PI-resistant Multiple Myeloma Research Articles

Abstract 7191: Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma

Abstract Background: Resistance to proteasome inhibitors (PI) bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of multiple myeloma (MM); thus, identification of novel therapeutic options is an unmet medical need. ALK inhibitors have been shown to exhibit anti-MM activity in monotherapy or in combination with CFZ. Our data showed that MM cells were negative for ALK; thus, we aimed to identify the mechanism of action of the ALK inhibitor ceritinib in MM cells and the mechanism of synergy between ceritinib and CFZ. Methods: A set of PI-naïve and PI-resistant MM cells was used in this study. Genome-wide CRISPR/Cas9-based screening using the Brunello library was performed in the AMO-1 cell line. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30 database. RNA sequencing was used to determine expression changes after treatment, and RNA-seq data from patients included in the CoMMpass study were analyzed. Western blotting was performed to assess the levels of total and phosphorylated protein. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and CFZ on the intracellular metabolites. An in vivo mouse model based on the orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Among approved ALK inhibitors, ceritinib has been identified as the most synergistic cytotoxic combination with CFZ in PI-naïve and PI-resistant MM cells. An in silico search of the ChEMBL database identified InsR and IGF1R as receptor tyrosine kinase that are inhibited by ceritinib. PI-naïve, PI-resistant cell lines, as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and the transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, FOXO1 inhibition using a selective chemical inhibitor protected the cells from ceritinib-induced cytotoxicity. Next, ceritinib inhibited mTORC signaling and induced the expression of genes related to cell cycle arrest, which are downstream of FOXO1. Likewise, ceritinib, by targeting the InsR/IGF1R, impaired purine-pyrimidine and amino acid homeostasis, suggesting metabolic and proliferation shut-down and amino acid starvation due to downstream mTORC and Akt inhibition and FOXO1 induction. The combination of ceritinib and CFZ was superior to CFZ in PI-resistant MM in vivo and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by inhibiting InsR/IGF1R and downstream Akt signaling. Therefore, ceritinib is a promising treatment option for PI-resistant MM. Supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Andrej Besse, Tiberiu Totu, Marianne Kraus, Anthonius P. Janssen, Jana Veprkova, Max Mendez Lopez, Ondrej Slaby, Marija Buljan, Mario van der Stelt, Christoph Driessen, Lenka Besse. Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7191.

MUC20 regulated by extrachromosomal circular DNA attenuates proteasome inhibitor resistance of multiple myeloma by modulating cuproptosis

BackgroundProteasome inhibitors (PIs) are one of the most important classes of drugs for the treatment of multiple myeloma (MM). However, almost all patients with MM develop PI resistance, resulting in therapeutic failure. Therefore, the mechanisms underlying PI resistance in MM require further investigation.MethodsWe used several MM cell lines to establish PI-resistant MM cell lines. We performed RNA microarray and EccDNA-seq in MM cell lines and collected human primary MM samples to explore gene profiles. We evaluated the effect of MUC20 on cuproptosis of PI-resistant MM cells using Co-immunoprecipitation (Co-IP), Seahorse bioenergetic profiling and in vivo assay.ResultsThis study revealed that the downregulation of Mucin 20 (MUC20) could predict PI sensitivity and outcomes in MM patients. Besides, MUC20 attenuated PI resistance in MM cells by inducing cuproptosis via the inhibition of cyclin-dependent kinase inhibitor 2 A expression (CDKN2A), which was achieved by hindering MET proto-oncogene, receptor tyrosine kinase (MET) activation. Moreover, MUC20 suppressed MET activation by repressing insulin-like growth factor receptor-1 (IGF-1R) lactylation in PI-resistant MM cells. This study is the first to perform extrachromosomal circular DNA (eccDNA) sequencing for MM, and it revealed that eccDNA induced PI resistance by amplifying kinesin family member 3 C (KIF3C) to reduce MUC20 expression in MM.ConclusionOur findings indicated that MUC20 regulated by eccDNA alleviates PI resistance of MM by modulating cuproptosis, which would provide novel strategies for the treatment of PI-resistant MM.

Targeting IGF1R/Insr Pathway with Approved ALK-Inhibitors Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Background: The treatment of multiple myeloma (MM) has advanced rapidly with the discovery of proteasome inhibitors (PIs) bortezomib (BTZ) and carfilzomib (CFZ). PIs disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in MM cells, leading to apoptosis. However, despite this success, essentially all patients with MM eventually develop resistance to PIs. Therefore, identifying strategies to overcome PIs resistance is clinically important. ALK inhibitors exhibit anti-MM activity ex vivo. Previously, the ALK inhibitor ceritinib has been shown to inhibit IGF1R/InsR signaling. Signaling through the IGF1/IGF1R axis contributes to acquired resistance to BTZ, and PI activity is reduced in the presence of IGF1, suggesting that IGF1R inhibitors may enhance the cytotoxic effect of PIs. To date, IGF1R/InsR inhibitors have not been successful as single agents in clinical trials and no formally approved drugs are available. Importantly, the drug repurposing approach offers a promising strategy for drug development, particularly for MM. Therefore, we aimed to identify i) whether ALK inhibitors are cytotoxic in MM by targeting the IGF1R/InsR pathway, and ii) whether they can be combined with PIs to overcome PI resistance in vitro and in vivo. Methods: A set of PI-naïve and PI-resistant cells was used in this study. The cytotoxicity of drugs was determined by CCK8 assay in cell lines and by CellTiter-Glo assay in primary cells. Genome-wide CRISPR/Cas9-based loss-of-function screening using the Brunello library was performed in the AMO-1 cell line to identify the mechanism of action of ceritinib. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30. Western blotting was performed to assess the levels of total and phosphorylated proteins. RNA sequencing was used to determine the number of transcripts in PI-sensitive and PI-resistant cells, and RNA-seq data from patients included in the CoMMpass study were analyzed. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and carfilzomib on intracellular metabolites. An in vivo mouse model based on orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Initially, seven ALK inhibitors were tested in PI-naïve and PI-adapted MM cell lines. Based on IC 50 values, the most effective ALK inhibitors to induce cytotoxicity in MM were ceritinib > brigatinib > entrectinib. The combination of ceritinib, brigatinib, and entrectinib showed synergistic cytotoxicity with PI BTZ and CFZ and overcame PI-resistance in four different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling and impaired purine-pyrimidine and amino acid production pathways, suggesting a metabolic and proliferation shut-down and amino acid starvation due to targeting of the upstream receptor tyrosine kinases (RTK), which are essential for cell viability and thus not identified by CRISPR screening. Further search in the ChemBL database identified InsR and IGF1R, as RTK that was potently inhibited by ceritinib. PI-naïve and PI-resistant cell lines as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. Accordingly, co-treatment of PI-resistant cells with a combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Importantly, the combination of ceritinib and CFZ was superior to CFZ in an orthotopic mouse model bearing PI-resistant MM cells and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by targeting InsR/IGF1R signaling, which is essential for PI resistance in MM. Therefore, ceritinib represents a promising, potential option for the treatment of PI-resistant MM.

Abstract 2851: ALK-inhibitors as a novel potential therapy for proteasome inhibitor-resistant multiple myeloma

Abstract Background: Proteasome inhibitors (PI) disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in multiple myeloma (MM) cells, leading to apoptosis. Resistance to PI bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of MM. The drug repurposing approach offers a promising strategy to classical drug development in particular in MM. Previously, ALK-inhibitors were shown to have anti-MM activity ex vivo. Therefore, we aimed to identify the mechanism of action of ALK-inhibitors against MM cells and to identify promising drug therapy combinations of ALK-inhibitors for refractory MM. Methods: A set of PI-naïve and PI-resistant cells was used in the study. Viability of the cells was determined by CCK8 viability assay. Genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library was used in MM AMO-1 cell line to identify mechanism of action of ceritinib. Western blot was used to assess levels of total and phosphorylated proteins. RNA-seq was used to determine the amount of transcripts in PI-sensitive and PI-resistant cells. Results: Initially, 7 different ALK-inhibitors were tested across PI-naïve and PI-adapted MM cell lines. Based on the IC50 values, the most effective ALK-inhibitors to induce cytotoxicity in MM were ceritinib > brigatinib > crizotinib > entrectinib. The combination of ceritinib, brigatinib and entrectinib showed synergistic cytotoxicity with the PI BTZ and CFZ and overcame PI-resistance in 4 different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in negative regulation of mTORC signalling (NPRL2, NPRL3, TSC1, TSC2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling, suggesting a shut-down of cellular proliferation due to targeting of the upstream receptor-tyrosine kinases (RTK), essential for cell viability and thus not identified by the screening. Further search identified InsR and IGF1R as the overlapping RTK inhibited by ceritinib and brigatinib. Co-treatment of PI-resistant cells with the combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Our data further show that PI-naïve and resistant cells are negative for ALK, but positive for InsR and IGF1R expression. Conclusion: Crizotinib inhibits InsR/IGF1R signaling in MM, which is essential for MM survival and together with CFZ it overcomes PI-resistance. Therefore, it represents a promising and already available therapy for PI-resistant MM patients. Citation Format: Lenka Besse, Andrej Besse, Marianne Kraus, Matej Jasik, Ondrej Slaby, Christoph Driessen. ALK-inhibitors as a novel potential therapy for proteasome inhibitor-resistant 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 2851.

Combination therapy for treating drug-resistant multiple myeloma

Abstract Proteasome inhibitors (PIs) have remarkably improved the survival of multiple myeloma (MM) patients but dose-limiting toxicities and the development of drug resistance limit their long-term utility. Elevated TGF-β levels in MM patient sera correlate with chemoresistance, disease progression, metastasis and poor prognosis. Therefore, we evaluated the anti-MM therapeutic potential of a TGF-β type I receptor kinase inhibitor, Vactosertib. In vitro treatment of Vactosertib synergistically inhibited the growth of PI-resistant MM cells in combination with the ixazomib by suppressing TGF-β activation of Smad2/3 and expression of PSMB5, which encodes the proteasome 5 catalytic subunit targeted by PIs. Oral administration of Vactosertib as a single agent inhibited MM progression and induced a decrease in mortality and an increase in body weight of the mice bearing MM. Vactosertib alone also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) in bone marrow tumor microenvironment, and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of Vactosertib plus ixazomib was exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by greatly prolonged survival and significant a reduction in both MDSCs and Tregs. Taken together, our data provide the rationale for clinical evaluation of Vactosertib in MM and demonstrate proof-of-concept that combination of Vactosertib and PIs may overcome chemoresistance and enhance durable patient responses. Supported by 1R03CA259901-01A1

Essential Drug Targets and Pathways in PI-Resistant MM Cells

BackgroundProteasome inhibitors (PI) have emerged as a powerful, cell biology-based treatment option for multiple myeloma (MM) and build a central backbone for MM treatment with three proteasome-inhibiting drugs currently approved: bortezomib (BTZ), carfilzomib (CFZ) and ixazomib. However, despite the high anti-MM activity of PI, MM cells adapt to the selective pressure of PI treatment in most cases to date and most MM patients relapse during or after treatment with PI, develop PI-refractory disease and ultimately die. Therefore, understanding and overcoming PI resistance is a key challenge for MM therapy. Our previous in vitro studies on PI-resistant MM suggest that PI-adapted, MM cells show very distinct features of general metabolism and cell biology that differentiate them from PI-sensitive MM, derived from the same cell line. We hypothesize that this highly specialized and adapted nature of PI-resistant MM offers novel areas of vulnerability, that differ from the therapeutic targets in PI-sensitive MM. The aim of our study was to identify essential drug targets and pathways in PI-resistant MM using genome-wide functional screening with the CRISPR/Cas9 system that could serve as novel therapeutic targets in PI-resistant MM.MethodsWe used genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library in L363-BTZ and RPMI-8226-BTZ cells, adapted to grow in the presence of 90 nM BTZ. The overlapping bortezomib genetic sensitivity candidates were further validated in the set of BTZ-resistant cells (L363-BTZ, RPMI-8226-BTZ, MM1S-BTZ and AMO-BTZ) cells using shRNA silencing or single-gene specific knockout or genetic overexpression using CCK8 viability assay. Subsequent functional analysis of the highest ranking BTZ sensitivity candidates in BTZ-adapted cells included apoptosis and cell cycle analysis, qPCR and western blotting, SILAC, proteasome activity determination using activity-based probes and FRAP analysis.ResultsCRISPR/Cas9-screening identified two candidate genes for BTZ sensitivity, ECPAS (KIAA0368; Ecm29 Proteasome Adaptor and Scaffold protein) and PSME1 (an 11S regulator complex subunit), as consistent screening hits in two independent BTZ-adapted MM cell lines. Both genes are related to proteasome, but do not build the proteasome core particle and do not have a proteolytic activity. Specific knock-down or knock-out of ECPAS sensitized PI-naïve cells to BTZ and CFZ, while significantly more sensitizing BTZ-adapted cells to both PI. Likewise, overexpression of PSMF1, an inhibitor of 11S regulator complex, sensitized BTZ-resistant as well as sensitive cells to BTZ. ECPAS-depleted BTZ-adapted cells showed accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, cell cycle arrest and induction of apoptosis, together with changes in protein synthesis after the treatment with 50 nM bortezomib, in contrast to BTZ-adapted control cells. FRAP analysis of cells with GFP-tagged PSMD6 revealed that the intracellular mobility of proteasomes in ECPAS-depleted cells was reduced. Importantly, proteasome activity determined by activity-based probes was not impaired in ECPAS-depleted cells.ConclusionIn conclusion, BTZ-resistant MM cells uniquely show a high dependency on the proteasome adaptor and scaffold protein ECPAS, which has been shown to be involved in coupling of proteasome in different compartments and promotes proteasome dissociation under oxidative stress. Specifically in PI-resistant MM, ECPAS is important to ensure functional proteasome, is involved in controlling the intracellular mobility of proteasomes, likely to ensure high proteasome turnover. ECPAS therefore represents a novel candidate that may be targeted to specifically re-sensitize PI-resistant MM cells to proteasome inhibitor treatment. DisclosuresNo relevant conflicts of interest to declare.

P-091: Genome-wide CRISPR/Cas9 screening identifies proteasome-related specific vulnerabilities as potential treatment options of proteasome inhibitor-resistant multiple myeloma

P-091: Genome-wide CRISPR/Cas9 screening identifies proteasome-related specific vulnerabilities as potential treatment options of proteasome inhibitor-resistant multiple myeloma

Defining the Most Effective Proteasome Inhibition in Multiple Myeloma: Head-to-Head Comparison of Currently Available Proteasome Inhibitors

Introduction: Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (β1, β2 and β5 of the constitutive (c) and the immunoproteasome (i), respectively). They differ in substrate preferences and affinity for PIs. β5c was identified as the rate-limiting proteolytic proteasome activity, while β2 and β1 activity received little attention. Consecutively, all proteasome-inhibiting drug candidates (bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, marizomib) by design target β5c, but differ in co-inhibition of non-β5 activities. Current chemical biology tools allow highly selective inhibition and visualization of individual proteasome subunits in living cells. We lack data that identify the most effective pattern of β5 and non-β5 type of proteasome inhibition in MM cells, as well as a head to head comparison of PIs and drug candidates, and their proteasome subunit inhibition profile in relation to pharmacodynamic activity. Such data shall allow rational selection and positioning of proteasome-inhibiting drugs.Methods: Head to head comparison of bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib and marizomib was performed in MM cells. Non-commercial, highly subunit-selective PIs and activity-based fluorescent chemical probes (ABP) were used to assess the most effective pattern of inhibition of proteasome subunits, in conjunction with a fluorescent ubiquitin Ub-G67V-GFP construct, and related to the viability of MM.Results: All commercial PI tested target primarily β5 activity and in higher concentrations co-targeted β2 and/or β1 (Table 1). In equimolar comparison, most effective PI was marizomib, followed by bortezomib, delanzomib, carfilzomib, ixazomib and oprozomib. MM cell viability inversely correlated with functional proteasome inhibition. Surprisingly, during short term, 1h pulse exposure mimicking the pharmacokinetics of i.v. proteasome inhibiting drugs, β5-selective proteasome inhibition was not sufficient to functionally inhibit protein degradation or induce cytotoxicity, even in PI-sensitive MM cells. Only co-inhibition β5 with either β1 or β2 resulted in accumulation of polyubiquitinated protein and decreased MM cell viability. Long time (48h) continuous β5-selective inhibition likewise induced cell death in PI-sensitive MM, however, only co-inhibition of β5 and β2 activity induced cytotoxicity in PI-resistant MM.Conclusion: All commercial PIs by design target β5c activity and differ in their co-targeting of non-β5 activities. Co-inhibition of β1 and β2 determines cytotoxicity. Selective inhibition of β5 by pulse exposure is not sufficient, but co-inhibition of β5 and β2 is the most effective combination to induce cytotoxicity, in particularly in PI resistant cells. This inhibition pattern is provided only by marizomib and high dose carfilzomib. By contrast, low dose of carfilzomib does not deliver co-inhibition of β2 and therefore has no advantage over bortezomib. Together, these results give a rational basis to select individual proteasome inhibitors for the treatment of MM: cytotoxicity in PI-sensitive MM is achieved by co-targeting of β5 together with β1 or β2 with a pulse treatment, or by prolonged β5 inhibition (s.c. bortezomib, orally administered ixazomib). In PI-resistant MM only dual β2/β5 inhibition is able to achieve meaningful cytotoxicity, which is provided by carfilzomib or marizomib. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nelfinavir Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma By Modulating Membrane Lipid Bilayer Composition and Fluidity

INTRODUCTION Nelfinavir is a highly lipophilic, first generation HIV-protease inhibitor (HIV-PI) approved for HIV treatment. It has largely been replaced by next-generation HIV-PI with increased specificity and efficacy for HIV therapy, partly reflecting the significant rate of the off-target activity of nelfinavir. Increasing preclinical and clinical evidence shows that nelfinavir has broad anti-cancer activity as a single agent and in combination, potentially related to its off-target activity in mammalian cells. Nelfinavir is particularly effective in the treatment of proteasome inhibitor-refractory multiple myeloma (MM), where the combination of nelfinavir+bortezomib+dexamethasone yielded an overall response rate (ORR, PR or better) > 65% in a Phase II clinical trial. The targets and molecular mechanism of action of nelfinavir in MM are unknown. This hampers both, a rational clinical repositioning and development of nelfinavir as antineoplastic drug, as well as the design, synthesis and testing of next generation nelfinavir-like compounds with optimized antineoplastic activity and improved specificity or pharmacologic properties. We therefore aimed to take an unbiased target-identification approach to identify molecular targets of nelfinavir in human malignant cells and link them to cell biological processes and mechanisms that mediate sensitivity or resistance to nelfinavir treatment. METHODS Proteome-wide affinity-purification of targets binding the nelfinavir active site was combined with genome-wide CRISPR/Cas9-based screening to identify protein partners interacting with nelfinavir and candidate genetic contributors affecting nelfinavir cytotoxicity. Multiple intracellular reporter systems including RUSH system, ATP/ADP constructs; FRAP microscopy, Seahorse measurements, flow cytometry, qPCR, metabolic labelling, lipidomics and viability assays were used to dissect functional alterations in pathways related to nelfinavir targets. RESULTS We identified a common set of proteins interacting specifically with the active site of nelfinavir. These proteins are embedded in intracellular, lipid-rich membranes of mitochondria (VDAC1,2,3, ANT2), endoplasmic reticulum (BCAP31, CANX, SRPRB) and nuclear envelope (PGRMC2) and are consistent across multiple cancer cell types. ADIPOR2, a key regulator gene of membrane lipid fluidity, was identified as a key nelfinavir resistance gene, while genes involved in fatty acids (FAs) and cholesterol metabolism, vesicular trafficking and mitochondria biogenesis are candidate sensitivity genes. We further show that via binding to proteins in lipid-rich membranes nelfinavir affects membrane composition and reduces membrane fluidity, leading to induction of FAs synthesis and the unfolded protein response (UPR). Via its structural interference with membrane fluidity, nelfinavir impairs the function and mobility of a diverse set of membrane-associated proteins and processes, such as glucose flux and processing, mitochondria respiration, energy supply, transmembrane vesicular transport and ABCB1-mediated drug efflux, as we show in different reporter systems in live MM cells. These functional effects are prevented by addition of metabolically inert lipids to be incorporated in membranes, supporting a direct structural activity of nelfinavir. The adaptive biology of proteasome inhibitor (PI)-resistant myeloma relies on metabolic reprogramming and changes in lipid composition, drug export and down-modulation of the UPR. Modulation of membrane fluidity and depletion of FAs/cholesterol is synergistic with proteasome inhibitors in PI-resistant MM. Thus, the mechanism of action of nelfinavir perfectly matches with the biology of PI-resistant MM, serving as a molecular rational for its significant clinical activity. CONCLUSION We here demonstrate in vitro that the activity of nelfinavir against MM cells is triggered through changes in lipid metabolism and the fluidity of lipid-rich membranes. Pharmacologic targeting of membrane fluidity is a novel, potent mechanism to achieve anti-cancer activity, in particular against PI-refractory MM. This mechanism explains the clinical activity of nelfinavir in MM treatment as well as the key side effects of nelfinavir during antiretroviral therapy. Disclosures No relevant conflicts of interest to declare.

Profiling the Cell Surface Landscape of Proteasome Inhibitor-Treated and -Resistant Multiple Myeloma to Guide Immunotherapy Targeting, Diagnosis, and Biology

Introduction: Proteasome inhibitor (PI) resistance remains a major clinical challenge in multiple myeloma (MM). As the proteasome plays a central role in cellular protein homeostasis, we hypothesized both PI treatment and resistance might rewire protein transport and recycling pathways, thereby leading to broad changes in cell surface protein expression. Defining the cell surface proteome has become increasingly important in MM to quantify immunotherapy target expression, identify potential biomarkers of drug response or resistance, and reveal proteins mediating interaction with the tumor microenvironment. Unbiased mass spectrometry approaches allow for profiling of hundreds of surface proteins simultaneously, allowing for novel protein discovery and extending beyond the limits of flow or mass cytometry. Methods: PI-resistant cells derived from cell lines AMO-1, L363, and RPMI-8226, and ARH-77 were grown in 90nM Bortezomib or Carfilzomib as previously described (Soriano et al, Leukemia (2016)). N-glycoproteomics was performed in triplicate on PI-resistant and parental lines. Briefly, glycosylated cell surface proteins were biotinylated prior to enrichment with Neutravidin beads and downstream LC-MS analysis. For perturbation studies, AMO-1 and RPMI-8226 cells were treated for 48 hours with Bortezomib, Lenalidomide, or CB-5083 prior to N-glycoproteomics. Flow cytometry was performed to validate surface protein abundance changes in PI-resistant lines, as well as with MM cell lines and ex vivo MM patient bone marrow mononuclear cells (PBMCs) treated with bortezomib and lenalidomide. For ex vivo MM patient cell surface proteomics, plasma cells were isolated from PBMCs with anti-CD138 magnetic beads prior to N-glycoproteomic analysis. Mass spectrometry data was analyzed in Maxquant and statistical analysis was performed in Perseus and R. Results: Supporting our hypothesis, we found that the MM surfaceome is broadly re-wired in the PI-resistant state. Using N-glycoproteomics, we identified EVI2B, CD53, CD50, and ITGB7 as downregulated and CD151, CD10, and SERC3 as upregulated in PI-resistant myeloma cell lines (up and downregulated defined as at least a +/- log2 fold change of 1.5 and p < .05). Notably, ITGB7 is under development as an immunotherapy target for MM (Hosen et al, Nat Med (2017)). To understand how surface remodeling in the PI-resistant state compares with short-term exposure to PI, we treated MM cells with bortezomib and again found broad surfaceome remodeling, including loss of immunotherapy target BCMA. We identified GITR, CD53, and ITGB7, among other proteins, as downregulated in both bortezomib-exposed and PI-resistant MM, suggesting that expression of proteins involved in therapeutic resistance might be rapidly modulated in initial cycles of therapy. To compare surfaceome changes identified in PI-exposure and resistance and those seen under anti-MM therapeutics with alternative mechanisms of action, we performed N-glycoproteomics on MM cells treated with the clinical immunomodulating agent Lenalidomide and the cytotoxic p97/VCP inhibitor CB-5083. Interestingly, we found MUC1, a proposed myeloma oncoprotein, to be upregulated under lenalidomide in AMO-1 but downregulated in the PI-resistant state. We validated surface protein abundance changes in PI-resistant MM cell lines and drug-treated MM cell lines or ex-vivo MM patient samples by flow cytometry. For additional validation, we are currently optimizing methods for N-glycoproteomics on CD138+ cells isolated from primary MM patient bone marrow aspirates. Initial results suggest several markers identified in cell lines are also highly expressed in primary PI-resistant MM. Conclusions: Unbiased N-glycoproteomics reveals broad cell surface protein remodeling in PI-resistant MM. Short-term treatment with bortezomib or lenalidomide also leads to significant surface alterations, with differential expression of proteins including MUC1, GITR, and BCMA potentially playing a role in drug resistance and informing rational combination with immunotherapies. Disclosures Martin: Roche and Juno: Consultancy; Amgen, Sanofi, Seattle Genetics: Research Funding. Wong:Celgene: Research Funding; Janssen: Research Funding; Roche: Research Funding; Fortis: Research Funding. Wiita:Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; UCSF: Patents & Royalties.

Trial in Progress: Phase I Dose-Escalation and Dose-Expansion Trial of a Novel Glutaminase Inhibitor (CB-839 HCl) in Combination with Carfilzomib and Dexamethasone in Relapsed and/or Refractory Multiple Myeloma

Background: c-MYC activation is an early event of myeloma pathogenesis. It upregulates the expression of the glutaminase 1 (GLS1) enzyme which converts glutamine to glutamate in the mitochondria. Glutamate is required for the biosynthesis of various molecules in the tricarboxylic acid (TCA) cycle (i.e., glutamine anaplerosis). CB-839 HCl is a first-in-class, orally available, selective, noncompetitive inhibitor of GLS1. This inactivation of GLS1 results in an increase of glutamine and a decrease of glutamate and several TCA cycle intermediates within cancer cells, leading to a decrease in their proliferation and/or an increase in cell death. In the phase 1 study, CX-839-002, the safety and tolerability of CB-839 HCl was evaluated in patients with hematological tumors (multiple myeloma (MM) and non-Hodgkin's lymphoma), either as monotherapy or in combination with pomalidomide and dexamethasone or with dexamethasone alone. It was determined to be well tolerated and the maximal tolerated dose (MTD) was not reached. Proteasome inhibitors (PI) are the cornerstone agents in the treatment of myeloma. They disrupt normal protein homeostasis causing an induction of cellular proteotoxic stress, thus, making it an effective strategy against myeloma plasma cells, which naturally mass-produce large quantities of immunoglobulin proteins. PI-resistant MM cells are associated with changes in cellular bioenergetics that favor the increased use of mitochondrial respiration for energy production. Given the increased reliance of PI-resistant MM cells on mitochondrial respiration, and the critical role of glutamine for cellular respiration, inhibition of glutamine metabolism is a rational molecular strategy for the treatment of PI-resistant MM. Furthermore, pre-clinical studies demonstrate the in vitro and ex vivo synergism of CB-839 HCl with carfilzomib (CFZ) in terms of its cytotoxicity and anti-proliferation capacity in various primary human myeloma cell lines and primary patient myeloma cells respectively. As a result, this novel combination of glutaminase inhibition with proteasome inhibition appears promising as a therapeutic combination in MM and warrants further clinical investigation. Methods: This study is a phase 1, multicenter clinical trial of CB-839 HCl in combination with carfilzomib and dexamethasone for patients with relapsed and/or refractory myeloma. Part A of this trial is a 3+3 dose escalation design and Part B is a dose expansion cohort at the RP2D determined in Part A. Up to a maximum of 42 patients will be enrolled at participating ETCTN sites. CFZ will be administered in its usual weekly dosing schedule of days 1, 8 and 15 of a 28 day schedule along with dexamethasone on days 1, 8, 15 and 22. CB-839 will be started at a dose level of 400 mg twice daily and will be investigated to a maximum dose of 800 mg twice daily. Prior to day 1 of Cycle 1, we will administer a 7 day lead in of CB-839 monotherapy before combining it with CFZ. Key inclusion criteria are having relapsed/refractory myeloma with at least 2 prior lines of therapy and prior exposure to PIs, immunomodulators and Anti-CD38 monoclonal antibodies, having measurable disease, adequate hematologic reserve, kidney function and liver function. Key exclusion criteria are being refractory or intolerant to CFZ, adverse cardiac history, central nervous system disease and AL amyloidosis. The primary objective of this trial is to determine the MTD or recommended phase II dosing (RP2D) of CB-839 HCl in combination with carfilzomib and dexamethasone. The secondary objective is to evaluate the safety and tolerability as well as the overall response rate (ORR) associated of CB-839 HCl in combination with carfilzomib and dexamethasone. Correlative objectives will evaluate plasma pharmacokinetic profiles of CB-839 HCl and carfilzomib when used in combination. They will also evaluate potential predictive and prognostic biomarkers as well as resistance mechanisms using genomic DNA, RNA, flow cytometry, immunohistochemistry and metabolomics-based assessment platforms. Recruitment is ongoing and this trial is registered on clinicaltrials.gov: NCT03798678. Disclosures Baz: Bristol-Myers Squibb: Research Funding; Sanofi: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Merck: Research Funding. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding.

In silico Prediction Followed By in Vitro validation Identifies the Survivin Inhibitor YM155 As a Potent Secondary Drug Against Pi-Resistant Myeloma

Drug resistance is a major cause of concern in cancer chemotherapy that not only reduces efficacy but also causes toxicity through overdosing. Multiple myeloma (MM) is the second-most common haematological malignancy in the USA that remains a challenging disease to cure due to the presence of significant complexity and heterogeneity at molecular level with high treatment cost. Proteasome inhibitor, the standard of care drug, has proven to remarkably improve myeloma patient outcomes but is associated with dose limiting toxicities and drug resistance. Therefore, new combination treatment regimens are urgently needed for the treatment of PI-resistant MM that can lower the required dose of standard-of-care drug without compromising on treatment efficacy. We have created an optimization-regularization based computational prediction method called secDrug that uses large-scale pharmacogenomics databases to identify novel secondary drugs against PI-resistance when used as single agent or in combination. We applied secDrug to PIs-resistant cell lines (>100) in the Genomics of Drug Sensitivity in Cancer (GDSC) database and predicted the top drugs that can be best combined with PIs to overcome PI-resistance in MM. These include: Survivin inhibitor (YM155), Nicotinamide phospho ribosyl transferase or Nampt inhibitor (FK866), PIKfyve inhibitor (YM201636), Raf inhibitor (PLX-4720), Bcl2 inhibitor (Navitoclax),HSP90 inhibitor (17-AAG), AKT inhibitor (KIN00102), HDAC inhibitors (Panobinostat, SAHA), S6K1-specific inhibitor (PF-4708671), and the neddylation inhibitor (MLN4924). To validate our prediction results, we treated human multiple myeloma cell lines (HMCLs) highly resistant to the proteasome inhibitors Bortezomib, Carfilzomob, Oprozomib and Ixazomib and >10 clonally derived acquired resistant cells with the top predicted best secondary drugs. Next, we treated three pairs of parental PI-sensitive (P) and clonally-derived PI-resistant (R) cell lines from PI-sensitive (P) HMCLs representing acquired PI-response with the top predicted drug combinations. Our preliminary in vitro data confirmed our in silico predictions of secondary drugs based on secDrug analysis. Among these, YM155 and YM155 + Ixazomib showed strikingly very high in vitro cytotoxicity against PI- resistant MM cell lines (Figure 1). The results, based on Chou-Talalay's combination index (CI) theorem, show that this combination works highly synergistically where the IC50 of ixazomib in MM cells was significantly reduced in presence of YM155. Similar results were obtained for the other top predicted combination regimens. Our findings provide a strong case for combining YM155 with ixazomib to enhance sensitivity or overcome resistance to ixazomib and thereby improve patient outcome. Further, this study provides additional support towards using secDrug for predicting secondary drugs in cancers resistant to the standard-of-care therapy. Currently, we are exploring the molecular mechanisms behind the synergism between proteasome inhibitors and the top drugs identified by our algorithm as candidates for secondary combination therapies using next-gen transcriptomics and single-cell analysis approaches. Earlier studies have shown that YM155 potentially induces selective cell death in human pluripotent stem cells. Further, the presence of cancer stem cells sub population/ MM-CSCs in drug-resistant tumors have been shown to significantly contribute to the development of PI-resistance in MM. Hence, we propose that YM155 reverses PI-resistance by reducing the stem cell load in multiple myeloma cell line model system. Therefore, our future plan is to assess the effects of YM155 on MM-cancer stem cells like subclones/ sub population/ MM-CSCs including CD19- CD138- quiescent stem cells, ALDH+ and side populations/SP) in drug-resistant tumors. Overall, our in vitro validation results corroborated with our in silico prediction of secondary drugs. Disclosures No relevant conflicts of interest to declare.

Repurposing Clofazimine As a Novel Drug for the Treatment of PI-Resistant Stem Cell-like Subclones in Myeloma

Multiple myeloma (MM) is the second-most common hematopoietic malignancy in the United States with estimated new reported cases of 32,110 and deaths of 12,960 in 2019. Despite recent improvement in therapies, including proteasome inhibitors (PIs) - the standard-of-care drug, MM still remains a difficult-to-cure disease with significant complexity and heterogeneity at the molecular level. Recent studies have shown that the presence of cell sub populations with potential cancer stem cell-like properties (MM-CSCs), including CD19- CD138- quiescent stem cells, dormant cells, ALDH+ cells and side populations/SP, in drug-resistant tumors significantly contribute to the development of PI resistance in myeloma. However, no study so far has attempted to develop drugs specifically targeting MM-CSCs involved in drug resistance. Further, the gene signature underlying these sub cellular populations are yet to be revealed. We have previously demonstrated that Clofazimine (CFZ), an anti-leprosy drug, could be successfully repurposed for the treatment of Chronic myeloid leukemia (CML) by specifically targeting quiescent stem cell population (CD34+CD38-, CFSE-bright) in drug resistant patient samples. Here, we hypothesized that we will repurpose CFZ as a novel drug for the treatment of PI-resistant MM by targeting the sub-cellular stem cell-like subclones. Therefore, in the current study we utilized our panel of >50 human myeloma cell lines (HMCLs) representing innate PI-response/resistance and >10 pairs (parental and PI-resistant lines generated using dose escalation over a period of time) of clonally derived PI-resistant HMCLs representing acquired resistance to repurpose CFZ for the management of PI-resistant MM. Cells were treated with CFZ and the PIs Bortezomib (Bz), Carfilzomib (Cz), Ixazomib (Ix) and Oprozomib (Opz) as single agents or in combination and in vitro cytotoxicity was determined using CellTiter-Glo assay. Synergy between CFZ and PIs was analyzed by Calcusyn software based on Chou-Talalay's combination index (CI) theorem. Drug-induced apoptosis, ROS generation, mitochondrial membrane potential and cell cycle arrest were evaluated by Flow cytometry. Sub-cellular stem cell-like populations were isolated using cell sorting by Flow Cytometry (FCM/FACS). Tumorigenic potential of purified MM-CSCs was determined using colony forming assay, carboxyfluorescein succinimidyl ester (CFSE) assay, Aldeflour activity and analysis of side population in resistant MM cells. We found very promising preliminary results where CFZ alone showed very potent inhibition of cell viability in HMCLs. Further, treatment with CFZ + PIs significantly improved the therapeutic index of PI administration to the cells. Similar results were observed in parental (P) vs clonally derived PI-resistant (VR) cell lines. We next evaluated if CFZ alone or in combination with PIs could erode quiescent CD138+ cells in parental-P/clonally derived PI-resistant cells-VR. Hence, we labelled RPMI-8226 (parental cells), RPMI8226-VR with CFSE and treated them with drugs for 96h. While PIs failed to reduce CFSE-bright (non-dividing) cells, CFZ alone or in combination with Ixazomib significantly reduced their number and increased CFSE-dim (dividing cell) population. Evaluation of apoptosis in these cells revealed that CFZ alone caused apoptosis in both CFSE-bright and CFSE-dim cells while combining CFZ +PI caused a more robust effect amounting to their near-obliteration. We next assessed side populations/SP in resistant cells. We found that % SP was higher in resistant cells as compared to parental cells. CFZ alone or in combination (CFZ+PI) reduced %SP in PI- resistant cell lines. Based on these results, we propose that CFZ may have strong potential to increase the therapeutic efficacy of PIs when used in combination by specifically targeting MM-CSCs sub cellular population in MM - which we are investigating further. Currently, we are: 1) validating the potency of CFZ (alone or CFZ+PI combination therapy) in primary myeloma cells/PMCs from newly diagnosed patients, followed by 2) bulk mRNA sequencing and 3) single-cell transcriptomic analysis of MM-CSCs in HMCLs and PMCs to evaluate molecular pathways involved in stem-cell based PI-resistance and to characterize PI-resistant sub-cellular stem cell populations based on gene expression signatures using our prediction analysis software SCATTome. Disclosures No relevant conflicts of interest to declare.

Preclinical Validation of Ubiquitin Receptor PSMD4 As Therapeutic Target in Multiple Myeloma

Background and Rationale Our preclinical studies have focused on Identification and validation of components within the ubiquitin proteasome system which can be targeted with novel therapies to overcome proteasome-inhibitor (PI)-resistance in multiple myeloma (MM). Our siRNA screening studies identified ubiquitin Receptor (UbR) PSMD4/Rpn10 as a mediator of MM cell growth and survival. Rpn10 is localized on the 19S regulatory lid of the proteasome, and plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. Here, we show that inhibition of Rpn10 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. Materials and Methods Rpn10 knockout 293 cell line was generated using CRISPR/Cas9. Rpn10-inducible knockdown (KD) MM.1S cell line was generated using shRNA. Drug sensitivity, cell viability, and apoptosis assays were performed using WST/CellTiter-Glo assay, and Annexin V staining, respectively. Cell signaling and caspase activity were determined by western blotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2012, 22:345-358). A xenograft human MM model was used to characterize the role of Rpn10 on tumor progression. Statistical significance was assessed with Student's t test. Results 1) Analysis of MM patient gene expression profiling database showed that Rpn10 expression inversely correlates with overall survival (n=175; p= 0.00064). 2)Real-time PCR, immunoblotting, and immunohistochemistry of MM patient bone marrow showed higher Rpn10 levels in patient MM cells and MM cell lines versus normal cells. 3)Transient transfection of MM cells with Rpn10-siRNA decreased their viability; conversely, transfection with Rpn10-WT specifically rescued cells from growth-inhibitory activity of Rpn10-siRNA. Immunoblot analysis confirmed significant knockdown of Rpn10 by Rpn10-siRNA versus scrambled (scr)-siRNA, and restoration of Rpn10 levels in cells transfected with Rpn10-WT versus Rpn10-siRNA. 4)Genetic blockade of Rpn10 decreased viability in bortezomib-resistant MM cells. 5)CRISPR/Cas9-mediated knockout (KO) of Rpn10 decreased cell growth. 5)Both Rpn10-KO and inducible Rpn10-KD cells showed elevated levels of polyubiquitylated proteins. 6)Inhibition of Rpn10 induced apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling. 7)Reduced tumor growth was noted in mice xenografted with Rpn10-KD MM.1S cells versus mice engrafted with WT-MM.1S cells. Conclusion Our data demonstrate the therapeutic potential of targeting ubiquitin receptor Rpn10/PSMD4, and provide the preclinical basis for development of Rpn10 inhibitors to overcome PI-resistance in MM. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Nelfinavir interacts with mitochondrial VDACs and disrupts oxidative phosphorylation in proteasome inhibitor resistant myeloma

Proteasome inhibitor (PI) resistance remains a problem in multiple myeloma (MM) therapy. Metabolic reprogramming towards oxidative phosphorylation (OXPHOS) and high mitochondrial ATP supply provide proteasome inhibitor resistance (Tsvetkov, NatureChemBiol 2019). The anti-HIV drug nelfinavir (NFV) overcomes PI-resistance in combination with bortezomib. We identified the molecular targets of NFV and addressed the effect of NFV on the metabolic reprogramming of PI-resistant MM.

Abstract 285: Ixazomib combined with the nuclear export inhibitors selinexor or eltanexor for the treatment of multiple myeloma

Abstract Introduction In the past decade, response and survival rates in multiple myeloma (MM) patients have been greatly increased by newer therapies. Nevertheless, most patients with MM eventually die from relapsed/refractory disease. Previous studies in our lab and in others have shown that exportin 1 inhibitors (XPO1i) are potent anti-MM drugs, whether used as single agents or in combination with proteasome inhibitors (PIs), immunomodulator drugs (IMiDs), anthracyclines, or alkylating agents. Ixazomib (IXA), an orally available PI, has been shown to be effective when combined with IMiDs, dexamethasone, alkylating agents, and/or prednisone for the treatment of patients with MM. We investigated whether the clinical XPO1i selinexor (SEL) or eltanexor (ELT) combined with IXA could overcome PI resistance in MM cell lines and in ex vivo bone marrow aspirates from PI-refractory patients. Materials and Methods PI-resistant MM cell lines 8226B25 and U226PSR and their respective parental cell lines, 8226 and U266, were treated in vitro and in vivo (mice) with SEL/IXA or ELT/IXA combinations or with each drug as a single agent. Bone marrow aspirates from newly diagnosed and PI-refractory MM patients were treated ex vivo with SEL/IXA or ELT/IXA and assayed for apoptosis. Mechanistic studies included NFkB and IkBα protein assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in SEL/IXA- or ELT/IXA-treated MM cells. Results SEL and ELT each sensitized parental and, to a lesser extent, PI-resistant MM cells to IXA, as measured by apoptosis. XPO1i/IXA treatments inhibited U266 and 8226 MM tumor growth and increased survival in mice; however, PI-resistant tumors were not sensitized by XPO1i/IXA treatment. Myeloma cells isolated from both newly diagnosed and PI-refractory MM patients were sensitized by XPO1i to IXA, as compared to untreated and single agent-treated MM cells, without affecting non-MM cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increased nuclear IkBα with the XPO1i/PI treatments. Proximity ligation found increased IkBα-NFkB complexes in treated MM cells. IkBα knockdown abrogated XPO1i/PI-induced cytotoxicity in MM cells. XPO1i/PI treatment decreased NFkB transcriptional activity. Conclusions In previous studies, we found that SEL sensitized PI-resistant cell lines to carfilzomib and bortezomib; however, IXA was less effective in these same MM cells. SEL or ELT used in combination with IXA have the potential to overcome PI drug resistance in MM patients. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Further investigation is underway to examine these mechanisms. The results presented in this study support combinatorial clinical trials in relapsed and refractory MM patients who use PI therapies. Citation Format: Joel G. Turner, Jana Dawson, Alexis Bauer, Juan Gomez, Erkan Baloglu, Yosef Landesman, Daniel M. Sullivan. Ixazomib combined with the nuclear export inhibitors selinexor or eltanexor for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 285.

Abstract LB-266: A novel computational combination-therapy prediction algorithm (secDrug) identifies the Nampt inhibitor FK866 reverses PI-resistant multiple myeloma

Abstract Drug resistance is a major cause of concern in cancer chemotherapy that not only reduces efficacy but also causes toxicity through overdosing. For example, Ixazomib, an oral proteasome inhibitor, has proven to remarkably improve myeloma patient outcomes but is associated with dose limiting toxicities and drug resistance. Therefore, new combination treatment regimens are urgently needed for the treatment of cancers, including multiple myeloma, which can lower the required dose of standard-of-care drug without compromising on treatment efficacy. We will present an optimization-regularization based computational prediction method called secDrug that uses large-scale pharmacogenomics databases to identify novel secondary drug combinations to reverse drug resistance. In this study, we applied secDrug to Ixazomib-resistant cell lines (>100) in the Genomics of Drug Sensitivity in Cancer (GDSC) database and predicted that the following FDA-approved drug classes can be best combined with Ixazomib to overcome resistance in MM: nicotinamide phospho ribosyl transferase or Nampt inhibitor (FK866), HDAC inhibitors (Panobinostat, SAHA), neddylation inhibitor (MLN4924), Raf inhibitor (PLX-4720) and HSP90 inhibitor (17-AAG). Our results corroborate with earlier studies that have indicated these drugs could potentially be used in MM, alone or in combination. To validate our prediction results, we treated our panel of >50 human multiple myeloma cell lines (Ix-sensitive and Ix-resistant HMCLs) with the top predicted drug combination - FK866 + Ixazomib. The results, based on combination index theorem, show that this combination works highly synergistically where the IC50 of ixazomib in MM cells was significantly reduced in presence of FK866. Similar results were obtained for the other top predicted combination regimens. Our findings provide a strong case for using secDrug for predicting secondary drugs in cancers resistant to the standard-of-care therapy. Furthermore, this provides additional evidence in support of combining FK866 with ixazomib to enhance sensitivity or overcome resistance to ixazomib and thereby improve patient outcome. Our future aim is to investigate the mechanism behind the synergism between proteasome inhibitors and the top drugs identified by our algorithm as candidates for secondary combination therapies using next-gen transcriptomics and single-cell approaches. Citation Format: Harish Kumar, Ujjal Kumar Mukherjee, Sayak Chakravarti, Li Chen, Brian Van ness, Amit Kumar Mitra. A novel computational combination-therapy prediction algorithm (secDrug) identifies the Nampt inhibitor FK866 reverses PI-resistant multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-266.

Proteasome Inhibition in Multiple Myeloma: Head-to-Head Comparison of Currently Available Proteasome Inhibitors

Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (β1, β2, β5), while β5 was identified as rate-limiting and is a primary target of clinically available PIs. The most effective pattern of subunit inhibition provided by these PIs for cytotoxic activity in MM is unknown. A head-to-head comparison of clinically available PIs shows that in the clinically relevant setting only the co-inhibition of β1 or β2 with β5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective β2/β5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of β5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the β5/β2 co-inhibition is the most cytotoxic in PI-resistant MM. These results give a rational basis for selecting individual PIs for the treatment of MM.

Glutaminase inhibitor CB-839 synergizes with carfilzomib in resistant multiple myeloma cells.

Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib), which are cornerstone agents in the treatment of MM. In isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. PI resistant cells exhibited increased mitochondrial respiration driven by glutamine as the principle fuel source. To target glutamine-induced respiration in PI resistant cells, we utilized the glutaminase-1 inhibitor, CB-839. CB-839 inhibited mitochondrial respiration and was more cytotoxic in PI resistant cells as a single agent. Furthermore, we found that CB-839 synergistically enhanced the activity of multiple PIs with the most dramatic synergy being observed with carfilzomib (Crflz), which was confirmed in a panel of genetically diverse PI sensitive and resistant MM cells. Mechanistically, CB-839 enhanced Crflz-induced ER stress and apoptosis, characterized by a robust induction of ATF4 and CHOP and the activation of caspases. Our findings suggest that the acquisition of PI resistance involves adaptations in cellular bioenergetics, supporting the combination of CB-839 with Crflz for the treatment of refractory MM.

Combination Therapy with Bortezomib or Carfilzomib and Selinexor Induces Nuclear Localization of Ikbα and Overcomes Acquired Proteasome Inhibitor Resistance in Human Multiple Myeloma

Introduction: Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired-resistance in MM.Materials and Methods: PI-resistant myeloma cell lines, RPMI8226-B25 and U226 PSR, and their respective parental cell lines RPMI8226 and U266, were treated both in vitro with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. In vivo studies using U266 and U266PSR tumors were performed in NOD/SCID-gamma (NSG) mice. Mice were treated with selinexor/bortezomib and single agents. Bone marrow biopsies from refractory myeloma patients were treated ex vivo with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. Mechanistic studies included NFkB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in selinexor/bortezomib treated MM cells.Results: We found that selinexor restored sensitivity of PI-resistant RPMI8226-B25 and U266PSR MM cells to bortezomib (P = 0.00055) and carfilzomib (P = 0.0017). Bortezomib, when combined with selinexor reduced U266 MM tumor growth versus single-agent bortezomib (P = 0.022) in NSG mice. NSG mice challenged with PI-resistant U266PSR MM tumors also had reduced tumor growth with selinexor/bortezomib as compared to single agent bortezomib (P = 0.0006). Combining bortezomib and selinexor improved survival in mice with U266 MM tumors (P = 0.0072) and PI-resistant U266PSR when compared to single-agent bortezomib (P = 0.0072). Myeloma cells from PI-refractory MM patients (n=14) were sensitized by selinexor to bortezomib (P = 0.002) and carfilzomib (P = 0.001) without affecting non-myeloma cells. Immunofluorescence microscopy of PI-resistant human MM cell lines found a greater than 212% increase in IkBα when compared to untreated cells (confirmed by Western blot). A similar increase in IkBα immunofluorescence was found in newly diagnosed, relapsed and refractory patient MM cells. ImageStream analyses of MM cells showed an increase in total and nuclear IkBα from selinexor/bortezomib exposure. Proximity-ligation assays showed that IkBα-NFkB-complexes were increased 12-fold in bortezomib/selinexor treated MM cells. IkBα knockdown abrogated selinexor/bortezomib induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFkB transcriptional activity in addition to a reduction of NFkB induced IAP-1, IAP-2, BCL-2, cyclin D2 and c-myc protein expression..Conclusions: Selinexor, when used with bortezomib or carfilzomib has the potential to overcome proteasome-inhibitor drug-resistance in MM. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Selinexor, an orally active selective inhibitor of XPO1-mediated nuclear export (SINE), is currently undergoing phase I/II studies in a variety of indications, including a combination with carfilzomib, in both relapsed and refractory MM patients (NCT02199665). The results presented in this study support combinatorial clinical trials in relapsed and refractory MM that utilize PI therapies. DisclosuresKashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shain:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Signal Genetics: Research Funding; Novartis: Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder.