Resistant Multiple Myeloma Cell Lines Research Articles

Bortezomib resistance in multiple myeloma is associated with increased serine synthesis

BackgroundThe proteasome inhibitor bortezomib (BTZ) is successfully applied in the treatment of multiple myeloma, but its efficacy is restricted by the wide-spread occurrence of resistance. Metabolic alterations play an important role in cancer development and aid in the cellular adaptation to pharmacologically changed environments. Metabolic changes could therefore play an essential role in the development of drug resistance. However, specific metabolic pathways that can be targeted to improve bortezomib therapy remain unidentified.MethodsWe elucidated the metabolic mechanisms underlying bortezomib resistance by using mass spectrometry-based metabolomics and proteomics on BTZ-sensitive and BTZ–resistant multiple myeloma cell lines as well as in a set of CD138+ cells obtained from multiple myeloma patients.ResultsOur findings demonstrate that a rewired glucose metabolism sustains bortezomib resistance. Mechanistically, this results in higher activity of both the pentose phosphate pathway and serine synthesis pathway, ultimately leading to an increased anti-oxidant capacity of BTZ-resistant cells. Moreover, our results link both serine synthesis pathway activity and expression of 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of serine synthesis, to bortezomib resistance across different BTZ-resistant multiple myeloma cell lines. Consistently, serine starvation enhanced the cytotoxicity of bortezomib, underscoring the importance of serine metabolism in the response to BTZ. Importantly, in CD138+ cells of clinically bortezomib refractory multiple myeloma patients, PHGDH expression was also markedly increased.ConclusionsOur findings indicate that interfering with serine metabolism may be a novel strategy to improve bortezomib therapy and identify PHGDH as a potential biomarker for BTZ resistance.

Raman spectroscopy differentiates between sensitive and resistant multiple myeloma cell lines

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific and biologically perturbing. Here, we show that single-cell micro-Raman spectroscopy can be used to discriminate between resistant and sensitive multiple myeloma cell lines based on their highly reproducible biomolecular spectral signatures. In order to demonstrate robustness of the proposed approach, we used two different cell lines of multiple myeloma, namely MM.1S and U266B1, and their counterparts MM.1R and U266/BTZ-R subtypes, resistant to dexamethasone and bortezomib, respectively. Then, micro-Raman spectroscopy provides an easily accurate and noninvasive method for cancer detection for both research and clinical environments. Characteristic peaks, mostly due to different DNA/RNA ratio, nucleic acids, lipids and protein concentrations, allow for discerning the sensitive and resistant subtypes. We also explored principal component analysis (PCA) for resistant cell identification and classification. Sensitive and resistant cells form distinct clusters that can be defined using just two principal components. The identification of drug-resistant cells by confocal micro-Raman spectroscopy is thus proposed as a clinical tool to assess the development of resistance to glucocorticoids and proteasome inhibitors in myeloma cells.

Tyrosine kinase inhibitors potentiate anti-myeloma activity of proteasome inhibitor carfilzomib in resistant multiple myeloma cell lines and patient samples

Tyrosine kinase inhibitors potentiate anti-myeloma activity of proteasome inhibitor carfilzomib in resistant multiple myeloma cell lines and patient samples

Marcks Peptide Inhibitor Displays Synergistic Cytotoxicity with Bortezomib in Drug Resistant Multiple Myeloma Cells but Enhances Autophagic Effect

Myristoylated alanine-rich C kinase substrate (MARCKS) is a protein which has recently been connected to the progression of multiple cancer types including multiple myeloma (MM). MARCKS can be phosphorylated and then activated by several PKCs in its phosphorylation site domain (PSD). All known biological functions of MARCKS including its oncogenic effects are conducted by its phosphorylated form. Although in an earlier study we have shown that MARCKS knocking down could lead to cell cycle arrest and apoptosis in drug resistant MM cell lines, whether it is possible to antagonize drug resistance of MM cells by inhibitors of MARCKS phosphorylation is still unclear.Here we investigated the anti-myeloma activity of MARCKS inhibitors in MM cells by applying MARCKS PSD peptide inhibitor (MPS), a validated 25-amino-acid peptide targeting the MARCKS PSD. Two drug resistant MM cell lines MM1R and 8226R5 as well as normal peripheral blood mononuclear cells (PBMCs) derived from three healthy volunteers were exposed to MPS for 48 hours. Compared with its mutated (inactive) form, treatment of this inhibitor led to a dose dependent cytotoxic effect on drug resistant MM cells, while it did not affect cell viability of normal PBMCs. Furthermore, MPS treatment significantly sensitized MM1R and 8226R5 cells to bortezomib exposure after 48 hrs. Co-treatment of these two agents exhibited synergistic effect on both of the above resistant MM cell lines (on 8226R5, the combination index (CI) = 0.79; on MM1R, CI=0.88).We next investigated the mechanisms by which MPS may induce cell death and overcome bortezomib resistance. Following MPS treatment, accumulation of p27Kip1as well as dampened phosphorylation of Akt1 were identified both in MM1R and 8226R5 MM cell lines, indicating that MPS treatment may lead to cell cycle arrest and growth suppression. Interestingly, the level of LC3BII, a marker of autophagy, was also significantly upregulated, which suggested an enhancement of autophagic activity after MARCKS inhibition. To confirm the correlation between MARCKS inhibition and autophagy induction, we knocked down MARCKS by transfecting shRNA lentiviral vectors into above drug resistant MM cells. Western blots showed a significantly upregulated LC3BII in both cell lines with MARCKS silencing, compared with cells transfected by scrambled shRNA. Accumulation of lysosomes as well as autophagosomes was also observed separately via cellular staining with acridine orange and florescence-labeled LC3BII antibody.Upregulation of autophagy has been tightly related to the induction of drug resistance in cancer cells. Thus, drug resistant MM cells with impaired MARCKS activity may become more dependent on the pro-survival outcome of autophagy when exposed to anti-tumor drugs like bortezomib. To examine this hypothesis, both MARCKS-silenced MM1R and 8226R5 cells as well as negative control cells expressing scramble shRNA were co-treated with bortezomib and autophagy inhibitor chloroquine (CQ) for 48hrs. MTT assays showed that the combination of CQ and bortezomib resulted in more cell death in MARCKS-silenced drug resistant MM cells than negative control cells (~40% and ~30% more cell death in MM1R (p<0.01) and 8226R5 (p<0.05) cell lines, respectively). In MARCKS-silenced cells, synergistic effects of CQ and bortezomib were also more significant than in negative control cells. Hence depletion of MARCKS activity makes MM cells more vulnerable to bortezomib when cellular autophagic activity is intercepted. We are currently examining the in vivo activity of MPS using xenograft mice models. Taken together, our results indicate that MPS peptide is effective in killing myeloma cells by inhibition of phosphorylation of MARCKS but also enhances autophagic response. This previously unappreciated correlation between MARCKS suppression and autophagy enhancement suggests that the combination of bortezomib, MARCKS inhibitor, as well as autophagy antagonists like chloroquine may contribute to the development of novel MM therapies. DisclosuresNo relevant conflicts of interest to declare.

Glutaminase Inhibitor CB-839 Enhances Proteasome Inhibitor Sensitivity in Multiple Myeloma Cells

Multiple myeloma (MM) remains largely incurable due to the emergence of therapeutic resistance. We therefore set out in this study to identify druggable molecular mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib/VELCADE, carfilzomib/KYPROLIS), which are cornerstone agents in the treatment of MM. In comparing isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. While glycolysis rates between cell lines were similar, PI resistant cells exhibited increased mitochondrial respiration characterized by higher basal oxygen consumption rates (OCR) and overall respiratory capacity. Additionally, PI resistant cells were found to have lower activation of the AMP kinase (AMPK) energy stress pathway and increased levels of NAD(P)H, which serve as electron carriers in the mitochondrial process of oxidative phosphorylation. We determined that glutamine was the principle source of fuel driving mitochondrial respiration as removal of glutamine completely inhibited OCR as well as cell proliferation, whereas glucose and pyruvate were dispensable. Given the propensity for mitochondrial respiration in PI resistant cells and the dependence on glutamine for this process, we hypothesized that targeting glutamine utilization by PI resistant cells would restore their sensitivity to the cytotoxic effects of PIs. To test this possibility, we inhibited glutamine metabolism using the small molecule GLS1 inhibitor CB-839. CB-839 repressed basal OCR and total respiratory capacity and reduced cell viability to varying degrees in a panel of PI sensitive and resistant MM cell lines. Most notably, we found that CB-839 synergistically enhanced the cytotoxic activity of multiple PIs, including bortezomib, carfilzomib, ixazomib, and oprozomib, in a genetically diverse panel of 15 PI sensitive and resistant MM cell lines. The effects of CB-839 were the most apparent in combination with carfilzomib (Crflz), where it enhanced Crflz-induced death by >4-fold. CB-839 enhanced Crflz-induced apoptosis as measured by the activation of caspase 3, 7, 8 and the cleavage of the caspase-3 substrate PARP. Mechanistically, the combination of CB-839 and Crflz induces a strong and synergistic ER stress response, characterized by the induction of ATF4 and CHOP. Our findings suggest that the acquisition of PI resistance may involve adaptations in cellular bioenergetics that may be exploited therapeutically by targeting glutamine metabolism. Furthermore, our results support the combination of clinical stage compound CB-839 with PIs, particularly Crflz, for the treatment of refractory MM. DisclosuresNo relevant conflicts of interest to declare.

Anti-Cancer Effects of Clofazimine As a Single Agent and in Combination with Cisplatin in Multiple Myeloma

Multiple myeloma (MM) is a malignant neoplasm of bone marrow plasma B cells with high morbidity. Clofazimine (CLF) is an FDA-approved leprostatic, anti-tuberculosis, and anti-inflammatory drug that was previously shown to have growth suppression effects on various cancer types such as hepatocellular, lung, cervix, esophageal, colon, and breast cancers as well as melanoma, neuroblastoma, and leukemia cells. The objective of this study was to evaluate the anticancer effect of CLF on U266 resistant MM cell line.The relative cell viability of a panel of hematological cell lines (Jurkat, U266, Namalwa, K562, HL60) treated with 10 µM CLF after 24 h of treatment significantly reduced the viability in all cell lines, with percentages ranging between 28% (U266) and 38% (Jurkat) (p<0.001). IC50 value of CLF was found as 9.8 ± 0.7 µM on the U266 cell line. Previous studies showed that this level of CLF does not inhibit growth of healthy cells, which supports safety of CLF. CLF had both dose (2, 5, 10 µM) and time (12, 24, and 48 h) dependent growth inhibitory effect. Combination chemotherapy is an approach to increase the effectiveness of chemotherapeutics as well as overcome drug resistance and suppresses side effects of drugs. Therefore, we evaluate the combination effect of CLF in U266 cells and showed that combination with cisplatin led to a synergistic interaction between two compounds in all tested dose regimes, resulting in a 2.5-7.1 fold marked increase in cell death. Importantly this synergism was observed in U266 cells, which have mutant p53 at A161T showing resistance to cytotoxic agents such as platinum analogs (cisplatin etc.). <>Depolarization of the mitochondrial membrane is one of the first events in apoptosis. JC-1 is a lipophilic and cationic dye that reversibly changes color from green to red as the mitochondrial membrane potential increases (depolarization). JC-1 assay used in both flow cytometry analyses and fluorescence microscopy images have shown that relative to the control, CLF treatment results in the depolarization of mitochondrial membrane 15, 20.5, 14.3 fold respectively at 12, 24, and 48 h in U266 cell line (Figure 1).The caspase family of cysteine proteases plays an important role in apoptosis. Caspase-3 is a major protease activated during the early stages of programmed cell death. 10 µM CLF was applied for 12, 24, and 48 h and anti-active caspase-3 PE stained U266 cells were analyzed by flow cytometry. Caspase-3 activity is enhanced 5.6, 24.5 and 13.6-fold relative to untreated controls at 12h, 24h and 48 h respectively.Phosphatidylserine (PS) translocation to the outer leaflet of the cellular membrane is one of the key steps in early stages of apoptosis. To support our previous findings on apoptotic effect of CLF, we employed Annexin-V assay. CLF treatment caused a significant increase in the percentage of early and late apoptotic cells at 12 h (2.1 and 1.8 fold respectively), 24 h (4.1 and 12.3 fold) and 48 h (10.1 and 11.5 fold). Fluorescence microscopy images also supported flow cytometry data (Figure 2). Collectively, all three apoptosis assay results show that CLF significantly induces apoptosis in U266 cells.Our study is the first to show apoptotic and growth inhibitory effects of CLF on a p53-mutant resistant MM cell line U266. Our results also proved that combined therapy employing CLF together with chemotherapeutics seems to be a possible future therapeutic approach for MM. Further in vivo and clinical studies are warranted to evaluate its therapeutic potential for resistant MM treatment. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Repurposing Leflunomide for Multiple Myeloma Treatment

Background: Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide, a commercially available oral immunosuppressive agent that has been FDA-approved since 1998 for the treatment of rheumatoid arthritis (RA) was evaluated as a potential MM therapy. The primary mechanism of action is de novo inhibition of pyrimidine synthesis by targeting dihydroorotate dehydrogenase (DHODH), and thus achieving an anti-proliferative effect in B- and T-lymphocytes. A secondary mechanism of action is inhibition of cytokine and growth factor receptor-associated tyrosine kinase activity. Methods and Results: Pre-clinical studies of teriflunomide, the active metabolite of leflunomide, showed that it inhibited cell growth and induced apoptosis in MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226) and primary MM patients’ (CD138+) plasma cells at clinically achievable concentrations (50-200 uM) in a time- and dose-dependent manner. We also found that teriflunomide induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <200 uM. In addition, teriflunomide and dexamethasone synergized in the in vitro growth inhibition of MM cell line MM.1S. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon teriflunomide exposure, MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary plasma cells from two MM patient samples were treated with 200 µM teriflunomide or DMSO control for 24 h before extraction and purification of mRNA and microRNA. mRNA-seq and miRNA-seq analysis from teriflunomide-treated MM samples revealed that similar changes were present between patient samples and cell lines. A total of 382 genes were found to be differentially expressed (225 upregulated, 157 downregulated). Upregulated genes included those that participate in defense response and negative regulation of cell growth. Genes involved in mitosis, rRNA biogenesis/processing, and immune response were generally downregulated. Analysis of microRNA-seq data from these samples revealed five differentially expressed, mostly newly discovered miRNAs that have unknown function.Conclusions: Leflunomide and its analogues demonstrated anti MM effects in vitro as well as synergy with dexamethasone. Based on our promising pre-clinical results we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory MM. DisclosuresNo relevant conflicts of interest to declare.

Abstract 541: Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance

Abstract Introduction: Multiple myeloma (MM) is a malignancy of plasma cells that is still considered incurable due to the 90% relapse rate in patients. Despite the advent of new agents the majority of MM patients relapse secondary to therapy resistance. The potential of reovirus (RV) as a novel therapeutic agent for MM under in vitro, in vivo and ex vivo conditions has been demonstrated by us and a phase I clinical trial of MM with RV therapy has shown indications of efficacy. Recently we have shown that RV could synergize with several standard of care MM drugs such as bortezomib (BTZ), carfilzomib (CFZ) and dexamethasone and importantly, enhance its therapeutic potential in therapy resistant human MM cell lines in vitro. Utilizing the syngeneic Vk*MYC BTZ resistant transplantable MM mouse model, we demonstrate that mice harbouring BTZ insensitive MM tumors significantly respond to RV treatment. Our data indicate that this RV treatment sensitivity is manifested via, direct oncolysis in conjunction with immune modulatory effects. Methods: Vk*MYC myeloma cells (Vk12598) were injected via tail vein (IV) into 3 groups of C57BL/6 wt recipient mice. Seven days post tumor injection, mice were treated with PBS (vehicle) or live (LV) or dead reovirus (DV) (5×10⁁8 PFU) administered IV every 6 days in a total of 5 doses. Serum gamma globulins (M-spike) were assessed weekly by serum protein electrophoresis. Mice were sacrificed on day 35 post tumour injection and their spleens and bone marrow (BM) were harvested. Splenic and BM cells were stained for CD45+/CD45R-/CD138+ (MM tumors), CD4+/CD8+ (T cells), CD3+/NKG2D+ (NKT cells), CD11b+Ly6C+ (monocytic myeloid derived suppressor cells (Mo-MDSC) and CD11b+Ly6G+ (polymorphonuclear (PMN) - MDSCs) and analyzed by flow cytometry. Results: Mice treated with RV demonstrated highly significant (P&amp;lt;0.001) reductions in their M-spikes at 4 and 5 weeks post treatment. RV treatment led to a significant NKT cell accumulation in splenic and BM of RV treated mice in comparison to vehicle treated mice suggestive of immune activation due to RV. Further, RV treatment led to a significant increase in BM Mo-MDSC number but not PMN-MDSC numbers. Interestingly, significant down regulation of splenic PMN-MDSCs was seen with RV treatment. Conclusions: Our results indicate reovirotherapy is successful in a validated syngeneic BTZ resistant MM model. These results have important implications for future clinical trials. Currently we are conducting further experiments to assess the immune modulatory (such as T - memory and T - regulatory cells) contributions of RV in this model. Citation Format: Chandini M. Thirukkumaran, Zhong Qiao Shi, Joanne Luider, Karen Kopciuk, Marta Chesi, Leif Bergsagel, Yuan Dong, Chunfen Zhang, Ahmed Mostafa, Kathy Gratton, Satbir Thakur, Don Morris. Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 541.

Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers

Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patient's serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers. Biological significanceExtracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.

Combination Therapy of Selinexor with Bortezomib or Carfilzomib Overcomes Drug Resistance to Proteasome Inhibitors (PI) in Human Multiple Myeloma

Purpose: Drug resistance is the greatest obstacle to the successful treatment of multiple myeloma (MM). We investigated whether the clinical XPO1 inhibitor selinexor (KPT-330), when combined with bortezomib or carfilzomib, could overcome proteasome inhibitor (PI) resistance in myeloma.Experimental Design: PI-resistant human MM cell lines 8226-B25 and U266-PSR were treated with the XPO1 inhibitors selinexor or KOS-2464 in combination with bortezomib or carfilzomib and assayed for apoptosis and viability. Mice challenged with PI-resistant human MM cells (U266-PSR) were treated with selinexor +/- bortezomib. CD138+/light-chain+ MM cells from PI-refractory MM patients were treated with selinexor +/- bortezomib or selinexor +/- carfilzomib and assayed for apoptosis. All experiments were compared to the standard of care, bortezomib therapy. IkBα-protein was assayed by Western blot and immunofluorescence microscopy and IkBα-NFkB-complex formation by proximity ligation assay. IkBα protein knockdown in human MM cells by siRNA was performed to determine the mechanism of selinexor inhibitor action. Further analysis of selinexor/bortezomib treatment on intra-cellular protein levels and intra-cellular localization was performed by lysine and N-terminal labeling with six-plex tandem mass tags (heavy isotope) and assayed by LC-MS/MS discovery proteomics.Results: Selinexor in combination with bortezomib or carfilzomib decreased viability and induced apoptosis in PI-resistant MM cells. Resistant MM cell lines were up to 10-fold resistant to single agent bortezomib or carfilzomib when compared to parental cells. The combination of the XPO1 inhibitors selinexor or KOS-2464 sensitized drug resistant cells to bortezomib (P < 0.02) and carfilzomib (P < 0.005) when compared to single agents. Selinexor and bortezomib inhibited PI-resistant MM tumor growth and increased survival with minimal toxicity in NOD/SCID-g mice. Bone marrow mononuclear cells isolated and treated with selinexor or KOS-2464 and bortezomib or carfilzomib from newly diagnosed (n=8), relapsed (n=5), and bortezomib (n=8) and carfilzomib (n=6) refractory MM patient samples were all sensitized by selinexor and KOS-2464 to bortezomib (P < 0.043) and carfilzomib (P < 0.044) as shown by increased apoptosis. Normal, non-myeloma CD138/light-chain double-negative patient cells were not sensitized to apoptosis by XPO1 inhibitors. Immunofluorescence microscopy of IkBα in 8226-B25 PI-resistant cells showed an increase in IkBα after treatment with selinexor/bortezomib as compared with vehicle control or single agent bortezomib or selinexor. Nuclear IκBα was also increased by selinexor treatment. IkBα protein expression was increased by bortezomib (70%) and selinexor (50%) versus control. The selinexor/bortezomib combination increased IkBα protein (212%) as compared to vehicle control or single agent bortezomib or selinexor. Similar results were found in drug-naïve 8226 and U226 cells, as well as PI-resistant 8226-B25 and U225-PSR cells. The increase in nuclear IkBα after selinexor treatment was confirmed by ImageStream flow cytometry.Selinexor/bortezomib therapy significantly increased IkBα-NFkB-complexes in PI-resistant MM cells. Selinexor in combination with bortezomib increased proximity co-localization of NFkB and IkBα without affecting XPO1 protein expression after 4 hours of drug treatment. Analysis of the number of NFkB-IkBα foci/binding showed that selinexor/bortezomib increased the number of foci in the nucleus versus untreated control or single agent selinexor or bortezomib (P ≤ 0.00077). IkBα knockdown reduced selinexor-induced cytotoxicity in both IM-9 (9.5-fold) and 8226 (12.3 to 25.4-fold) human MM cells. Intracellular protein analysis by heavy isotope labeling and LC-MS/MS showed changes in several signaling pathways including p53, MAPK, VEGF and angiopoietin, IL-1, HMGB1/TLR and APRIL and BAFF as well as those related to NFkB signaling.Conclusion: Selinexor, when used in combination with bortezomib or carfilzomib has the potential to overcome PI drug resistance in MM. DisclosuresKashyap:Pharma: Employment. Landesman:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership.

Proteomic Characterization of Circulating Extracellular Vesicles Identifies Novel Serum Myeloma Associated Markers

Introduction: Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells (PCs) in the bone marrow (BM). MM cells are dependent on the BM microenvironment (e.g. BM stromal cells, macrophages etc) and create a network with surrounding cells. These cells play a pivotal role in the regulation of MM cell survival and drug resistance bydirect interactions through adhesion molecules causing cell adhesion mediated drug resistance (CAM-DR) or soluble factors including supportive cytokines (e.g. IL-6, IL-8, and VEGF) or exosomes (or extracellular vesicles). Exosomes (EV) are endosome-derived membrane-covered cell fragments, which contain specific protein and RNA cargo. The protein content of EVs in MM has not been readily explored. Recently our group established the use a global label-free quantification method to determine the relative amount of proteins identified from EVs obtained from MM cell lines. Here we are reporting a systematic proteomic analysis of EVs derived from MM cell lines, blood from MM patients, and BM from MM patients. We also report preliminary data showing the biological importance of specific EV enriched proteins in MM cell lines and MM patients.Methods: Liquid Chromatography Mass Spectrometry (MS) and Label-free relative quantitation were used to assess the protein content of MM EV and cellular lysates. Cryo-Transmission Electron Microscopy (cryo-TEM) and Nanoparticle Tracking Analysis were used to assess size distribution and specific EV surface markers in EV isolated from the supernatant of MM cell lines and from the serum of non-cancer, MGUS, smoldering MM and active MM donors. Western blot and enzyme-linked immunosorbent assays were used to validate MS data in a bigger cohort of primary MM patients and to assess the biological effect of MM derived EV in the BM stromal cells.Results: Our data show that the Major Histocompatibility Complex Class I (MHCI) and its associated binding protein β2-microglobulin (β2-MG) are the most abundant communally enriched proteins in the EV derived from MM cell lines and from the serum of MM patients. Although it is well known that the serum level of β2-MG is an important prognostic factor in MM, our data indicate that β2-MG represents only a small percentage of the total serum β2-MG, suggesting that two separate β2-microglobulin populations coexist in the serum of patients. Additionally, we show that the main receptor of hyaluronic acid, CD44, is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R, and is a protein that is differentially expressed in EVs isolated from newly diagnosed MM patients and which seems to play a key role in communicating with the BM microenvironment. By using a large cohort of serum obtained from MM patients prospectively treated on a randomized phase 3 trial (233), we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as easily accessible MM biomarker.Conclusions: Our results generate a foundation for the potential use of circulating EVs as novel serum markers of MM and provide the rationale to further explore previously unconsidered molecular players associated with MM disease. DisclosuresPalumbo:Array BioPharma: Consultancy; Onyx Pharmaceuticals: Consultancy; Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Genmab A/S: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Sanofi Aventis: Consultancy.

Targeting Redox Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Proteasome inhibitors (PIs) like bortezomib (Btz) and carfilzomib (Crflz) induce an oxidative stress response in Multiple Myeloma (MM) cells. Oxidative stress is a key effector pathway in PI-induced cell death, and altered redox signaling has been implicated in the acquisition of PI resistance. The potential of redox as a therapeutic target/pathway for PI resistant MM has not been realized due to the absence of a precise molecular targeted strategy that exploits redox signaling in a way that attacks PI resistant cells while sparing normal cells. Therefore, we set out in this study to characterize redox adaptations that contribute to PI resistance in MM, and to use drug screening platforms to identify specific redox-targeted small molecules that restore PI sensitivity. Using multiple isogenic pairs of PI sensitive and resistant MM cell lines, we found that resistant cells exist under high basal levels of reactive oxygen species (ROS) and oxidation of protein thiols (i.e., oxidative damage). Resistant cells induce significantly higher relative levels of ROS following PI treatment, but exhibit no further increase in oxidative damage. By comparison, their PI sensitive counterparts have relatively low levels of basal and PI-induced ROS levels, but undergo significantly higher levels of oxidative damage following PI treatment. These findings demonstrate that PI resistance is associated with alterations in redox balance; they further suggest that PI resistant cells have acquired adaptations that allow them to survive under high basal levels of oxidative stress, and that provide protection from PI-induced oxidative damage. We also identified significant changes in cellular bioenergetics that are typical of PI resistant cells. Generally, PI resistant cells appear to be more metabolically efficient, relying on mitochondrial respiration as their primary source of ATP production. Specifically, PI resistant cells have higher basal oxygen consumption rates (OCR), expanded respiratory capacity, increased NAD(P)H levels and pyruvate dehydrogenase (PDH) activity, and nearly absent activation of the AMP kinase energy stress signaling pathway. Thus, the acquisition of PI resistance is associated with significant changes in redox balance as well as in cellular bioenergetics. Given these findings, we next used a cell-based drug screening method to screen for redox-targeted small molecules capable of restoring PI sensitivity to resistant cells. We screened a compound collection of known pro- and anti-oxidant small molecules with wide-ranging mechanisms of action. From this screen we identified compound E61, which demonstrated strong synergy with multiple PIs, including Btz, Crflz, ixazomib, and oprozomib. E61 induced an oxidative stress response characterized by a burst of ROS generation and oxidation of protein thiols, and synergistically enhanced the PI-induced oxidative stress response in resistant cells. The synergistic cytotoxic response to E61 and PI co-treatment was dependent on ROS, and was evident across several models of PI resistance, representing cells of diverse genetic backgrounds. While E61 enhanced PI-induced cell death in resistant MM cells, its effects were protective in normal cell types, including peripheral blood mononuclear cells (PMBCs) and lymphocytes from normal human donors. These findings suggest that compound E61 will have a wide therapeutic index in combination with PI therapy in preclinical mouse models of MM, a hypothesis that we are currently testing. All together, our findings identify specific redox and bioenergetics changes that are acquired by PI resistant MM cells. Furthermore, our work offers a novel redox-targeted small molecule, E61, to be used in combination with PI-based therapeutic regimens in refractory MM. DisclosuresNo relevant conflicts of interest to declare.

Metabolomic, Proteomic and Genomic Profiling Identifies Biomarakers of Sensitivity to Glutaminase Inhibitor CB-839 in Multiple Myeloma

IntroductionGlutaminase is a mitochondrial enzyme that converts glutamine to glutamate to support several metabolic processes including amino acid and nucleotide synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. Selective glutaminase inhibitors BPTES and CB-839 have anti-proliferative activity in several pre-clinical cancer models including breast, pancreatic, lung, renal, brain, leukemia, and lymphoma. Across a panel of twenty-nine multiple myeloma cell lines, we found that glutaminase inhibition with CB-839 caused tumor cell death or growth inhibition in only a subset of cell lines. To identify biomarkers that predict sensitivity to CB-839 in multiple myeloma cells, we profiled cellular metabolites, mRNA transcripts, and signaling pathways in eight multiple myeloma cell (four CB-839-sensitive and four CB-839-resistant).ResultsProteomic analysis showed that CB-839 treatment suppressed the activity of the amino-acid sensing kinase mTORC1 in CB-839-sensitive cells, leading to down regulation of protein synthesis and expression of metabolic genes. Analysis of steady-state levels of intra-cellular metabolites revealed that CB-839-sensitive cells had more profound decreases in nucleotide levels and less pronounced increases in essential amino acids upon CB-839 treatment compared to CB-839-resistant cells. This suggests that the metabolic response to glutaminase inhibition is fundamentally different in sensitive versus resistant multiple myeloma cell lines. Consistent with the in vitro data, in a xenograft model with the CB-839-sensitive cell line RPMI8226, CB-839 treatment produced a 71% reduction in tumor growth that was associated with reduced levels of intratumoral nucleotides and no changes in the levels of essential amino acids. We next explored protein biomarkers that predict resistance to CB-839 and found that pyruvate carboxylase (PC) expression strongly correlated with resistance. siRNA-mediated knockdown of PC reduced TCA cycle activity and sensitized cells to CB-839 treatment, suggesting that PC can rescue cells from glutaminase inhibition by supporting anapleurotic utilization of glucose. This hypothesis was further substantiated by the observation that treatment of CB-839-resistant cells with the AKT inhibitor MK2206 led to a decrease in glucose utilization, and when combined with CB-839, produced a significant decrease in TCA cycle activity and a profound synergistic anti-proliferative response.ConclusionMultiple myeloma cells show varying anti-proliferative responses to glutaminase inhibition by CB-839. CB-839 treatment inhibits mTORC1 pathway signaling and causes decreases in nucleotides in sensitive multiple myeloma cells. Multiple myeloma cells that are resistant to glutaminase inhibition have high expression of PC, which may allow these cells to utilize glucose instead of glutamine to resupply TCA cycle intermediates. Knockdown of PC or treatment with an AKT inhibitor causes cells to utilize less glucose and sensitizes resistant cells to glutaminase inhibition with CB-839. CB-839 is currently being evaluated in Phase 1 clinical trials for the treatment of various solid and hematological cancers including multiple myeloma. We are exploring the utility of PC and mTORC1 pathway signaling biomarkers to identify multiple myeloma patients that may respond to CB-839 treatment. DisclosuresMacKinnon:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Li:Calithera Biosciences: Employment, Equity Ownership. Rodriquez:Calithera Biosciences: Employment, Equity Ownership. Wang:Calithera Biosciences: Employment, Equity Ownership. Zhang:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.

Vaccine based immunotherapy as a strategy to bypass drug resistance in multiple myeloma

Clonal evolution under therapy leads to drug resistance and relapse in multiple myeloma (MM), requiring new treatment mo- dalities to control disease. Immunotherapy and specifically vacci- nation to induce anti-tumor CD8+ cytotoxic T-cells (CTLs) provides one such strategy, but susceptibility of drug resistant MM cells to CTL attack is as yet not defined. To examine this, a first requirement is identification of tumour-associated antigens in drug refractory MM. To this end, we had previously specifically evaluated cancer testis antigens (CTAs) in MM for retention at relapse post- therapy, and identified SPAG9 and MAGEC1 as frontline antigen targets for vaccines. Of these, SPAG9 is generic to relapse MM (expressed in w100% of cases) and MAGEC1 is essentially tumour-specific (expressed w60% of cases). An optimal strategy for vaccination as therapy is in disease remission to attack re-emerging MM cells that escape drug therapy. A second requirement is to model drug resistance in MM, and accordingly we have now generated derivatives of the XG2 MM cell line that are resistant to melphalan (XG2 M) or Bortezomib (XG2 600R3); the RPMI8226-derivative R5 line with multiple drug resistance was also available. A third requirement is to show effective lysis of drug resistant MM cells by antigen specific CTLs. To generate antigen-specific CTLs, we utilized pDOM-CTAepitope DNA vaccine delivery in HLA-A2 transgenic HHD mice. We examined two epitopes in respective vaccines, pDOM-SPAG9p1 and pDOM-MAGEC1p2. Both vac- cines elicited epitope specific T-cell responses in HHD measured by IFN release assays following prime and boost with electroporation. Significantly, using MM cell lines made chimeric for HHD MHC class I as targets, both vaccines induced CTLs in HHD mice that were able to effectively lyse primary (XG2 and others) and drug resistant MM cell lines (XG2M, XG2 600R3, R5) effectively. These data demonstrate that drug resistant MM cells are susceptible to CTL killing, substantiating a role for vaccination in MM to over- come drug resistance and hence prevent relapse.

Abstract 4426: A 13 mer LNA miR-221 inhibitor restores drug sensitivity in melphalan-refractory multiple myeloma cells

Abstract The onset of resistance to melphalan (MF) in Multiple Myeloma (MM) patients adversely affects disease outcome. Presently there is increasing evidence on the pivotal role of microRNA (miRNAs) in mediating drug-resistance. We here investigated the effect of an original LNA inhibitor of microRNA-221 (LNA-i-miR-221), in restoring sensitivity to MF in MM cells. LNA-i-miR-221 has been previously demonstrated as anti-tumor agent in preclinical models of MM. We studied the MF-sensitizing effect of LNA-i-miR-221 on MM cells in vitro and in vivo. MiR-221/222 expression inversely correlated with MF-sensitivity in a panel of 6 MM cell lines. Noteworthy, LNA-i-miR-221 overcame MF-resistance in vitro significantly enhancing apoptotic cell death as demonstrated by activation/cleavage of caspase-8, caspase-9 and caspase-3, as well as by PARP cleavage and by histone H2AX (γ-H2AX) phosphorylation. Importantly, the synergistic activity of LNA-i-miR-221 with MF affected cell survival even in the presence of bone marrow stromal cells (BMSCs). Gene expression profiling (GEP) comparing treated and untreated MF-sensitive and -resistant MM cell lines was performed to investigate genes and pathways significantly affected by combinatory treatment. Based on the GEP findings, we confirmed by Western Blot that the growth impairment occurred together with up-regulation of pro-apoptotic BBC3/PUMA protein, a target of miR-221/222 which we validated by luciferase assay in our system. Moreover, GEP analysis showed modulation of drug-influx -efflux transporters such as L-type amino acid transporter 1 (LAT1) and multidrug resistance associated protein 1 (MRP1/ABCC1) which was confirmed by Western Blot analysis. Indeed, LNA-i-miR-222 led to a reduction of the Side Population in MF-resistant cells as assessed by Hoechst dye exclusion assay. Finally, treatment of human MM xenografts in SCID/NOD mice with LNA-i-miR-221 plus MF induced a marked antitumor effect, which again occurred together with BBC3/PUMA and MRP1/ABCC1 proteins modulation in retrieved tumors. Taken together, our findings provide proof-of-concept that LNA-i-miR-221 overcomes MF-resistance of MM cells, providing the rationale for clinical translation of LNA-i-miR-221/MF combination in MM patients. Citation Format: Annamaria Gulla, Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Eugenio Morelli, Nicola Amodio, Cirino Botta, Maria Rita Pitari, Santo Giovanni Lio, Pierosandro Tagliaferri, Pierfrancesco Tassone. A 13 mer LNA miR-221 inhibitor restores drug sensitivity in melphalan-refractory multiple myeloma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2015-4426

Abstract 4434: Selinexor and melphalan combination therapy for the treatment of multiple myeloma

Abstract Introduction High-dose melphalan (MEL) followed by autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma (MM). However, patients eventually develop drug resistance and die from progressive disease despite newer therapies using proteasome inhibitors and immunomodulatory drugs. The incurable nature of MM demonstrates the need for novel treatments. Our aim was to investigate whether MEL therapy could be improved by the addition of the XPO1 inhibitor selinexor (SEL) in drug-resistant and parental MM cells both in vitro and ex vivo. Materials and Methods SEL/MEL-treated human MM cells were assayed for cell viability (CT-Blue) and apoptosis by flow cytometry (activated caspase 3). Proximity ligation assays (PLD) were used to assess if XPO1-p53 binding was inhibited by SEL. Western blots of SEL-treated MM cells were performed for nuclear and total p53. MEL-resistant U266 (LR6) and 8226 (LR5) MM cell lines were developed by incremental exposure to MEL. MEL resistant and parental MM cells were treated in vitro with SEL +/- MEL and assayed for apoptosis and cell viability. Cells isolated from patients with newly diagnosed or relapsed MM, were treated with SEL +/- MEL and assayed for apoptosis. Results MM cell viability was decreased synergistically by SEL when used in combination with MEL, as shown by combination index (CI) values. Drug sequencing assays showed that concurrent treatment with MEL (10 μM) and SEL (300 nM) for 48 hours produced synergistic results in human H929 MM cells (CI value 0.079, n = 3). Sequential treatment, SEL for 24 hours followed by MEL for an additional 24 hours or the reverse sequence, also demonstrated synergy with CI = 0.208 (n = 3) and 0.142 (n = 3), respectively. Normal PBMCs (control) were unaffected by SEL/MEL treatment as shown by viability and apoptotic assays. PLD demonstrated that SEL blocks XPO1/p53 binding. Western blot data showed that the SEL treatment of MM cells increased nuclear and total p53. Drug-resistant LR5 and LR6 MM cells were found to be resistant to MEL when compared to parental cell lines. Both resistant MM cell lines were sensitized by SEL to MEL as shown by apoptosis assay (20-fold). CD138+/light chain+ MM cells derived from newly diagnosed and relapsed MM patients were also sensitized (5 to 10-fold) by SEL to MEL as demonstrated by apoptosis assay. Conclusions SEL synergistically improved the response of drug-resistant and parental MM cells to MEL in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by SEL and the reported activation of p53 by MEL. Ongoing studies include in vitro experiments to investigate whether this drug combination reverses MEL resistance by the Fanconi Anemia/BRCA pathway, in vivo treatment of MM in NSG mice with SEL/MEL and a clinical trial using high-dose MEL in combination with SEL. Combination therapies using SEL and MEL may significantly improve the treatment outcomes of MM. Citation Format: Joel G. Turner, Jana L. Dawson, Christopher Cubitt, Taiga Nishihori, Claudio Anasetti, Melissa Alsina, Rachid C. Baz, Michael Kauffman, Sharon Shacham, William S. Dalton, Daniel M. Sullivan. Selinexor and melphalan combination therapy for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2015-4434

Ikaros Degradation Efficiency Correlates with Response of Multiple Myeloma (MM) Cells to IMiD Therapy and Is Blocked By Proteasome Inhibitors

Background: It is now well established that the immune modulator (IMiD) drugs thalidomide (Thal), lenalidomide (Len) and pomalidomide (Pom) bind a specific pocket in the protein, cereblon (CRBN) that is required for MM cytotoxicity after IMiD treatment. Recently, we reported 46 CRBN binding proteins which were altered by Len, two of which were the transcription factors Ikaros (IKZF1) and Aiolis (IKZF3), important regulators of B and T cell development. Others had previously reported the same finding and went further by demonstrating that following binding of IMiD drugs, CRBN accumulates and accentuates the ubiquitination and subsequent proteasomal degradation of these two binding proteins. The resulting degradation and depletion of IKZF proteins has the simultaneous effect of downregulating IRF4 and MYC leading to MM cell death while simultaneously enhancing transcription of interleukin 2 and activating the immune compartment.Methods: To further explore this phenomenon and its relationship to drug sensitivity we first created two stable MM cell lines (H929IKZF1Luc, 8226IKZF1Luc) expressing an IKZF1-luciferase (luc) fusion gene and tested for sensitivity to Len alone and in combination with dexamethasone (Dex), bortezomib (Bor) or the histone deacetylase inhibitor SAHA. Next, we constructed an adenoviral vector expressing IKZF1-luc and used this to transfect 16 MM cell lines, confirmed expression and tested sensitivity to Len by MTT (Figure 1).Results: We first examined the stable cell lines and measured luc expression after exposure to all three IMiD drugs. With all IMiD drugs luc dramatically decreased in sensitive H929 cells. IKZF1 depletion was both time and dose dependent. Using this surrogate marker cell line assay, luc is most efficiently degraded by Pom which is twice as potent as Len, but a thousand times more potent than Thal (Concentration of drug required to reduce luc expression by 50%, Pom: 4.9nM, Len: 10.2 nM, and Thal: 4,795 nM). We then tested all 16 cell lines with Len 2uM for 5 hours and show a strong correlation of IKZF-luc depletion with sensitivity. [Display omitted] We next performed western blots on 9 MM cell lines and demonstrate that IKZF1 levels were lowest in 3 of 5 resistant MM cell lines and normal in all 4 sensitive lines studied. The cell line (FR4) with the lowest levels of IKZF1 harbors a damaging mutation of IZKF1 as well as a translocation which upregulates IRF4, an IKZF target. Of the remaining two resistant lines one has low levels of Cereblon. In one line the mechanism of IMiD resistance could not be established. Finally, we were curious about the dichotomy of the IMiD biology which seemingly has an absolute requirement for a functional proteasome for successful IMiD cytotoxicity which is at variance with clinical reports of synergy between the IMiDs and the proteasome inhibitors (PIs) Bor and carfilzomib (Car). To explore this, we treated the 8226IKZF1Luc cell line with Len alone or in combination with increasing concentrations of Bor (6 nM) or Car (40nM) and found that both totally blocked IKZF1 fusion protein degradation promoted by Len, beginning as soon as 1 hour at 100nM Bor or after 3 hours at 40nM treatment. Conversely, in low concentration of Bor (<4nM) no IKZF1 degradation blocking was observed in the Luc assay and Bor remained lethal to the MM cells. In that light with lower levels of Bor (<3nM) even varying the sequencing of MM cell exposure to the two drug classes did not block cytotoxicity. Other MM active drug Dex or SAHA, blocking the aggresome, had no significant inhibition for Len-induced IKZF1 fusion protein degradation.Conclusion: These results validate the central role for the CRBN-IKZF-IRF4 axis proteins in IMiD response and show a strong correlation of IZKF1 expression and the ability to degrade IKZF1 with sensitivity. Our data provide interesting and perhaps disconcerting biologic data suggesting that the timing and dosing of clinical use of IMiDs and PI treatment in combination should be carefully evaluated to prevent antagonism of effect but suggest in vitro that a therapeutic window exists at which proteasome inhibitors can be active without blocking IMiD activity. DisclosuresStewart:Sanofi: Consultancy; Array BioPharma: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding.

Selinexor Demonstrates Marked Synergy with Dexamethasone (Sel-Dex) in Preclinical Models and in Patients with Heavily Pretreated Refractory Multiple Myeloma (MM)

Introduction: The nuclear export protein exportin 1, (XPO1) is overexpressed in a wide variety of cancers including MM and often correlate with poor prognosis. Selinexor (KPT-330) is an oral Selective Inhibitor of Nuclear Export (SINE) XPO1 antagonist in Phase 1 and 2 clinical studies. Selinexor forces nuclear retention and reactivation of tumor suppressor proteins (TSPs) and reduction of many proto-oncogenes, including MDM2, MYC and Cyclin D. In addition, selinexor potently deactivates NF-κB, through forced nuclear retention of IκBα. Together these effects induce selective apoptosis in MM cells and inhibition of NF-κB dependent osteoclast activation. XPO1 is also responsible for nuclear export of the glucocorticoid receptor (GR). We hypothesized that selinexor will enhance the activity of dexamethasone (DEX)-bound GR, resulting in synergistic tumor cell killing. Methods: In vitro tumor cell viability measurements were based on MTT (CellTiter 96¨/Promega) and combination indices were calculated using CalcuSyn software. For xenograft studies, utilized NOD-SCID mice with subcutaneous inoculation of MM.1s cells. GR nuclear localization was measured with immunofluorescent anti-GR (phosphor-S211) antibody and quantitative imaging. To assess GR transcriptional activation, GR binding to a GCR consensus sequence was measured in nuclear extracts using an ELISA method (GR ELISA kit/Affymetrix). Patients (pts) with heavily pretreated refractory MM were dosed with oral selinexor at doses of up to 60 mg/m2 (8-10 doses/4 wk cycle) as part of a Phase 1 program in advanced hematological malignancies. Response we defined based on the IMWG criteria. The effect of combining DEX with selinexor was analyzed in all pts who received selinexor at moderate to high doses (30-60 mg/m2). Safety and efficacy were analyzed separately in three groups: no DEX, Disclosures Chen:Celgene: Honoraria; Janssen: Honoraria. Off Label Use: Lenalidomide maintenance therapy after ASCT. Gutierrez:Senesco: PI Other. Siegel:Celgene, Millennium, Onyx: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau. Baz:Celgene: Research Funding; Millennium: Research Funding; Bristol Myers Squibb: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding. Kukreti:Celgene: Honoraria. Azmi:Karyopharm Therpeutics: Research Funding. Kashyap:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Marshall:Karyopharm Therpeutics: Employment. McCartney:Karyopharm Therpeutics: Employment. Saint-Martin:Karyopharm Therpeutics: Employment. Norori:Karyopharm Therpeutics: Consultancy. Savona:Karyopharm Therpeutics: Membership on an entity9s Board of Directors or advisory committees. Rashal:Karyopharm Therapeutics: Employment. Carlson:Karyopharm Therapeutics: Employment. Mirza:Karyopharm Therpeutics: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics: Employment, Equity Ownership. Reece:Millennium: Honoraria, Research Funding; Millennium: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Merck: Research Funding; Merck: Research Funding; BMS: Research Funding; BMS: Research Funding; Novartis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen : Honoraria; Amgen : Honoraria.

Targeting Aberrant Non-Homologous End Joining in Multiple Myeloma: Role of the Classical and Alternative Pathways in Genomic Instability

Multiple Myeloma (MM) is characterized by the growth of malignant plasma cells harboring numerous genomic aberrations. The molecular basis driving MM genomic instability is still largely unknown. The ability to repair DNA damages is essential for the maintenance of its integrity, especially the double-strand breaks (DSBs) which are mainly repaired by Non Homologous End Joining (NHEJ). We have investigated NHEJ pathway in myeloma and observed a significant association between up-regulated NHEJ pathway-related gene expression and poor overall survival in two large datasets (IFM and Arkansas) in myeloma. We have also observed a higher end joining (EJ) activity in MM cell lines compared to normal cells using a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene to measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency. Moreover, we confirmed an increased NHEJ activity in several primary patient myeloma cells at different disease stage. Based on this rationale, since an altered NHEJ has been linked to genomic instability and its inhibition leading to eventual cell death, we hypothesized that the aberrant NHEJ can be used as a potential therapeutic target in MM.To address the relevance of NHEJ inhibition in MM cell proliferation and survival, we used SCR7, an inhibitor of Ligase IV (Lig-IV) which is essential for ligation of the double strand breaks following their recognition by the KU70/KU80 heterodimer and the recruitment of DNA-PKcs. We tested 4 different MM cell lines (U266, R8226, MM1s and Dox40), however, except for some level of inhibition in Dox40 (IC50, between 50 and 100 uM at 72 hours), the other cell line growth was not significantly affected (R8226 - IC30 at the concentration; and U266 and MM1s did not reach IC30). The same data were confirmed by Annexin V/7AAD staining and Caspase assay. Interestingly, expression of Lig-IV estimated by western blot analysis, inversely correlated with MM cells sensitivity suggesting that higher protein concentration may require higher drug levels for inhibition. Consistent with this result, we observed a strong inhibition of the NHEJ pathway by ku86-directed shRNAs, which was able to induce cell death in the more resistant MM cell line u266. Subsequently we used the dual gene plasmid-based assay to evaluate the effect of sub-lethal dose (20 uM) of SCR7 on NHEJ in 3 MM cell lines (u266, R8226 and MM1s) and observed an increased recombination activity in 2 of them. We also confirmed these data with another NHEJ inhibitor, NU7441, which target DNA-PK; and by using ku86-shRNA in U266 cell line. Moreover we observed an accumulation of unrepaired DSBs at the genome level as demonstrated by an increased γ-H2AX by western blotting. These results suggested the possibility that the inhibition of the NHEJ by blocking Lig-IV could activate the alternative NHEJ pathway (a-NHEJ), which is more error-prone compared to the classical NHEJ (c-NHEJ). To confirm this hypothesis further, we treated MM cell lines with sub-lethal dose of NU7441 (2.5 uM), Benzamide (2.5 uM), an inhibitor of PARP, which is one of the main protein involved in the a-NHEJ, or both. The different modulation observed with single and combination treatments, along with the ability of NU7441 to revert sensitivity to Benzamide in R8226 cells, suggested that inhibition of the classical pathway could switch on the a-NHEJ and indicated its basal activity at least in this cell line. Ongoing study is assessing the influence of such compounds on NHEJ in primary MM cells and their impact on acquisition of new genomic changes.In conclusion, our data confirm the aberrant activation of NHEJ in MM, and suggest the potential role for both classical and more error-prone a-NHEJ pathways in inducing genomic instability, which may require a dual inhibition to trigger myeloma cell death. DisclosuresNo relevant conflicts of interest to declare.

Targeted Disruption of USP14 and UCHL5 with the Novel Deubiquitinase Enzyme (DUB) Inhibitor, VLX1570, Induces Immense Proteotoxicity and Cell Death in Malignant Plasma Cells

Background: Deubiquitinating enzyme (DUB) inhibitors are an emerging class of compounds, which are increasingly being regarded for their anti-cancer activity and therapeutic potential. The 19S regulatory particle of the proteasome contains two DUBs, USP14 and UCHL5, which are critical for the proper unfolding and deubiquitination of proteins prior to their entry into the 20S proteasome β-catalytic core. In B/plasma cell malignancies such as Waldenströms macroglobulinemia (WM) and multiple myeloma (MM), protein homeostasis and optimal proteasome functionality are paramount for tumor survival. This is evidenced by the success of 20S proteasome inhibition with bortezomib and carfilzomib, which have demonstrated remarkable clinical benefit in patients with WM or MM. As such, the proteasome degradation pathway represents a highly attractive and clinically validated, yet still largely unexplored therapeutic target. Here, we provide first evidence of the cytotoxic effects and molecular sequelae associated with a novel 19S proteasome DUB inhibitor, VLX1570, which selectively targets USP14 and UCHL5, in preclinical models of B/plasma cell cancers and their drug resistant tumor subclones.Materials: VLX1570 was obtained from Vivolux AB, Sweden. Bortezomib was purchased from Sellekchem, Houston TX. Human myeloma cell lines (OPM2, U266, KMS11), human WM cell lines (BCWM.1, MWCL-1 and RPCI-WM1), their respective bortezomib-resistant (BR) subclones as well as ibrutinib-resistant (IR) derivatives of BCWM.1 and RPCI-WM1 were used in experiments (total n=12). Peripheral blood mononuclear cells (PBMCs) from healthy donors were used to assess for cytotoxicity of VLX1570 in non-tumor cells.Results: A 72hr MTS assay first established sensitivity of plasma cell cancer models towards VLX1570, including drug resistant subclones (EC50 range, 20 – 90nM). Next we assessed whether loss of viability was due to apoptosis and observed dose-dependent annexin-V staining in ~50 – 70% of wild-type MM and WM cells, ~45 – 55% in corresponding BR models and ~50% in IR subclones when exposed to VLX1570 (100nm – 500nM) for 24hrs. VLX1570 was tested at similar concentrations in PBMCs and induced minimal cell death. To understand if VLX1570-induced apoptosis was mitochondrial mediated, we assessed mitochondrial outer membrane permeability (MOMP) in all plasma cell cancer models treated with the DUB inhibitor (100 – 500nM) and found a dose-dependent increase in MOMP (p<0.00025 – 0.000001). Activation of the mitochondrial/intrinsic apoptotic pathway was also evidenced by cleavage of caspase-9 and 3 on immunoblot analysis. As inhibition of protein deubiquitination results in accumulation of polyubiquitinated protein conjugates, we examined global ubiquitinated protein levels and found increasing amounts of high molecular weight poly-ubiquitinated protein in VLX1570 treated tumor cells in a dose-dependent manner. This prompted us to further delineate the potential cellular response mechanisms that relay the antitumor activity of VLX1570 in malignant plasma cells. Immunoblot analysis of drug sensitive and (bortezomib or ibrutinib) resistant MM and WM cell lines revealed several changes in ER stress response proteins, notably induction of HSP70 and splicing of XBP1; the latter event which was less prominent in BR cell lines treated with bortezomib. This suggested the ability of VLX1570 to overcome resistance through a rapid and potent proteotoxic effect, which overwhelmed the tumor cells stress response mechanisms beyond its capacity for survival. Finally, as abnormalities in the NFkB signaling pathway are implicated in malignant plasma cell survival, we found markedly decreased nuclear translocation of NFkB (p-P65) protein in tumor cells treated with VLX1570.Conclusions: The DUBs of the 19S proteasome cap offer an attractive target within the proteasome degradation system and whose inhibition by VLX1570 is able to circumvent mechanisms that support resistance to b-catalytic PI (bortezomib) or BTK inhibitor (ibrutinib) therapy. The studies undertaken thus far, coupled with more rigorous mechanistic and in vivo efficacy analyses currently underway, provides rational for the clinical development of DUB inhibitors for use in malignant B/plasma cell malignancies. DisclosuresMartin:Janssen: Honoraria.