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Introduction. Multiple myeloma (MM) is an all but incurable plasma cell malignancy without predictive biomarkers for approved therapies. Selinexor (SELI), a nuclear export inhibitor targeting exportin 1 (XPO1), is approved with dexamethasone (DEX) with promising SELI-combination studies ongoing. We investigated SELI combinations ex vivo to identify synergistic combinations and companion biomarkers. Methods. We established a platform to perform parallel RNA/exome sequencing and ex vivo drug sensitivity assessment on CD138+ cells from MM patient bone marrow aspirates. At the time of this analysis, 844 different samples with clinical, WES and RNA sequencing data were treated ex vivo with the following agents: SELI (n=75), DEX (192), pomalidomide (POM, 268), elotuzumab (ELO, 21), daratumumab (DARA, 117), SELI+DEX (22), SELI+POM (20), SELI+ELO (21), SELI+DARA (27). Cells were cultured with autologous macrophages, stroma, collagen matrix and patient-derived serum. Cell death (LD50 and AUC) was assessed through digital image analysis. Sequencing was performed through ORIEN/AVATAR. Links between non-synonymous mutations in coding genes and cell death were calculated using T-tests with multiple test correction. Results. Our analysis identified SELI+DEX (number of samples=60, p Conclusions. We observed ex vivo synergy between SELI and DEX, POM, ELO and DARA, and identified expression signatures and mutations associated with response to these agents. Ongoing analysis of additional samples is being performed to validate these results. Citation Format: Praneeth Reddy Sudalagunta, Mark B. Meads, Rafael Renatino Canevarolo, Maria Coelho Silva, Christopher Cubitt, Gabriel DeAvila, Raghunandan Reddy Alugubelli, Constantine N. Logothetis, Amit Kulkarni, Qi Zhang, Oliver Hampton, Christopher J. Walker, Yosef Landesman, Kenneth Shain, Ariosto Siqueira Silva. Characterization of synergistic selinexor combinations with dexamethasone, pomalidomide, elotuzumab, and daratumumab in primary MM cells [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 1061.

Introduction Certain cancers have been shown to express a specific type of drug-resistance involving the export of drug target proteins from the nucleus of the cell to the cytoplasm. This mechanism of drug-resistance has been shown to be important in multiple myeloma (MM) and recently a nuclear export inhibitor, selinexor, has received approval by the FDA in MM. However, general nuclear export signal (NES) inhibitors like selinexor exhibit off-target toxicities when used in patients. We have begun to develop novel drugs that specifically prevent the export of the drug-target topoisomerase IIα (TOP2A). Methods In this study, we 1) generated an atomic homology model of human TOP2A to identify lead compounds targeting the NES of TOP2A; NES inhibitors (NESi), 2) examined binding specifically to TOP2A and inhibit nuclear export by immunofluorescence microscopy (IF) , nuclear-cytoplasmic fractionation, immunoprecipitation (IP), proximity ligation assay (PLA), and Biacore kinetic/affinity assay, and 3) tested the anticancer activity of these compounds in MM in vitro, ex vivo and in vivo MM. Results The atomic homology model of human TOP2A provided the basis for in silico molecular docking screenings of 139,735 small molecules (NCI) (MW < 500) to identify lead compounds targeting the NES of TOP2A. NESi compounds with the highest docking scores were assayed for their ability to induce apoptosis in human MM cells when used with the TOP2 inhibitor doxorubicin (DOX). We isolated four lead compounds that induced apoptosis when used with DOX and that inhibited nuclear export of TOP2A, as shown by IF and nuclear-cytoplasmic fractionation, IP, a PLA and Biacore kinetic/affinity assays. Inhibition by the lead compound, NCI-9138, was specific to TOP2A because p53 trafficking was unaffected and the inhibitor did not affect TOP2A protein expression or enzymatic function (decatenation). This NESi was found to sensitize human MM cells in vitro, in vivo mouse models, and patient MM cells exposed ex vivo to DOX but did not affect normal cell lines. These TOP2A-specific nuclear export inhibitors may potentially lead to a new approach in circumventing drug resistance. Conclusions Our previous studies have shown that TOP2A is exported from the nucleus of MM cells and that this is a significant cause of resistance to DOX therapy. In this study, we have shown that blocking export of TOP2A can sensitize human MM cells to DOX both in vitro, ex vivo in MM cells from patients, and in in vivo mouse models of MM. A drug that specifically inhibits the export of a single cancer-related protein has not been published. We intend to show that this unique approach to cancer-treatment may significantly advance the treatment of MM. This paper also demonstrates a proof of concept for the development of highly specific small molecule inhibitors that could be used as cancer therapeutics. Citation Format: Joel G. Turner, David Ostrov, Nicholas Lawrence, Jana Dawson, Juan Gomez, Alexis Bauer, Mark Meads, Alex Achille, Kenneth Shain. Novel small molecule inhibitors that target the exportin binding pocket of TOP2A for the treatment of multiple myeloma [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 1228.

Multiple myeloma remains treatable but incurable. Despite a growing armamentarium of effective agents, choice of therapy, especially in relapse, still relies almost exclusively on clinical acumen. We have developed a system, Ex vivo Mathematical Myeloma Advisor (EMMA), consisting of patient-specific mathematical models parameterized by an ex vivo assay that reverse engineers the intensity and heterogeneity of chemosensitivity of primary cells from multiple myeloma patients, allowing us to predict clinical response to up to 31 drugs within 5 days after bone marrow biopsy. From a cohort of 52 multiple myeloma patients, EMMA correctly classified 96% as responders/nonresponders and correctly classified 79% according to International Myeloma Working Group stratification of level of response. We also observed a significant correlation between predicted and actual tumor burden measurements (Pearson r = 0.5658, P < 0.0001). Preliminary estimates indicate that, among the patients enrolled in this study, 60% were treated with at least one ineffective agent from their therapy combination regimen, whereas 30% would have responded better if treated with another available drug or combination. Two in silico clinical trials with experimental agents ricolinostat and venetoclax, in a cohort of 19 multiple myeloma patient samples, yielded consistent results with recent phase I/II trials, suggesting that EMMA is a feasible platform for estimating clinical efficacy of drugs and inclusion criteria screening. This unique platform, specifically designed to predict therapeutic response in multiple myeloma patients within a clinically actionable time frame, has shown high predictive accuracy in patients treated with combinations of different classes of drugs. The accuracy, reproducibility, short turnaround time, and high-throughput potential of this platform demonstrate EMMA's promise as a decision support system for therapeutic management of multiple myeloma. Cancer Res; 77(12); 3336–51. ©2017 AACR.

Multiple myeloma (MM) remains treatable but incurable. Despite a growing armamentarium of effective agents, choice of therapy, especially in relapse, still relies almost exclusively on clinical acumen. We have developed a system, EMMA (Ex vivo Mathematical Myeloma Advisor), consisting of patient-specific mathematical models parameterized by an ex vivo assay that reverse engineers the intensity and heterogeneity of chemosensitivity of primary cells from MM patients, allowing us to predict clinical response to up to 31 drugs within 5 days post-bone marrow biopsy. From a cohort of 52 MM patients, EMMA correctly classified 96% as responders/non-responders and correctly classified 79% according to IMWG stratification of level of response. We also observed a significant correlation between predicted and actual tumor burden measurements (Pearson r=0.5658, P

The bone marrow microenvironmental components interleukin (IL)-6 and fibronectin (FN) individually influence the proliferation and survival of multiple myeloma (MM) cells; however, in vivo, these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via β1 integrins, stimulated with IL-6, or treated with the two combined. Although G1-S cell cycle arrest associated with FN adhesion was overcome when IL-6 was added, the cell adhesion–mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Concomitant exposure of MM cells to IL-6 and FN adhesion revealed a dramatic increase in signal transducers and activators of transcription 3 (STAT3) phosphorylation, nuclear translocation, and DNA binding, compared with either IL-6 or FN adhesion alone in four MM cell lines. Importantly, this increase in STAT3 activation correlated with a novel association between STAT3 and gp130 in cells adhered to FN before stimulation with IL-6, relative to nonadherent cells. Taken together, these results suggest a mechanism by which collaborative signaling by β1 integrin and gp130 confers an increased survival advantage to MM cells. [Cancer Res 2009;69(3):1009–15]

Multiple myeloma remains an incurable malignancy due to the influence of the bone marrow microenvironment on survival, drug response, and resultant minimal residual disease (MRD). Therefore, identification of microenvironment-specific survival signaling determinants is critical for the rationale design of therapy and elimination of MRD. The pleiotropic cytokine IL-6 has been shown to mediate drug resistance in both solid and hematopoietic tumors. We and others have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin (FN) and Interleukin-6 (IL-6) confers a more malignant phenotype via amplification of STAT3 activation. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. Further characterization of the post-translational events modulated under these culture conditions with quantitative phospho-tyrosine profiling identified 193 differentially phosphorylated peptides after adhesion to FN, treatment with IL-6, and the combination. Phosphopeptides represented proteins involved in signal transduction, cytoskeleton assembly/adhesion, transcription, RNA processing, metabolism, and the cell cycle. Seventy-seven phosphorylations were up-regulated upon adhesion, including PYK2/FAK2, paxillin, CASL, and p130CAS consistent with focal adhesion (FA) formation. In addition to the enhanced phosphorylation of PYK2 at Y580 identified by proteomic screening, western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN or bone marrow stromal cells (BMSC), correlating with the amplification of IL-6-induced STAT3 and JAK1. We hypothesized that the collaborative signaling between β1 integrin and the IL-6 signal transducer, gp130, was mediated by FA formation and PYK2. Although PYK2 has been linked to downstream signaling events of other JAK-dependent cytokine receptors, it has not been associated with a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation in adhered cells. Further, myeloma cells co-cultured with patient bone marrow stromal cells (BMSC) showed similar gp130 and β1 integrin-specific enhancement of PYK2, JAK1, STAT3 signaling when adhered to patient BMSCs, demonstrating that PYK2 modulates co-stimulation specific STAT3 signaling in more complex, clinically relevant models of the microenvironment. Importantly, molecular and pharmacological targeting of PYK2 induced cell death solely in BMSC-adherent myeloma cells. These data identify a novel PYK2-mediated survival pathway activated only within the context of microenvironmental cues and suggest PYK2 may be a specific target in MRD. Citation Format: Mark B. Meads, Bin Fang, Linda Mathews, Jennifer Gemmer, Liang Nong, Robert MacCleod, Lori Hazlehurst, John Koomen, Kenneth H. Shain. PYK2 mediates microenvironment-specific myeloma survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4892. doi:10.1158/1538-7445.AM2014-4892

Initially studied mainly for its role as a regulator of neural cell differentiation and development, expression of HDAC11 was once thought to be restricted exclusively to brain, kidney and testes. Hence, our recent discovery that HDAC11 acts as an important modulator of antigen presentation and T cell activation, downregulating IL-10 transcription via interactions with the IL-10 promoter at the chromatin level, exposes a previously unknown capacity and tissue specificity for this enzyme. Transgenic mice harboring an eGFP reporter construct driven by the HDAC11 promoter (Tg-HDAC11-eGFP) (Heinz, N Nat. Rev. Neuroscience 2001) clearly illustrate the dynamic changes in HDAC11 gene expression in hematopoietic cell lineages, additionally unveiling an important role for HDAC11 in B cell lymphopoiesis and plasma cell biology. While common lymphoid progenitors appear to be devoid of HDAC11 transcriptional activation as indicated by minimal detectable eGFP expression, eGFP intensity markedly increases in the B-1 stage of differentiation in the periphery. Interestingly, examination of both the bone marrow (BM) and peripheral blood (PB) plasma cell compartment demonstrates an increase in expression of eGFP/HDAC11 mRNA at the steady-state, and these results are consistent with HDAC11 expression measured in PB from healthy human subjects. Furthermore, mice globally deficient in HDAC11 expression (HDAC11KO mice) exhibit a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. The concordance of HDAC11 expression and plasma cell differentiation leads us to hypothesize that HDAC11 may also be critical to malignant plasma cell survival. A comparison of normal bone marrow and malignant plasma cells isolated from multiple myeloma patient samples reveals a significantly higher level of HDAC11 expression associated with malignancy. Similar results are observed in 8 of 12 myeloma cell lines suggesting that HDAC11 expression may provide a distinct survival advantage to malignant plasma cells. Further stratification of patients into “newly diagnosed” and “proteasome inhibitor resistant” categories defines a positive correlation between HDAC11 expression and refractory disease. Treatment of the myeloma cell lines with Quisinostat, a second-generation HDAC inhibitor with enhanced selectivity for HDAC 1, 2, 4, 10 and 11 induces growth retardation at low nanomolar concentrations. Future studies will entail direct targeting of HDAC11 in myeloma cell lines and patient specimens to determine the contribution of HDAC11 to Quinsinostat activity. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The demonstration of HDAC11 overexpression in primary human myeloma cells provides a framework for therapeutics targeting this HDAC in multiple myeloma. Citation Format: Eva Sahakian, Jason Brayer, John Powers, Mark Meads, Susan Deng, Allison Distler, Melissa Alsina, Taiga Nishihori, Rachid Baz, Alejandro Villagra, Javier Pinilla-Ibarz, Eduardo Sotomayor, Kenneth Shain. Histone deacetylase 11 (HDAC11) regulates B cell lymphopoiesis and potentiates plasma cell survival in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5537. doi:10.1158/1538-7445.AM2014-5537