S myeloma cell line, which has only moderate in vitro response to M3258 compared to other blood cancer cell lines tested in our "DNA-barcoded" pooled analysis. M3258 significantly increased the overall survival of mice with diffuse MM-bone lesions established after tail vein injection of MM.1S cells; and of mice harboring MM.1S lesions within a subcutaneously implanted ceramic scaffold-based system engineered to establish a bone marrow-like histological and functional unit with a "humanized" stromal compartment. The anti-tumor activity of M3258 in this scaffold model exhibited a trend for increased efficacy compared to twice weekly subcutaneous treatments with bortezomib. These results highlight that in vivo administration of M3258 exhibits anti-tumor activity against clinically-relevant models of MM lesions, even from cell lines that have modest in vitro responsiveness to this immunoproteasome inhibitor. Furthermore, our studies with a large panel of "DNA-barcoded" cell lines indicates that the anti-tumor effects of M3258 may extend beyond MM and into other classes of hematologic malignancies, including subsets of leukemias and lymphomas, identifying previously underappreciated therapeutic implications for the class of selective immunoproteasome inhibitors. Disclosures Walter: Merck Healthcare KGaA: Employment. Friese-Hamim:Merck Healthcare KGaA: Employment. Goodstal:EMD Serono Research and Development Institute: Employment. Sanderson:Merck Healthcare KGaA: Employment. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

The discovery that thalidomide derivatives recruit the E3 ligase CRBN to induce neomorphic degradation of proteins critical for multiple myeloma (MM) cells stimulated the research into proteolysis-targeting chimeric compounds (PROTACs), led to development of several CRBN- or VHL-based PROTACs against various oncoproteins and put a new spotlight on the biology and therapeutic targeting of E3 ligases in human neoplasias. However, so far only a few of the ~600 known/presumed E3 ligases have been leveraged for generation of PROTACs. The mechanisms regulating the function of most E3 ligases have not been systematically examined. Because the function of an E3 ligase is considered essential for anti-tumor activity of its respective PROTACs, we applied CRISPR knock-out (KO) systems to identify candidate regulators of E3 ligase function, via characterizing the the network of genes which modulate MM cell responses to PROTACs. We thus performed genome-scale CRISPR-based gene editing (for loss-of-function, LOF) studies in MM.1S cells treated with PROTACs targeting BET bromodomain proteins through MDM2 (A1874), CRBN (dBET6) or VHL (ARV-771 or MZ-1) or targeting CDK9 through CRBN (Thal-SNS-032); and validated key hits with individual sgRNAs in different MM cell lines. The top individual LOF events conferring resistance to PROTACs did not involve a compensatory mechanism or "work-around" the loss of the respective oncoprotein, but were predominantly associated with LOF of the respective E3 ligase; or with LOF for genes with known or plausible role in regulating the respective E3 ligases. For instance, sgRNAs against members of the COP9 signalosome complex decreased MM cell responses to CRBN- and (to a lesser extent) VHL-, but not MDM2-based PROTACs. PROTACs leveraging different E3 ligases were regulated by different cullin ring ligase (CRL) complex members (e.g. CUL2, RBX1, TCEB1, TCEB2 for VHL- vs. DDB1 for CRBN- vs. no CRL member for MDM2-based PROTACs) or E2 conjugating enzymes (UBE2R2 vs. UBE2G1 for VHL- vs. CRBN-based PROTACs). Collectively, these results suggest that MDM2 regulation is largely CRL- and COP9-signalosome independent; while VHL regulation is less COP9 signalosome-dependent compared to CRBN. These mechanistic differences suggest that PROTACs targeting the same oncoprotein through different E3 ligases should not be associated with cross-resistance, a result which we validated in experiments involving sequential administration of different PROTACs against BRD4/3/2. In turn, this observation implied that developing PROTACs that leverage a more extended spectrum of E3 ligases may facilitate sequential uses of existing and these new PROTACs to delay or prevent treatment resistance. Building on results of our genome-scale CRISPR essentiality screens, we examined the dependency landscape of known E3 ligases of MM (n=20 cell lines) and 500+ non-MM cell lines. CRBN is redundant for nearly all MM or non-MM cell lines tested, while most other E3 ligases leveraged for PROTACs (e.g. MDM2, BIRC2, DCAF15, DCAF16, RNF114) are essential for only modest or small subsets of human cancer cell lines, suggesting that resistance to respective PROTACs may readily emerge through LOF of these E3 ligases without major fitness cost to tumor cells. We thus sought to identify E3 ligases which are highly expressed in subsets of human tumor cell lines (at levels well above the large majority of normal tissues) and are major dependencies for these "high expressor" cell lines: we identified MDM2 as a major dependency for p53-wild-type cell lines (consistent with MDM2 role as E3 ligase for p53) and we validated this result by documenting the preferential activity of a MDM2-based PROTAC for BRD4/3/2 against p53 wild-type cells. We also identified other E3 ligases genes with well-known roles in tumor cell biology (e.g. members of anaphase promoting complex/cyclosome); as well as E3 ligases (e.g. KCMF1, RNF4) which, to our knowledge, have not been leveraged for design of PROTACs, but warrant consideration given their patterns of essentiality in "high expressor" tumor cells. Our study provides insights on differential regulation and distinct patterns of essentiality for different E3 ligases and informs the design of new PROTACs which leverage different E3 ligases to help delay/overcome treatment resistance in MM and beyond. Disclosures Schlossman: Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

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.

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.

Natural killer (NK) cell-based therapies are considered promising future approaches for multiple myeloma (MM) treatment, but immune evasion mechanisms are poorly understood. To determine the mechanisms regulating MM cell response to NK cells, we performed genome-wide (GW) and targeted CRISPR screens in MM cell lines. To further investigate the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed additional pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform and integrated these findings with data from phenotypic assessment of NK cell response of pooled "DNA-barcoded" cell line including 15 MM cell lines (PRISM platform) and molecular profiling of MM patient samples. Our loss-of-function (LOF) and gain-of-function (GOF) GW-CRISPR screens were performed in the MM1.S, LP1, KMS11 MM lines treated with ex vivo expanded NK cells vs untreated control. The top LOF hits were validated using a focused library of ~600 genes. As expected, LOF of class I HLA /antigen presentation machinery genes, transcriptional regulators of HLA and IFNg pathway genes sensitized MM cells to NK cells, confirming that these pathways represent prominent suppressors of NK cell killing. Moreover, LOF of death receptors or downstream effectors was associated with NK resistance; while LOF of the negative regulators of death receptor signaling (e.g. CFLAR, and XIAP) sensitized to NK cell killing. To mechanistically dissect the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed scRNAseq using the CROP-seq platform. Pools of MM1.S and LP1 expressing single-guide (sg)RNAs targeting 31 select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection, differential gene-expression analysis and patient data correlation. The single cell transcriptional profiling of each CRISPR-based LOF for genes of interest documented that disruption of TRAF2, NFKBIA or NFKBIB enhanced NF-kB signaling and was associated with increased expression of the death receptor FAS, a key mediator of NK cell sensitivity (Figure), and changes in expression of BIRC3, CD70, and CXCL10(the latter may further increase immune reactivity through recruitment of T and NK cells). Interestingly, in our PRISM data, high transcriptional NF-kB signatures and/or presence of TRAF3 mutations (an activator of NF-kB signaling) correlated with decreased NK cell response. Among the individual genes most highly correlated with NK cell resistance was CFLAR, a known NF-kB target gene, and negative regulator of death receptor-mediated apoptosis. In contrast, PTEN and NLRC5 mutations were associated with enhanced NK cell sensitivity in several MM lines tested, consistent with their depletion in the GW LOF CRISPR screens and the HLA I regulatory function of NLRC5. We correlated our findings with patient-derived data from the CoMMpass study. Mutations of NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes, consistent with our CROP-seq data. NFKBIA mutations were also linked to reduced HLA-E expression in both MM patient and CROP-seq data, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. NLRC5 mutations were associated with lower HLA-E expression consistent with CROP-seq data, suggesting that, although rare, NLRC5mutations may represent a more NK-sensitive MM subset. TRAF3 alterations occurred both in a distinct TRAF3-altered cluster and in a subset of CoMMpass patients with WHSC1 translocations, consistent with the observations in our PRISM data set. CFLAR expression was enriched in patients with TRAF3 alterations belonging to both of these groups. The aggregate of molecular data from MM patient samples and PRISM or CRISPR-based functional studies preclinically raise the possibility that TRAF3 and WHSC1/t(4;14) alterations may contribute, at least in part through CFLAR, to decreased NK cell response in MM cells. Our data illuminate the complex mechanisms of response to NK cells in MM, highlighting the different effects of distinct molecularly defined subgroups of MM tumor cells with increased susceptibility to NK cell treatment, underlining the potential of such studies as a blueprint for identification of biomarkers individualized use of NK cell-based therapies in MM. Figure 1 Figure 1. Disclosures Mustjoki: Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Janpix: Research Funding. Mitsiades: H3 Biomedicine: Research Funding; FIMECS: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Nurix: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; BMS: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Arch Oncology: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals: Consultancy, Honoraria.

To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated ‘DNA-barcoded’ solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell–sensitive tumor cells tend to exhibit ‘mesenchymal-like’ transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell–sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. The use of natural killer (NK) cells in immunotherapy as an alternative to allogeneic T cells is gaining ground. Here, two genome-scale high-throughput platforms are used to identify genes that modulate the sensitivity of multiple solid tumor cell lines to NK-mediated killing.

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding.

Background: Endoplasmic reticulum (ER)-associated degradation (ERAD) for unfolded proteins represents an important biological vulnerability for multiple myeloma (MM) cells, given the high proteostatic stress in these cells due to immunoglobulin secretion. This helps explain the efficacy of proteasome inhibitors in MM compared to most other neoplasms. Aim: To systematically investigate ER-associated genes that mediate the essential role of ERAD in MM. Methods: We integrated results from (i) genome-scale studies for CRISPR-gene knockout in 19 MM cell lines (Avana library) and CRISPR-activation (Calabrese library) in 5 MM lines, to define ER-associated dependencies and drivers of increased MM cell growth; (ii) publicly-available CRISPR-based studies for genes regulating ERAD of substrates located in the luminal (ERAD-L), membrane (ERAD-M) or cytosolic (ERAD-C) side of the ER in non-MM models; (iii) functional characterization of candidate genes in MM models in vitro and in vivo. Results: While ER function in general is globally required for all normal and malignant cells, a surprisingly large proportion of ER-related genes are not "pan-essential" genes (e.g. CERES scores Conclusions: Our results provide functional insights into the role of ERAD in MM cells and nominate specific mediators which are not broadly essential across all cancers, but are recurrent or even preferentially essential for MM. Our study indicates a previously underappreciated role for the ER-to-cytosol dislocon complex that includes HERPUD1, SEL1L, SYVN1 and UBE2J1; distinct enzymes of the N-glycan-dependent ER glycoproteins quality control system; and ER chaperones in MM. These findings provide a rationale for development of agents selectively targeting these key regulators of ER protein folding processes in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Arch Oncology: Research Funding.

Chemo-persistent residual tumors are a major barrier for curative cancer therapy and provide a reservoir of cancer cells for eventual relapse. This clinically critical tumor cell population is poorly understood and lacks faithful in vitro models. We compared the transcriptional profiles of paired samples from patient with solid tumors or hematological neoplasias at the stage of post-treatment residual tumors or minimal residual disease vs. at their respective baselines. For a large proportion of patients, post-treatment tumor cells had a molecular signature that resembled that of embryonic diapause, a dormant stage of suspended development in early mammalian embryos triggered by stress and induced by suppression of Myc activity and overall biosynthesis. This Myc-inactivated molecular program was acquired during treatment and not pre-existing at baseline. Remarkably, baseline tumors with multiple MYC copy numbers maintained their MYC amplification in the post-treatment persistent tumor cells but suppressed Myc activity. We simulated this cancer cell state using preclinical models of breast and prostate cancer and multiple myeloma, including patient-derived cancer organoids and PDX models. Parallel approaches of genome sequencing and DNA barcode-mediated clonal tracking in vitro and in vivo independently indicated that the residual cancer cell subpopulations that persisted after treatment with common chemotherapeutic classes were not driven by rare pre-existing clones or de novo genetic events. Transcriptional analysis of the treatment-persistent cancer cells in 3D organoid cultures and in xenograft/PDX models indicated a diapause-like transcriptional adaptation with inactivated Myc, similar to that in patients. We functionally examined the role of Myc suppression in in vitro models of breast cancer and multiple myeloma using genetic (CRISPR/Cas9 editing and interference) and pharmacological (inhibition of Myc transcriptional co-activator Brd4) approaches. Suppression of Myc in cancer cells induced an embryonic diapause-like molecular profile and attenuated the acute chemotherapeutic cytotoxicity. Myc-inactivated cancer cells had reduced apoptotic priming and maintained low redox stress even in the presence of cytotoxic agents, which may contribute to cell survival during treatment. High-throughput screening of chemo-persistent cells revealed that inhibitors of CDK9 could revert the biosynthetic pause, reactivate Myc, attenuate the diapause-like state, and re-sensitize the residual cancer cells to chemotherapy. Overall, our study shows that cancer cells can co-opt the stress survival mechanism of embryonic diapause by dynamically suppressing Myc to enter transient drug-refractory dormancy. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Dhvanir Kansara, Caroline Vilas, Aziz Al'Khafaji, Juliette Bouyssou, Aedin Culhane, Constantine S. Mitsiades. Treatment-induced embryonic diapause-like adaptation through suppression of Myc activity as mediator of drug persistence in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 42.

During the last two decades, cell lines and patient-derived samples from multiple myeloma (MM) have been extensively profiled for alterations in their genome with the anticipation that those genes with the most recurrent lesions could represent attractive novel therapeutic targets or markers for aggressive disease. Yet for many of these genes, their functional significance for MM cells has not been formally evaluated. With the advent of new CRISPR/Cas9-based functional genomics platforms, it is possible to generate in genome- or subgenome-scale direct quantitative information on the impact that perturbation of these genes exerts on tumor cell survival, proliferation or other phenotypes. We therefore examined the landscape of our CRISPR-based functional genomic data for these recurrently dysregulated genes We specifically curated information from the MMRF CoMMpass study and multiple other publicly available studies, to identify genes which are recurrently identified to harbor nonsynonymous mutation (SNV or indel), DNA copy number loss or gain, or participation in chromosomal translocations. We then examined the patterns of results for these genes in our genome scale CRISPR-based gene-editing studies for loss-of-function in n=18 MM cell lines. We identified a subset of genes (e.g. FAM46C, CDKN2C, RASA2) which are considered targets for recurrent loss-of-function events and indeed exhibit, for large fractions of the cell lines tested enrichment, of their sgRNAs in CRISPR knock-out studies, consistent with a role of these genes as suppressors of tumor cell survival or proliferation. CRISPR KO of TP53 leads to increased survival/proliferation of only a small minority (2/18 of cell lines tested thus far), which reflects the fact that the overwhelming majority of MM cell lines already harbor LOF events for this gene. Interestingly, a substantial number of genes which have been considered to harbor recurrent LOF events in MM patient samples (e.g. NF1, NF2, CYLD) do not exhibit sgRNA enrichment in CRISPR KO screens in the MM cell lines tested so far. In addition, several other recurrently mutated genes for which their loss- or gain-of-function status had not been previously evaluated with extensive functional studies in MM (e.g. SP140, LTB, EGR1, ATM, PARK2, PRKD2, RAPGEF5, DOCK5, TGDS, TNFAIP8) exhibit in the majority of cell lines tested in in CRISPR knockout studies no significant enrichment or depletion of their sgRNAs. In contrast, PTPN11, CREBBP, EP300, KMT2B, KMT2C, SETD2, SF3B1 and UBR5, are notable examples of recurrently mutated genes which represent dependencies for large fractions of MM cell lines in vitro. These results highlight the value of interpreting results from next generation sequencing studies in the context of information provided by the genome scale by use of functional genomic characterization of available cell line models. We envision that, similar sub-genome scale assays were performed at the level of patient derived samples will also provide direct information about the relevance of some of these genes. In addition, functional studies conducted with context of tumor-microenvironemtn compartment interactions and tumor interface will be needed to evaluate several genes identified in the study. Disclosures Licht: Celgene: Research Funding. Mitsiades:Takeda: Other: employment of a relative; Janssen/ Johnson & Johnson: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; TEVA: Research Funding.

Natural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. To systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated “DNA-barcoded” solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach), correlating cytotoxicity scores for each cell line with the CCLE transcriptional data (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells. Based on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with an HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, we observe that transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Strikingly, comprehensive analysis of the CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells treatment, compared with cell lines of epithelial-like program. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA, suggesting a link between these two machieneries. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. Citation Format: Michal Sheffer, Emily Lowry, Nicky Beelen, Minasri Borah, Suha Naffar-Abu Amara, Chris C. Mader, Jennifer Roth, Aviad Tsherniak, Olga Dashevsky, Sara Gandolfi, Samantha Bender, Jordan Bryan, Cong Zhu, Li Wang, Ricardo De-Matos Simoes, Channing Yu, Yiguo Hu, Olli Dufva, Marios Giannakis, Todd Golub, Rizwan Romee, Satu Mustjoki, Aedin C. Culhane, Lotte Wieten, Constantine S. Mitsiades. Landscape of molecular events regulating tumor cell responses to natural killer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO041.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological “degraders” of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Background: CRISPR-based functional genomics studies in pooled format are increasingly used to define the mechanisms of tumor cells resistance to diverse therapies. Despite many advantages of CRISPR compared to prior functional genomics methodologies, controlling for off-target effects and other artifacts remains important, especially for focused screens, which involve limited numbers of genes and thus have room for relatively few controls. We reasoned that olfactory receptor (OR) genes can be applied as controls for both genome and sub-genome scale studies since these GPCR superfamily members are either not expressed or biologically inactive in most tissues beyond the olfactory epithelium; and their use may address limitations of non-targeting sgRNA controls that do not bind the human genome, and thus do not provide control for events occurring upon interaction of CRISPR nucleases with DNA/chromatin. Aim: To define the performance of olfactory genes as controls for CRISPR-based screens. Method: We examined the performance of a collection of 346 OR genes in a series of genome-scale and focused CRISPR studies including several releases of the Broad Institute Dependency Map dataset and a collection of screens performed to define mechanisms of tumor resistance vs. sensitization to pharmacological or immune-based therapies. Results: The large majority of OR genes exhibit in genome-scale CRISPR knockout studies metrics associated with non-essentiality. Specifically, ~90% of OR genes have CERES scores between -0.4 to 0.4 in ~90% of cell lines (e.g. 20Q1 data on 22 lymphoma, 19 multiple myeloma (MM) and 40 leukemia lines; and 658 solid tumor lines). Importantly, the average CERES score of all OR genes in a given cell line ranges between -0.08 to +0.05 for all hematologic or solid tumor lines tested. However, there are "outlier" OR genes for which CERES scores (and DNA copy number-uncorrected metrics) are in the same range as valid gene dependencies. Such "outlier" OR genes (e.g. OR7G2, OR8A1, OR1E2, OR4N2, OR2A1, OR4K1, OR2T29, OR7A10, OR7A17, OR2T5, OR1D2, OR11H1, OR4F4) have average CERES scores Conclusions: Collectively, these results indicate that in general OR genes, as an entire group or subsets of them, can serve as a control for CRISPR studies. The lack of expression or biological function of OR genes in most human cancers enhances their attractiveness as negative controls. The overwhelming majority of OR genes show limited deviation from the bulk of the distribution of "non-hits" from a variety of screening platforms and experimental conditions. Despite the existence of some outliers that warrant further investigation, even small subsets of OR genes are sufficient to establish a distribution of negative controls across hundreds of cell lines; different CRISPR platforms and experimental setups. We propose that OR genes can be applied as well-defined negative control genes across genome- and sub-genome scale functional genomics studies Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative; Sanofi: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; TEVA: Research Funding; FIMECS: Consultancy, Honoraria; EMD Serono: Research Funding; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding.

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. >10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Luo:Ionis Pharmaceuticals, Inc.: Current Employment. Kim:Ionis Pharmaceuticals, Inc.: Current Employment. MacLeod:Ionis Pharmaceuticals, Inc.: Current Employment. Mitsiades:Arch Oncology: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; Karyopharm: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; EMD Serono: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

The clinical success of combination therapies for multiple myeloma (MM) has led our group and others to perform pharmacological screens seeking to identify promising candidates for combination regimens. However, in our genome-scale CRISPR knockout (KO) studies, we have observed that the biological behavior of MM cells is driven by key transcription factors (TFs), including several that represent major preferential dependencies for MM cells compared to other blood cancers or to solid tumors. Most of these TFs are currently considered "undruggable", as they lack identifiable hydrophobic pockets that can be selectively engaged with small-molecule inhibitors and are not thalidomide derivative (IMID)-induced neo-substrates for CRBN. Therefore, pharmacological screens are inherently limited in their ability to probe the functional interactions of MM-related TFs. To bypass this limitation and interrogate in MM cells how key TFs may interact with each other or with other pathways, we performed combinatorial CRISPR-based gene editing studies, in which we examined how MM cell survival/proliferation is impacted by CRISPR KO of each gene individually vs. simultaneous KO of two genes. We applied a combinatorial CRISPR dual knockout (DKO) study in MM.1S or KMS-11 cells, which have been engineered to express 2 orthologous Cas9 nucleases (from S. pyogenes and S. aureus), to increase the efficiency and specificity of combinational KO. Synergy (or antagonism) of the KO of gene pairs was determined by comparing the average (+/-95% CIs) normalized log2-fold change (log2FC) of sgRNA readcounts for each gene pair vs. the sum of log2FC for the respective "singeletons" (pair of sgRNA for one gene and control sgRNA) vs. "double controls"; and focusing on genes pairs with concordant results for sgRNA pairs involving Sp.Cas9-Sa.Cas9 and vice versa. We performed a focused study on ~100 genes, including 45 TFs (including 20 TFs that represent MM-preferential dependencies); additional MM-preferential or broad-spectrum dependencies; tumor suppressors (e.g. TP53, PTEN); as well as TFs and other genes that are not major dependencies for MM cells in single KO studies. We also used this DKO system to study MM.1S cells implanted in "humanized" scaffold-based bone marrow-like model in NSG mice. Both in vitro and in vivo, synergistic combinations of gene KOs were heavily enriched for presence of at least one TF, most often one of the MM-preferentially essential TFs, including IRF4, POU2AF1, TCF3, and NF-kappaB pathway members. Many of these synergistic combinations had greater effect on MM cell survival/proliferation than the combined KO of IKZF1/IKZF3 (IKZF1/ZFP91 or IKZF3/ZFP91 did not result in synergy): these observations suggest that many MM TFs which are not CRBN neo-substrates are involved in synergistic DKO combinations with more potent anti-MM activity compared to all DKOs of the main TFs (IKZF1, IKZF3, ZFP91) degraded by IMIDs. This observation suggests that efforts to advance the therapeutic targeting of currently "undruggable" MM TFs (alone or paired) which do not interact with CRBN may have therapeutic implications that do not overlap with the effect of IMIDs. The results of our DKO studies exhibited a time-dependent effect: in early time-points, strong dependencies (e.g. IRF4) are highly recurrent partners in synergistic pairs, while later time-points uncover interactions between genes with limited, if any, individual roles as essential genes, including TFs (e.g. ZBP1) but also other pathways (the translation regulator ELL2, a gene proposed to be associated with increased risk for myelomagenesis). MM cell "dedifferentiation" (e.g. through suppression of plasma cell-specific TFs such as XBP1) has been proposed as potential mechanism for proteasome inhibitor resistance. However, in our study, loss of XBP1 or other MM-related TFs (alone or in combination with other genes) was not associated with significant resistance to MM.1S or KMS11 cell treated with clinically relevant pulses of bortezomib, suggesting that perturbation of key MM-related TFs can be therapeutically compatible with proteasome inhibition. More broadly, our DKO studies revealed critical interactions between TFs with central roles in MM biology and also others with previously underappreciated effects, pointing to combinatorial effects that may be exploited in the future through novel therapeutic strategies. Disclosures Mitsiades: Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Takeda: Other: employment of a relative .

Immunotherapy has remarkably changed the treatment paradigm in hematologic malignancies and natural killer (NK) cell therapy represents an attractive option, as it has been feasible and safe in early clinical trials, without graft-versus-host effects. Nevertheless, the molecular markers determining cancer cell sensitivity or resistance to NK cells, especially in the context of tumor cell interaction with the bone marrow (BM) stromal microenvironment remain incompletely understood, but have major translational relevance since these tumor-stromal interactions have been known to attenuate the response of blood cancer cells to diverse classes of pharmacological agents. To address these questions, we performed NK cell treatment of a series of cell lines from hematologic malignancies by applying a pooled "DNA-barcoded" format of these cell lines (PRISM system). We specifically quantified the dose-dependent responses to primary NK cells for 70 molecularly-annotated blood cancer cell lines, including myeloid and lymphoblastic leukemia, diffuse large B cell lymphoma and 15 multiple myeloma (MM) lines, either in presence or in absence of BM stromal cells (BMSCs) and interferon gamma (IFNg), followed by integrated computational analyses to identify candidate molecular markers correlating with tumor cell sensitivity or resistance to NK cells. NK cell cytotoxicity, quantified by the relative abundance of barcodes in treated cells compared to controls, was correlated with the transcriptional, mutational and other molecular features of each of the 70 cell lines from publicly available databases. Furthermore, data from MM cell lines were compared to our genome-wide loss of function (LOF) and gain of function (GOF) CRISPR screen data in the MM cell line MM.1S. Two distinct clusters of cell lines, sensitive and resistant to NK cell treatment, were identified. Such clusters retained distinct pattern of in vitro resistance in the presence of stroma, while showing an overall markedly decreased NK cell responsiveness, which underscores the protective effect of stromal microenvironment in blood malignancies, regardless of the addition of IFNg. RNA-seq data showed no differences in dependencies between the two clusters and no distinct gene expression patterns at baseline that clearly allows to predict NK cell response, which underscores the heterogeneity of resistance patterns at single gene level, across different hematologic malignancies. However, when comparing baseline RNAseq data to data obtained from previous GOF and LOF CRISPR screens in MM.1S, surface antigens such as PVR, ULBP1, ULBP3 were more frequently downregulated, whereas MUC1 was upregulated in resistant cells clusters. An important observation is that gene lesions such as TP53, PTEN, MMSET, commonly associated with high-risk diseases, do not affect NK cell responses in the cell lines tested. Interestingly, a gene set enrichment analysis (GSEA) showed that the cluster of resistant cells displays upregulation of class I MHC complex, class II MHC complex binding, IL7 pathway and a downregulation of transmembrane receptor protein serine/threonine kinase signaling pathway. GSEA also showed that baseline state of IFN-JAK-STAT signaling correlates with BMSCs-induced NK cell resistance, a result further confirmed by addition of IFNgto tumor-NK cocultures in the absence of BMSCs. No significant differences in NK cell response were observed when comparing cell lines of different hematologic neoplasms, suggesting that candidate markers from these studies may be relevant across different hematologic malignancies. In conclusion, this is the first study of this size correlating the molecular annotation of different concurrently-treated hematologic cell lines with their response to a NK-based treatment in the context of BMSC interaction. This study of a large panel of pooled "DNA-barcoded" cell lines provided complementary and orthogonal information to our LOF and GOF screens, expanding our potential to identify and validate molecular markers for individualized use of NK cell-based therapies in hematologic malignancies. Disclosures Mustjoki: BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding. Mitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Arch Oncology: Research Funding; Fate Therapeutics: Honoraria; Ionis Pharmaceuticals: Honoraria; Takeda: Other: employment of a relative ; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding.

Loss-of-function (LOF) studies (e.g. using RNAi or CRISPR) have historically been the main functional approaches to identify and study genes which drive the biology of Multiple Myeloma(MM) or other neoplasias. We hypothesized though that substantial complementary data can be derived from systematically examining the impact of transcriptional activation of endogenous genomic loci of different genes. To perform such gain-of-function (GOF) studies in an open-ended genome-scale manner, we applied CRISPR activation approaches in 4 MM lines (MM1S, KMS11, LP1, L363) which were transduced with the dCas9-P65-HSF transcriptional activation system and the Calabrese genome-scale sgRNA library (2 pooled sub-libraries; total of ~110,000 sgRNAs targeting promoters of ~18000 genes; initial coverage ~800-1000 cells/sgRNA). Next generation sequencing quantified the sgRNA abundance in MM cells at baseline and various time points (e.g. 2-12 weeks of culture), and rank aggregation algorithms identified genes with statistically significant enrichment or depletion of their sgRNAs, reflecting positive vs. negative, respectively, effects of GOF of these genes on MM cell survival/proliferation. These studies identified distinct groups of genes which serve as positive regulators of MM cell growth in at least 3 of the cell lines tested, including critical transcription factors (TFs)/cofactors, such as POU2F2, POU2AF1, IRF4, MYC; growth factor signaling mediators, such as IGF1R and IRS1; Ras family members (e.g. KRAS in MM1S cells); diverse members of the solute carrier family of transporters for amino acids and other bioactive small molecules. We also observed positive regulators of MM cell growth with distinct roles in certain cell lines, e.g. induction of IKZF3, IKZF1, RELA, CD48, MBNL1, PAX2 exhibited significantly more pronounced role as positive regulators of MM1S cells compared to the other cell lines tested. Notably, several positive regulators of MM growth identified in these CRISPR activation studies are not essential for MM cell survival/proliferation in CRISPR knockout studies in the same MM cell lines or many others. Examples of such genes include the TFs POU2F2 (Oct2) and PAX2, the TRAF interacting protein TIFA or the Toll-like receptor TLR4. We went on to validate these results for several such genes (e.g. POU2F2, POU2AF1) with individual sgRNAs for CRISPR activation and/or cDNA overexpression (vs. isogenic controls) in competition experiments and cell cycle analyses. We focused on further probing the mechanistic basis for the role of POU2AF1 as one of the top positive regulators of MM cell growth and performed RNA sequencing analyses of LP1 cells harboring induction of POU2AF1 expression using CRISPR activation. We observed upregulation of a distinct cluster of >50 genes that are themselves positive regulators of LP1 cell growth in our genome-scale CRISPR activation studies. While some of these are known drivers of tumor cell growth (e.g. AURKB, E2F1, FGFR3), the large majority of these POU2AF1-driven positive regulators of MM cell growth have not been previously studied in MM; and exhibit a distinct enrichment for transcriptional regulators and other genes which are not required for LP1 cell survival/proliferation in CRISPR KO studies of LP1 cells. These results indicate that CRISPR-based activation of endogenous gene expression provides data which complementary to those derived from CRISPR LOF studies. Indeed, we not only validated through our GOF studies the relevance of genes which with prominent essentiality in CRISPR LOF studies (e.g. IRF4, POU2AF1, etc.), but also identified promising new candidate regulators of MM cell biology which are not essential for baseline survival/growth of MM cells, but can induce MM cell growth when further activated. These results provide novel insights into the biology of MM cells and may have therapeutic implications towards suppressing the ability of MM cells to transition to states of advanced biological aggressiveness. Disclosures Mitsiades: Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Alzheimer's disease (AD) progressively destroys cognitive abilities in the aging population with tremendous effects on memory. Despite recent progress in understanding the underlying mechanisms, high drug attrition rates have put a question mark behind our knowledge about its etiology. Re-evaluation of past studies could help us to elucidate molecular-level details of this disease. Several methods to infer such networks exist, but most of them do not elaborate on context specificity and completeness of the generated networks, missing out on lesser-known candidates. In this study, we present a novel strategy that corroborates common mechanistic patterns across large scale AD gene expression studies and further prioritizes potential biomarker candidates. To infer gene regulatory networks (GRNs), we applied an optimized version of the BC3Net algorithm, named BC3Net10, capable of deriving robust and coherent patterns. In principle, this approach initially leverages the power of literature knowledge to extract AD specific genes for generating viable networks. Our findings suggest that AD GRNs show significant enrichment for key signaling mechanisms involved in neurotransmission. Among the prioritized genes, well-known AD genes were prominent in synaptic transmission, implicated in cognitive deficits. Moreover, less intensive studied AD candidates (STX2, HLA-F, HLA-C, RAB11FIP4, ARAP3, AP2A2, ATP2B4, ITPR2, and ATP2A3) are also involved in neurotransmission, providing new insights into the underlying mechanism. To our knowledge, this is the first study to generate knowledge-instructed GRNs that demonstrates an effective way of combining literature-based knowledge and data-driven analysis to identify lesser known candidates embedded in stable and robust functional patterns across disparate datasets.

Panobinostat is an oral broad-spectrum histone deacetylase inhibitor that alters gene expression via epigenetic mechanisms and function of key proteins through changes in their protein acetylation state. Panobinostate was approved a few years ago by the FDA and EMA for use in combination with bortezomib and dexamethasone in patients with multiple myeloma (MM) who have received ≥2 prior regimens, including bortezomib and an immunomodulatory drug. To study the mechanisms which regulate the response vs. resistance of MM cells to panobinostat, we performed genome-scale CRISPR activation screen of MM1S cells. MM1S cells were transduced with dCas9 and pooled lentiviral particles of the Calabrese P65-HSF CRISPR activation library consisting of 2 pooled sgRNA sub-libraries. After selection for viral transduction, cells were continuously cultured with (2 different concentration of 12.5 and 20 nM) or without panobinostat for 5 weeks, before being harvested. At that time point, dose-response curves for panobinostat treatment confirmed that the drug-exposed MM.1S cell populations of our study had become significantly less sensitive to panobinostat than treatment- naive MM.1S cells. Genomic DNA was extracted and next generation sequencing was performed to quantify the abundance of the sgRNA "barcodes" within the tumor cell populations of our study, while rank aggregation algorithms were performed to rank genes according to the magnitude and concordance of enrichment for its different sgRNAs. In MM.1S cells which had developed resistance to the lower dose (12.5 nM) of panobinostat, we observed major sgRNA enrichment (at least 4 of 6 sgRNAs, enrichment p-value 1.5) for sgRNAs for a variety of genes, of which the most prominently enriched ones encode for the cell surface ABC transporters ABCB1 (MDR1/p-glycoprotein), to a lesser extent ABCC4 (MRP4) and even less so for ABCG2. In addition, we observed sgRNA enrichment for transcription factors, such as IRF4, POU2AF1, AFF2, IKZF3, AFF3, and RELA, or the transcriptional coregulator MTA1; Bcl2 family members such as BCL2 and BCL2L1; and chromatin remodeling genes such as KAT6A. However, in MM.1S cells which had survived the treatment with higher concentration (25 nM) of panobinostat, the genes with significant and concordant sgRNA enrichment were restricted to ABCB1, ABCC4, and IRF4. These observations indicate that the most efficient mechanism for MM cells to develop resistance to both low and higher concentrations of panobinostat is by increasing its export from the cells, with ABCB1 as the primary, but not sole, transporter which can assume this role. When we transduced MM.1S cells, which already express high levels of IRF4 transcript and protein, with lentiviral construct for IRF4 cDNA, we observed a shift to the right for the panobinostat dose-response curve, further supporting the observation that modulation of IRF4 levels in MM cells can alter the degree of MM cell sensitivity to panobinostat. Proteasome inhibitors suppress the activity of RELA and NFkappaB more broadly, while thalidomide derivatives cause degradation of IKZF3 and can decrease the IRF4 transcript levels: these results may respectively explain, at least in part, the mechanistic basis for the enhanced combined effect of panobinostat with proteasome inhibitors and the favorable clinical results obtained with panobinostat and other broad spectrum HDAC inhibitors in combination with thalidomide derivatives. Our study provides unbiased genome-scale systematic characterization of the mechanisms regulating MM cell response vs. resistance to panobinostat and identify the pronounced and dose-dependent enrichment of these resistance mechanisms for genes contributing to panobinostat export from MM cells, as well as a role for IRF4 and (primarily at lower panobinostat concentrations) for other transcription factors, chromatin remodeling genes and anti-apoptotic BCL-2 family members. These observations also support the need for a more systematic characterization of the regulation of expression of ABC transporters in MM cells; and for development of novel strategies to disrupt more comprehensively IRF4 and other transcription factors for which gain-of-function is associated with decreased responsiveness to panobinostat, with the goal of improving the impact of this agent and potentially other broad spectrum HDAC inhibitors in MM. Disclosures Mitsiades: TEVA: Research Funding; Takeda: Other: employment of a relative; EMD Serono: Research Funding; Abbvie: Research Funding; Janssen/ Johnson & Johnson: Research Funding.