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We present a unique database of tumor samples from 399 multiple myeloma (MM) patients, whose functional genomic landscape was assessed by integrating ex vivo drug sensitivity to 138 drugs, clinical variables, cytogenetics, mutational profiles and transcriptomes. These analyses revealed a novel MM transcriptomic topology that, when associated with ex vivo drug sensitivity, generates “footprints” having both mechanistic and predictive applications. We validated the transcriptomic footprint for the nuclear export inhibitor selinexor in two clinical trials, BOSTON and MCC17814, and showed such a gene signature can accurately classify clinical response and suggest mechanism of resistance. Finally, we propose a novel evolutionary-based therapeutic strategy involving sequential therapy of monoclonal antibody daratumumab and selinexor, based on anti-correlative transcriptomic footprints, which was validated in two clinical trials (STOMP and XPORT-MM-028). Collectively, this database and computational framework can be leveraged to inform underlying biology, and to identify novel therapeutic strategies to improve treatment of MM.

Multiple myeloma (MM) is an incurable disease. Classical chemotherapeutics including bortezomib, melphalan, lenalidomide and thalidomide have greatly enhanced survival times. Unfortunately, patients typically relapse and become refractory with an average survival of 5 years post-diagnosis. Our emerging studies demonstrate a novel role for ULK3 in regulating autophagy in MM, a key program that sustains cell survival under times of stress and has been implicated as a major mechanism of proteasome inhibitor (PI) resistance. MM is known to be highly dependent on autophagy and, currently, specific ULK3 inhibitors are lacking. We posit that by targeting this marker in chemotherapy resistant MM patients, we can circumvent alternative metabolic routes and resensitize to standard of care proapoptotic therapy. We performed RNASeq analysis of CD138+ MM cells derived from patients across the disease stages spectrum (n=815) to confirm the role of ULK3 in disease progression and resistance to chemotherapy. We developed novel inhibitors SG3014/MA9060 that target multiple kinases including ULK3 (EC50 90nM) as well as BRD4. BRD4 is a known driver of MYC and its expression is increased in refractory MM. The BRD4 inhibitor, JQ1, effectively impairs the tumorigenic potential of MM but resistance has also been noted. We determined the efficacy of MA9060 for the treatment of CD138+ MM isolated from naive and refractory patients using a novel ex vivo high throughput platform developed at Moffitt.ULK3 is highly associated with MM stage of the disease. Refractory MM patients have increased autophagy activity with significantly higher expression of ULK3 in refractory patients and in drug resistant cell lines (immunoblotting U266 vs U266-PSR; RPMI-8226 vs RPMI-8226-B25; ABNL vs V10 resistant cells).Genetic ablation of ULK3 by siRNA in U266 and 8226 cell lines results in rapid cessation of the downstream autophagy proteins (ULK1, ATG13, pATG13) and MM cell death within 72h of transduction. Increased concentrations of autophagy inhibitors MA9060/SG3014 progressively decreased CMYC and ULK3 levels, as measured by immunoblotting in U266 cells. In vivo preclinical model of U266Luc tail vein injection proved our drugs are highly effective in reducing tumor dissemination and extending overall survival (CTRL untreated n=65 days vs MA9060 n=110). Importantly, we noted no overt toxicity and protected effect against myeloma-induced bone disease. This novel class of drug works synergistically with PI and can re-sensitize PI resistant disease to these effective therapies. We also show by EMMA ex vivo platform that MA9060 is highly effective for the treatment of CD138+ MM cells isolated from patients with refractory disease.ULK3 represents a novel target for treatment of MM refractory disease. Our dual inhibitors can increase overall survival in vivo and ex vivo, therefore we expect to quickly translate our novel molecules to the clinic. Citation Format: Marilena Tauro, Tao Li, Mark Meads, Praneeth R. Sudalagunta, Raghunandan R. Alugubelli, Nicholas J. Lawrence, Ernst Schonbrunn, Harshani Lawrence, Kenneth H. Shain, Conor C. Lynch. Novel autophagy inhibitory strategies to overcome chemotherapy resistance in multiple myeloma [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 452.

Small-scale regulatory networks can model known biological processes; while large-scale genome-wide datasets can identify novel mechanisms. We developed a biophysical modeling framework that combines the accuracy of small-scale networks with the power of large-scale datasets. As a proof of principle, we implemented this framework on a cohort of 844 multiple myeloma (MM) patients’ (and 1092 TCGA breast cancer patients) z-normalized RNAseq data using t-Distributed Stochastic Neighbor Embedding to construct a disease-specific transcriptomic map, where genes closer to each other co-express within the cohort. Fuzzy c-means clustering is carried out to identify clusters of genes that are likely regulated by a common transcription factor (TF). We construct a gene regulatory network (GRN) for each cluster of co-expressing genes on a disease-specific transcriptomic map by identifying upstream TFs for each cluster using publicly available databases ENCODE and ChEA, and kinases that phosphorylate these TFs using PhosphoSitePlus and PhosphoPoint. An exhaustive list of TFs and kinases are reduced to a few key predictor variables using regression tree modeling for each gene in that cluster. This leads to a cascading network of kinases that phosphorylate TFs, which regulate expression of genes in a cluster. We derived a mechanistic model from first-principles to define functional relationships governing the GRN; where transcription, translation, and post-translational modifications are modeled using first-order reversible reaction kinetic equations. The patient-specific rate constants of the model are parametrized by single sample gene set enrichment analysis scores of key KEGG pathways like ribosome, protein synthesis, RNA degradation, etc. The system of differential equations, under steady-state, reduce to an algebraic equation that can predict the expression of every gene in a cluster from the expression of its upstream TFs and kinases alone, which is fitted to RNAseq data of 422 MM patients to estimate undetermined parameters. The remaining patients’ data is used to estimate the accuracy of the model using Pearson’s correlation (model predicted vs actual) coefficient, r. Out of 16,738 genes, 7,936 were predicted accurately (r>0.5), while the remaining genes were shown to have a significant overlap (hypergeometric test; p-value Citation Format: Praneeth Reddy Sudalagunta, Rafael Renatino Canevarolo, Mark Meads, Maria Coelho Silva, Xiaohong Zhao, Raghunandan Reddy Alugubelli, Joon-hyun Song, Erez Persi, Mehdi Damaghi, Kenneth H. Shain, Ariosto Silva. A novel gene regulatory network model identifies master regulators in cancer. [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 4313.

Multiple myeloma remains an incurable bone marrow resident malignancy due to the eventual development of drug resistance and minimal residual disease linked to both acquired genetic changes and the dynamic influences of the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. The influence of these two effectors on drug resistance has primarily been studied separately; however, within the context of the bone marrow, myeloma cells are influenced by both soluble and physical effectors simultaneously. Using a reductionist model of the multivariate microenvironment, we demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype [2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6 and drug resistance was maintained. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. To further characterize post-translational events induced by beta1 integrin mediated adhesion and under multivariate conditions, we performed immune-affinity phosphotyrosine proteomic screening in RMPI8226 myeloma cells maintained in suspension or adhered to FN with or without IL-6 stimulation. Screening identified 179 differentially tyrosine phosphorylated peptides among four conditions (Suspension or FN, +/- IL-6). Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Among 67 peptides upregulated by adhesion to FN alone, proline-rich tYrosine Kinase 2 (PYK2/FAK2) was highly phosphorylated at Y580 in RPMI8226 myeloma cells, and focal adhesion formation in adhered cells was validated by phosphorylation of paxillin at pY118 and p130CAS at pY128/pY249. PYK2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation [3]. PYK2 has also been linked to downstream signaling events of JAK-dependent cytokine receptors, but not to a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families [4-7]. As such, we hypothesized that PYK2 may be an important modulator of the enhanced STAT3 activation following multivariate signaling between beta1 integrin and the IL-6 signal transducer, gp130. In addition to enhanced phosphorylation of PYK2 at Y580 identified by proteomic screening, western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3 and JAK1. Targeting PYK2 with RNA interference attenuated the adhesion-associated amplification of STAT3 and JAK1 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. PYK2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). We have also demonstrated that STAT3 activation is markedly enhanced in myeloma cell lines adhered to patient bone marrow stroma (BMS), but not cells grown in transwell coculture (no myeloma cell-BMS contact). The protective advantage afforded myeloma cells in coculture was attenuated knockdown of PYK2 or STAT3 expression in myeloma cell lines and patient specimens using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value < 0.05). Critically, inhibition of PYK2 with nanomolar amounts of the focal adhesion kinase inhibitor PF562.271 had a similar impact only on myeloma cells directly adhered to BMS, but did not impact BMS viability. These data demonstrate a novel PYK2-mediated JAK1/STAT3 signaling cascade within the context of multivariate stimulation. Moreover these data identify a novel PYK2-mediated survival pathway activated only within the context of multivariate stimulation in the bone marrow microenvironment, and suggests that PYK2 represents a MRD-specific target. Disclosures: No relevant conflicts of interest to declare.

Abstract 4021 Multiple myeloma remains an incurable bone marrow resident malignancy, in part, due to the eventual development of drug resistance linked to both acquired genetic changes and the dynamic influences of the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. The influence of these two effectors on drug resistance has primarily been studied separately; however, within the context of the bone marrow myeloma cells are influenced by both soluble and physical effectors simultaneously. Using a reductionist model of the multivariant microenvironment, we demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype [2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6 and drug resistance was maintained. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. To further characterize post-translational events induced under multivariant conditions, we performed immune-affinity phosphotyrosine proteomic screening in RMPI8226 myeloma cells maintained in suspension or adhered to FN with or without IL-6 stimulation. Screening identified 338 differentially tyrosine phosphorylated peptides. Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Proline-rich tYrosine Kinase 2 (Pyk2/FAK2) was highly phosphorylated at Y597 in FN-adhered RPMI8226 myeloma cells. Eighty-three phosphorylation events were up-regulated by adhesion, including paxillin at pY118 and p130CAS at pY249 indicating focal adhesion formation. Pyk2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation. As such, we hypothesized that Pyk2 may be an important modulator of the enhanced STAT3 activation following multivariant signaling between beta1 integrins and gp130 [3]. In addition to enhanced phosphorylation of Pyk2 at Y579 identified by proteomic screening, western blot analysis demonstrated Pyk2 autophosphorylation of Y402 following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3 and JAK1. Targeting Pyk2 with RNA interference attenuated the adhesion-associated amplification of STAT3 and JAK1 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. Pyk2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). Our previous results demonstrated that that the enhanced STAT3 signaling involved a FN-adhesion specific binding of unphosphorylated STAT3 with gp130 (independent of IL-6 stimulation). To determine if Pyk2 was similarly recruited to gp130 under co-stimulatory conditions we have used proximal ligation assay (PLA). Confocal imaging of the PLA reaction with antisera to Pyk2 and gp130 demonstrated colocalization of the two effectors upon adhesion of patient myeloma cells to FN, but not in those grown in suspension. We have also demonstrated that STAT3 activation is markedly enhanced in myeloma cell lines adhered to patient bone marrow stroma (BMS), but not cells grown in transwell coculture (no myeloma cell-BMS contact). Critically, the protective advantage afforded myeloma cells in coculture was attenuated only by dual knockdown of STAT3 and Pyk2 using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value 0.027). These data demonstrate a novel Pyk2-mediated JAK1/STAT3 signaling cascade within the context of multivariant stimulation. Moreover, these data suggest that strategies targeting of both lateral and vertical signaling (Pyk2 & STAT3) may be required to overcome therapy resistance conferred to tumor cells by the multivariant bone marrow niche. Disclosures: No relevant conflicts of interest to declare.