Abstract: Introduction: Innate and acquired resistance to anti-cancer therapies poses a major hurdle in effectively treating many cancers, especially an incurable cancer like multiple myeloma (MM). Rational combination therapies have shown improved efficacy and reduced toxicity in MM. Patient variability in response to single agents leads to variability in combination effects, which require quantification on a patient-to-patient basis. Conventional combination effect quantification methods rely on dose - response curves obtained from experiments involving cell lines. Such studies don't account for intratumoral and intertumoral heterogeneity that play an important factor in driving a patient's clinical response. Materials and Methods: We propose a framework that captures tumor-specific two-way combination effect in an ex vivo reconstruction of the tumor microenvironment using patient-derived primary multiple myeloma cells. The framework translates the data obtained from an ex vivo drug sensitivity assay to patient-specific combination therapy response predictions using mathematical modeling. MM cells (CD138+) extracted from fresh bone marrow aspirates are seeded in an ex vivo co-culture model with human stroma in multi-well plates, and tested with various drugs/combinations at several concentrations. Each well is imaged for at least 96 hours, once every 30 minutes to estimate percent viable cells. Such a platform facilitates measuring response with respect to dose and time, making this an ideal paradigm to capture pharmacodynamical interactions between drugs. An empirical mathematical model is used to measure the combination effects between two drugs, and when combined with their pharmacokinetic data obtained from Phase-I clinical trials the model predicts patient-specific response over a 90 day treatment period within five days post biopsy. Results: A total of 58 multiple myeloma patient samples were tested ex vivo with 19 two-drug combinations. The resulting ex vivo response data is fit to single agent (EMMA - Ex vivo Mathematical Myeloma Advisor) and combination (SAM - Synergy Augmented Model) mathematical models to estimate patient-drug/combination-specific LD50s and area under the curves (AUCs) from the dose-time-response curves (shown in Figs. 1a-f). The 96 hour single agent, additive (in the sense of Bliss), and combination LD50s for 19 patients tested with the combination Carfilzomib and Dexamethasone (CFZ+DEX) are presented as a box plot in Fig. 2a . A red dashed line signifies a patient who would see a benefit over additive LD50 (synergism), while a blue dashed line implies the opposite (antagonism). Similarly, Fig. 2b presents the AUCs as a box plot, where the "area" in AUC is in fact the volume under the dose-time-response curve. Inclusion of the time axis accounts for exposure-response effect in addition to the dose-response effect captured in LD50. The effect of accounting for exposure via AUC suggests greater synergy than LD50 as seen in Figs. 2a-b. In spite of being insightful, a decrease in LD50 and/or AUC doesn't always translate to a synergistic effect in patients. In order to predict the response observed in patients, the ex vivo models are integrated with pharmacokinetic data from Phase-I clinical trials to simulate patients' response over a 90 day treatment period (shown in Figs. 1j-l). The best response over a 90 day period for the single agents, additive, and the combination are presented in Fig. 2c as a box plot and the right y-axis classifies the response. However, additive effect is a theoretically computed quantity that may have pharmacological relevance but isn't significant clinically. A more clinically relevant reference model would be to compare the combination response with the better of the two single agents. Figure 2d presents the box plot comparing the predicted best single agent and combination responses. The model predictions indicate all of the 19 patients would benefit from the combination, although the extent of benefit varies from patient-to-patient. Conclusion: The proposed framework captures patient-specific combination effects using a pharmacodynamic model that can be used to screen for the most efficacious combination for a patient and across a cohort. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction: Despite some long-term remissions, eventual drug resistance in most patients remains a critical obstacle in the treatment of multiple myeloma (MM). The development of new drugs/drug combinations with novel mechanisms of action are needed for continued improvement in patient outcomes. Initiation of tumor cell death via activation of the intrinsic (mitochondrial) and/or extrinsic (death receptor) apoptotic signaling pathways has been shown to be an effective therapeutic strategy in MM. Venetoclax (Ven) is a selective, small-molecule inhibitor of BCL-2 that exhibits clinical activity in MM cells, particularly in patients harboring the t(11;14) translocation. Navitoclax (Nav) is a small-molecule that targets multiple antiapoptotic BCL-2 family proteins, including BCL-XL, BCL-2, and BCL-W to initiate the intrinsic apoptotic pathway. Eftozanermin alfa (Eftoza) is a novel, second generation TRAIL receptor agonist that induces cell death via death receptor pathways and is under investigation in multiple solid and heme malignancies. In addition, the pan-BET inhibitor mivebresib (Miv) and the BDII selective BET inhibitor ABBV-744 have shown synergistic activity with Ven in cell line models of multiple heme malignancies. Results reported here describe ex vivo drug sensitivities and functional genomic analyses of Ven, Nav, Eftoza, Miv, and ABBV-744 alone or in combination with standard-of-care agents, including bortezomib, carfilzomib, panobinostat, daratumumab, or pomalidomide. Methods: A high-throughput ex vivo drug screening assay using a coculture system of bone marrow (BM)-derived MM and stromal cells was used to assess the sensitivity of MM patient tumor cells (Figure 1A). Paired whole exome sequencing (WES) and RNA sequencing (RNA-seq) analyses were performed. Results: Primary MM patient specimens (n=52) were evaluated in the ex vivo platform, including treatment-naïve, early relapse (1-3 prior lines), and late relapse (4-8 prior lines) patients treated with proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies. As expected, t(11;14)-positive MM patient specimens were more sensitive than wildtype to Ven ex vivo (D AUC, -18.6, P=0.002), however MM cells harboring amp(1q) were more resistant than wildtype (D AUC, +5.07, P=0.032), suggesting MCL1 (1q21 gene locus) is a key resistance factor to Ven single-agent activity in MM. Gene set enrichment analysis identified B-cell receptor signaling (normalized enrichment score (NES), 1.96, adjusted P=0.010) and MYC pathway (NES, 1.95, adjusted P=0.010) overexpression as predictors of increased sensitivity to Ven ex vivo. A t(11;14) gene expression signature was also generated using a penalized regression model approach in an additional MMWG/ORIEN MM patient cohort (n=155). The t(11;14) predictive gene expression signature was confirmed by correlation with Ven AUC in the ex vivo model. Additional pathway analyses were performed to identify potential predictive markers of sensitivity/resistance for each single agent and drug combination. Although ex vivo activity of Nav was higher in t(11;14) specimens compared to non-t(11;14) (D AUC, -17.8, P=0.011), ex vivo activity in non-t(11;14) specimens was also observed, indicating additional anti-MM activity by cotargeting of BCL-XL and BCL-2. Both Miv and ABBV-744 showed single-agent activity ex vivo, however Miv demonstrated higher activity (median LD50=88.4nM), suggesting that pan-BET inhibition is more effective than BDII-specific BET inhibition in MM. Finally, a novel drug-combination effect analysis was used that identified novel synergistic ex vivo combinations including Ven and panobinostat (P=0.0013) and Eftoza with bortezomib (P=1.8E-7) or carfilzomib (P=7E-4). Additionally, single-agent induction of macrophage-mediated phagocytosis was observed in both Ven and daratumumab, which was synergistic when the 2 drugs were combined (Figure 1B). Conclusion: An ex vivo functional genomic screen of MM patient specimens demonstrated the usefulness of this approach to identify candidate drugs and potential predictive biomarkers for continued evaluation in clinical trials. This approach confirmed known mechanisms of drug sensitivity and identified new ones, including a novel characterized immune-mediated synergy between Ven and daratumumab, and potential combination strategy for Eftoza and proteasome inhibitors. Figure 1 Disclosures Siqueira Silva: Karyopharm: Research Funding; NIH/NCI: Research Funding; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Mitchell:AbbVie: Other: payment for bioinformatics analysis, Research Funding; M2GEN: Current Employment, Research Funding. Dai:Cygnal Therapeutics: Current Employment; M2GEN: Ended employment in the past 24 months. Hampton:M2GEN: Current Employment. Lu:AbbVie: Current Employment, Current equity holder in publicly-traded company. Modi:AbbVie: Current Employment, Other: may own stock or stock options. Motwani:AbbVie: Current Employment, Current equity holder in publicly-traded company. Harb:AbbVie: Current Employment, Other: may hold stock or stock options. Ross:AbbVie: Current Employment, Current equity holder in publicly-traded company. Shain:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: While this is a preclinical study, venetoclax for treatment of multiple myeloma is not an approved indication

Abstract: Introduction. Multiple myeloma (MM) is an incurable plasma cell malignancy with a growing list of anti-MM therapeutics. However, the development of predictive biomarkers has yet to be achieved for nearly all MM therapeutics. Selinexor (SELI), a nuclear export inhibitor targeting exportin 1 (XPO1), has been approved with dexamethasone (DEX) in penta-refractory MM. Clinical studies investigating promising SELI- 3 drug combinations are ongoing. Here, we have investigated potential synergistic combinations of SELI and anti-MM agents in terms of ex vivo sensitivity, as well as paired RNAseq and WES to identify companion biomarkers. Methods. MM cells isolated from fresh bone marrow aspirates were tested for drug sensitivity in an organotypic ex vivo drug sensitivity assay, consisting of co-culture with stroma, collagen matrix and patient-derived serum. Single agents were tested at 5 concentrations, while two-drug combinations were tested at fixed ratio of concentrations. LD50 and area under the curve (AUC) were assessed during 96h-exposure as metrics for drug resistance. Drug synergy was calculated as a modified BLISS model. Matching aliquots of MM cells had RNAseq and WES performed through ORIEN/AVATAR project. Geneset enrichment analysis (GSEA) was conducted using both AUC and LD50 as phenotypes for single agents and combinations. Both curated pathways (KEGG and cancer hallmarks) and unsupervised gene clustering were used as genesets. Student t-tests with multiple test correction were used to identify non-synonymous mutations in protein coding genes associated with single agent or combination AUC. Results. For this analysis, a cohort of specimens from 103 patients (48% female, 4% Hispanic, 11% African American) was tested with SELI and/or DEX. with a median of 2 lines of therapy (0-12). A smaller cohort of 37 have been examined with SELI, pomalidomide (POM), elotuzumab (ELO) and daratumumab (DARA). Within this cohort we observed synergy between SELI and DEX, POM and ELO as shown in Figure 1. The volcano plot illustrates the number of samples, maximum drug concentration, as well as magnitude (x- axis) and significance (y- axis) of synergy. Although the SELI-DARA combination trended toward synergy, statistical significance was not achieved. To identify molecular mechanisms and biomarkers associated with sensitivity to SELI and SELI- combinations, we investigated paired RNAseq and WES with ex vivo sensitivity. Initially, we conducted GSEA on two cohorts of primary MM samples tested with SELI alone at 5µM (n=53) and 10µM (n=50). Cell adhesion (KEGG CAMS), inflammatory cytokines (KEGG ASTHMA), and epithelial mesenchymal transition (HALLMARK EMT) were associated with resistance in both cohorts, while the HALLMARK MYC TARGETS was associated with sensitivity (FWER p & lt;0.05). Mutational analysis identified 46 gene mutations associated with SELI resistance and 100 associated with sensitivity at 5µM, and 87 and 27 mutations associated with SELI resistance and sensitivity, respectively, at 10µM. Two gene mutations were identified in both cohorts: BCL7A, involved in chromatin remodeling, was associated with sensitivity and CEP290, a microtubule binding protein, associated with resistance (p & lt;0.05). Analysis of both gene sequences (NetNES 1.1) identified nuclear export signal (NES) residues suggesting these may be XPO1 cargo. Additionally, translocation t(11;14) was associated with SELI resistance in the 5µM cohort (p=0.037). The completed set of 50 specimens ex vivo, RNAseq and WES analysis will be mature and updated for the potential presentation at ASH. Conclusions. We observed ex vivo synergy between SELI and DEX, POM and ELO. Molecular analysis of matched ex vivo drug sensitivity, transcriptome and mutational profile identified environment-mediated drug resistance pathways positively correlated with SELI single agent resistance, as well as MYC regulated genes associated with ex vivo sensitivity. We also identified a list of mutations associated with SELI drug resistance and sensitivity, with special emphasis on two novel NES-containing genes, CEP290 and BCL7A. The next step of this project is to analyze transcriptional and mutational patterns associated with ex vivo synergy in the combinations here described, as putative biomarkers for future clinical investigation. Disclosures Shain: Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; Karyopharm: Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Janssen: Honoraria, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Zhang:M2GEN: Current Employment. Hampton:M2GEN: Current Employment. Argueta:Karyopharm: Current Employment. Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Siqueira Silva:AbbVie: Research Funding; NIH/NCI: Research Funding; Karyopharm: Research Funding.

Abstract: Problem: Multiple myeloma (MM) is a treatable yet incurable hematologic cancer that lacks predictive biomarkers. Approach: Here we apply a systems biology approach to determine patient-specific mechanisms, as well as signatures of drug resistance in MM. To achieve this goal, we have combined ex vivo drug sensitivity data from 307 MM fresh primary samples tested with 162 drugs and combinations, with paired molecular data (RNAseq and mutational profiling) from a larger overlapping cohort of 606 MM samples from Moffitt's Multiple Myeloma Working Group (MMWG) repository in collaboration with M2Gen/Oncology Research Information Exchange Network (ORIEN). With the purpose of decoupling biological function from intracellular control mechanisms, we have re-constructed a MM-specific transcriptional regulatory network composed of clusters of co-expressing genes. We demonstrate how this gene cluster network regulates biology, and how different biological functions (e.g. Proteasome, Ribosome, Oxidative Phosphorylation) share common regulatory circuits. We have used gene set enrichment analysis (GSEA) to identify gene clusters with transcriptional profiles, and investigated mutations associated with drug resistance. Results: As a preliminary validation of this approach, we have confirmed established mechanisms of resistance (MOR) to targeted therapies, as well as proposed novel MOR to clinically relevant and experimental drugs in MM, as well as putative synergistic drug combinations. In addition, we have identified a list of low frequency mutations ( 〈 5%) indirectly involved in drug resistance (or sensitivity) through modulation of expression of gene clusters correlated with drug resistance (GSEA). This would suggest that low frequency mutations in a number of different genes, targeting a shared transcriptional regulatory mechanism, can drive drug resistance in MM, while been overlooked by statistical analysis of each individual gene. We have also explored evolution of drug resistance in sequential samples. Consistent with altered transcriptional programming in therapeutic escape, single sample GSEA demonstrated cumulative dysregulation of cancer-related genes with increasing lines of therapy. We have identified 60 MM-specific transcriptional core auto-regulatory circuits (CRC) correlated with ex vivo drug resistance, suggesting that characterization of transcriptional regulatory circuits is a critical approach to infer mechanisms of MM resistance, and providing a novel rationale for combination therapy. We hypothesized that identifying and targeting these transcriptional CRCs could facilitate patient-specific rational combination therapies, with the goal to overcome therapy resistance in the clinic. As proof-of-principle, we have identified a novel transcriptional network consisting of 3 of these CRCs (FOXP1, JUNB and JUN) associated with BCL2 inhibitor (BCL2i) response in MM. Our preliminary data suggests that this transcriptional regulatory circuit is associated to t(11;14) MM through CCND1 up-regulation, but is also present in non-t(11;14) BCL2i-sensitive primary samples, and can be modulated to induce BCL2i sensitivity in non-t(11;14) MM through HDAC inhibitors. Conclusion and next steps: Preliminary results confirm the potential of this combination of unsupervised and supervised, yet functionally testable approach, to infer novel, and patient-specific MOR for MM drugs. Disclosures Dai: M2Gen: Employment. Dalton:MILLENNIUM PHARMACEUTICALS, INC.: Honoraria. Shain:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Adaptive Biotechnologies: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction The use of proteasome inhibitors (PIs), such as bortezomib (BTZ), in multiple myeloma (MM) has markedly increased the survival of newly diagnosed patients. Although advancements in therapeutic regimens in the past decade have improved prognosis, we lack knowledge of the mechanisms that lead to drug resistance. To assess the contributors to BTZ-resistance, we integrated steady-state metabolomics, proteomics and gene expression from two naïve and BTZ-resistant cell line models. In addition, gene expression associated with ex vivo PI resistance has been analyzed. Potential predictive biomarkers of PI-resistance and novel targets for combination therapy will be investigated. Methods Parental cell lines, RPMI 8226 and U266, were acquired from ATCC. 8226-B25 and U266-PSR (kind gift from Dr. S. Grant) BTZ-resistant derivatives were selected from their respective parental naïve cell lines by chronic drug exposure. Untargeted metabolomics, activity-based protein profiling (ABPP), and expression proteomics data were acquired using liquid chromatography-mass spectrometry. Gene expression profiles of both cell lines and ex vivo patient specimens were derived from RNAseq. Metabolomics and proteomics data were normalized with iterative rank order normalization. Significantly different genes, proteins, and metabolites were integrated for pathway mapping and identification of biomarkers for PI resistance. Results Consistent with previous findings, kynurenine, a product of tryptophan catabolism, is significantly altered in both of our cell line models. In the 8226 and 8226-B25 pair, PI resistance was associated with increased kynurenine and positively correlated with TDO2 and IDO1 overexpression consistent with published literature (Li et al. Nature Medicine, 2019, 25, 850-60). As expected, PSMB2, a subunit of the proteasome, is overexpressed and has a higher activity in both 8226-B25 and U266-PSR in the ABPP and expression proteomics, and higher expression in 8226-B25 RNAseq data. PSMB2 is also overexpressed and significant in the RNAseq patient data, increasingly from newly diagnosed/pre-treatment to early relapse (p-value 2E-4) and late relapse (p-value 0.0052). In addition, CD38 is an enzyme responsible for conversion of NAD+ to nicotinamide and ADP-ribose. It has increased expression in MM cells and is significantly downregulated in ABPP (log2 ratio -4.25, p-value 2E-13), expression proteomics (log2 ratio -2.5), and RNAseq (log2 ratio -2.6, p-value 5E-6) in the 8226-B25 BTZ-resistant cells. In the steady-state metabolomics of the 8226-B25 cells, ADP-ribose (log2 ratio 4.11, p-value 2E-5) is the most upregulated known metabolite. This change suggests a downstream result of resistance within this interaction and a potential biomarker of PI resistance. However, gene expression of CD38 in patient samples was relatively unchanged. CD38 was not detected in the U266-PSR proteomics or RNAseq data and ADP-ribose (log2 ratio -0.63, p-value 0.06) was not significantly altered, suggesting a different mechanism of resistance in this cell line. Conclusions Though common mechanisms of PI resistance were identified, our data clearly show that BTZ-resistance arises by heterogeneous means in the two cell line models, promoting the need for biomarkers that can determine resistance and predict response in individual patients (or cohorts). Decreased expression of CD38 in 8226-B25 could elucidate mechanisms of PI resistance and immune response evasion strategies of MM cells. Further investigation of CD38 expression as a BTZ-resistance biomarker could lead to improving combination therapies with monoclonal antibodies, such as daratumumab, and PIs in newly diagnosed MM patients by predicting response prior to treatment. Further examination of ADP-ribose metabolism may lead to the mechanism of synergy between PARP inhibitors and proteasome inhibitors. Ultimately, we plan to integrate and utilize these multi-omics approaches in patient specimens and improve MM patient care by identifying PI resistance biomarkers to predict patient response. Disclosures Shain: Adaptive Biotechnologies: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.