Selinexor In Combination Research Articles

Abstract 4720: Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma

Abstract Introduction: Selinexor is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports over 200 cargos, including major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. XPO1 also mediates the export of key signaling molecules in multiple myeloma (MM) including c-MYC mRNA and NF-κB. Selinexor is currently being investigated in phase 2 clinical trials in MM in combination with dexamethasone, pomalidamide, lenalidomide, bortezomib and carfilzomib. In preclinical studies selinexor has been shown to synergize broadly with these anti-MM drugs making it an excellent candidate partner for combination therapies in MM. To identify additional synergistic pairings, we investigated the use of selinexor in combination with panobinostat, a pan-histone deacetylase inhibitor (HDAC) recently approved by the FDA in combination with bortezomib for 3rd line treatment of MM, as a potential treatment for MM both in-vitro and in-vivo. Methods: The effects of selinexor and panobinostat alone or in combination on cell viability were tested on MM1.S and NCI-H929 cell lines using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, MM.1S cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of selinexor and panobinostat alone or in combination and with the therapeutic dose of selinexor. Tumor growth was monitored for 17 days and% Tumor Growth Inhibition (%TGI) was determined. Xenografts were harvested and analyzed microscopically and by immunohistochemestry (IHC). Results: Selinexor-panobinostat combination was highly effective both in-vitro and in-vivo. In MTT assays, both selinexor and panobinostat demonstrated low IC50 values (nanomolar) and when combined, they synergistically/ additively inhibit proliferation. Gene expression and western blot analyses showed that the combination treatment leads to a synergistic reduction in c-MYC mRNA and protein levels. In addition, an overall reduction in expression of anti-apoptotic signaling molecules including NF-κB and p21 and an increase of expression of pro-apoptotic molecules including cleaved caspase 3 and BAX were observed. Importantly, in-vivo, while%TGI of sub-therapeutic doses of selinexor and panobinostat measured 58% and 52% respectively, the combination showed a synergistic effect, measuring 93%. Importantly this high%TGI exceeded the effect of the therapeutic dose of selinexor alone 79%. Conclusion: Selinexor-panobinostat combination synergizes to induce apoptosis in MM cells and amplifies anti-tumor effect in a MM xenograft model. These data provide rational support for study of selinexor/ panobinostat combination in clinical trials. Citation Format: Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma. [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 4720.

An investigator sponsored phase Ia/Ib trial of selinexor in combination with irinotecan in patients with adenocarcinoma of stomach and distal esophagus.

TPS176 Background: The karyopherin protein Exportin 1 (XPO1) is highly expressed and associated with poor prognosis in gastroesophageal tumors. XPO1 is responsible for nuclear export of major tumor suppressor proteins and other proteins including topoisomerases leading to carcinogenesis and drug resistance. KPT-330 (selinexor) is an oral, first in class, covalent Selective Inhibitor of Nuclear Export that specifically blocks XPO1. In gastric cancer xenograft models, treatment with selinexor resulted in > 75% inhibition of tumor growth. Synergistic activity has been observed with the combination of XPO1 inhibitors and topoisomerase inhibitors. Methods: This is a phase I study with cohort expansion that utilizes the standard 3+3 design for dose escalation. The primary endpoint is to evaluate the safety, tolerability and determine the maximally tolerated dose (MTD) of selinexor in combination with irinotecan. Patients with recurrent, metastatic or unresectable histologically confirmed gastric or distal esophageal adenocarcinomas are eligible for this study. Patients must have received at least one line but less than three lines of prior systemic therapies. Secondary endpoints are to explore the safety, tolerability and efficacy of this combination at the established MTD and characterize its pharmacokinetics. Correlative studies will be performed to assess if differential levels of serum and tissue biomarkers including XPO1, cytokine levels and changes in gene expression, before and after selinexor dosing, correlate with clinical outcomes. Circulating tumor cells will be collected in the expansion cohort. Up to 18 patients will be enrolled in 3 dose level cohorts (40, 50 and 65mg/m2) of selinexor in combination with irinotecan (125mg/m2). Selinexor will be given orally on days 1, 3, 8 and 10 of a 21 days cycle. Irinotecan will be given on days 1 and 8. The MTD is defined as the highest dose level at which 1 or less of 6 patients experience a DLT. An additional cohort of 15 patients will be accrued once the recommended dose for expansion is determined. As per September 2015, 3 patients have been included on cohort 1. Clinical trial information: NCT02283359 Clinical trial information: NCT02283359.

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.

Phase 1 MMRC Trial of Selinexor, Carfilzomib (CFZ), and Dexamethasone (DEX) in Relapsed and Relapsed/Refractory Multiple Myeloma (RRMM)

Phase 1 MMRC Trial of Selinexor, Carfilzomib (CFZ), and Dexamethasone (DEX) in Relapsed and Relapsed/Refractory Multiple Myeloma (RRMM)

Selinexor Is an Effective Cancer Treatment in Hypoxic Conditions and Synergizes with Proteasome Inhibitors to Treat Drug Resistant Multiple Myeloma

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) associated with lytic bone disease. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance. Low oxygenation (hypoxia) in the BM microenvironment, which was shown to decrease beyond physiologic BM conditions during the progression of MM, together with cellular and acellular components, plays a critical role in drug resistance. Increased levels of the nuclear exporter of tumor suppressors, exportin 1 (XPO1), was demonstrated in MM, and XPO1 inhibition showed a broad anti-tumor activity. In this study, we tested inhibition of XPO1 using the novel oral, covalent, slowly reversible inhibitor of nuclear export selinexor currently in Phase I/II clinical trials on sensitizing the hypoxia-inducible drug resistant MM cells both in vitro and in vivo.Methods: MM cell lines (MM1s, H929, OPM2 and U266) were treated with increasing concentrations of selinexor in normoxic (21% O2) and hypoxic (1% O2) conditions with or w/o bortezomib or carfilzomib. Assays included MTT proliferation Annexin V/PI staining for apoptosis, PI staining and immuniblots for cell cycle analysis.We tested the effects of selinexor as a single agent on tumor initiation in vivo, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and mice were treated instantaneously with selinexor (15mg/kg) or vehicle (PVP + F-68) three times a week. We also tested the effect of selinexor (15mg/kg) as a single agent on tumor progression and survival in mice with established MM tumor. Furthermore, we studied the effects of selinexor on sensitization to bortezomib-resistant MM cells in vivo. MM1s-Luc-GFP cells were injected IV into SCID mice and tumors were allowed to grow for three weeks. Animals were then treated with a high-dose of bortezomib (1.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were then randomly divided into 3 groups treated with (1) bortezomib (0.5mg/kg), (2) KPT-330 (10mg/kg), and (3) a combination of selinexor and bortezomib. Selinexor was administered by oral gavage three times a week (days 1, 3 and 5) and bortezomib was injected intraperitoneally twice a week (days 1 and 4). Tumor progression was imaged using bioluminescence imaging (BLI).Results: Selinexor, as a single agent, decreased proliferation, induced G1 cell cycle arrest and increased apoptosis of MM cells in both hypoxia and normoxia in a dose-dependent manner. Moreover, we found that the combination of selinexor with bortezomib or carfilzomib reversed the hypoxia-induced resistance in MM cells to proteasome inhibition and induced a synergistic effect on the inhibition of cell proliferation, G1 arrest and apoptosis, which was confirmed by response of respective cell signaling pathways as PI3K, MAPK and JNK.In vivo studies revealed that selinexor alone delayed tumor initiation and decreased by half existing tumor size as a single agent (p=0.0397), and significantly extended mice survival (53 days on vehicle versus 62 days on selinexor, p=000375). In addition, MM-bearing mice which developed resistance to bortezomib were resensitized by treatment of selinexor combined with bortezomib, decreasing tumor burden and showed considerably extended survival (half of the mice were alive at day 52) compared to mice treated with either selinexor (half of the mice were alive at day 49) or bortezomib alone (half of the mice were alive at day 40) (bortezomib versus selinexor p=0.0014, bortezomib versus combination treatment p=0.0004 as analyzed by Student t-test).Conclusions: The orally available inhibitor of nuclear export, selinexor, decreased survival of MM cells in vitro and delayed tumor initiation and tumor progression in vivo as a single agent. In addition, selinexor overcame hypoxia-induced resistance to proteasome inhibitors and resensitized MM cells to therapy both in vitro and in vivo, extending mice survival. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by inhibition of XPO1 with selinexor as well as combination studies of selinexor and proteasome inhibitors. A study of selinexor and carfilzomib is currently ongoing and early results that were published last year showed marked activity for this combination treatment in heavily pretreated MM patients. DisclosuresLandesman:Karyopharm: Employment. Azab:Karyopharm: Research Funding; Selexys: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Cell Works: Research Funding.

Selinexor, a Selective Inhibitor of Nuclear Export (SINE) Compound, Shows Synergistic Anti-Tumor Activity in Combination with Dexamethasone Characterized By Specific Pattern of Gene Expression in Multiple Myeloma (MM)

Background: SINE compounds are a family of small-molecule drugs that inhibit XPO1 mediated nuclear export, resulting in nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IκB and subsequently in specific cancer cell death. Selinexor is a clinical stage SINE compound currently in human phase I/II clinical trials in patients with solid and hematological malignancies. Glucocorticoid Receptor (GR) is an XPO1 cargo and dexamethasone (Dex) acts as a GR agonist and inhibits NFκB activity. The combination of selinexor with Dex (Sel-Dex) shows enhanced anti-tumor potency in vitro and in vivo. The current study aimed at deciphering the molecular changes that contribute to the synergism of Sel-Dex.Methods: Total RNA and whole protein cell lysates from MM cell lines treated with selinexor with or without dexamethasone were analyzed by quantitative PCR and by immunoblots. Localization of GR was evaluated by immunofluorescence microscopy. GR and NFκB transcriptional activity was analyzed using ELISA assays (Affimetrix and Thermo Scientific). RNA from naïve and drug treated cells was analyzed by deep sequencing (by Asuragen).Results: Dexamethasone, but not selinexor, induced phosphorylation of GR. Selinexor blocked nuclear export of phosphorylated GR, enhancing GR transcriptional activation. Deep sequencing revealed a set of genes, whose level of expression was synergistically modified by Sel-Dex. Those genes belong to several pathways including GPCR signaling, cell metabolism and ERK, TGF-β and PI3-AKT signaling. Among the genes that were synergistically up regulated by the combination were genes from the Early Growth Response family (EGR1, EGR3, EGR4). EGR1 is a tumor suppressor protein which down-regulates survivin and triggers Caspase signaling and cell death. Interestingly, EGR1 also mediates the cytotoxic effects of bortezomib and lenalidomide in multiple myeloma cells. The Glucocorticoid-Induced Leucine Zipper (GILZ) gene was also up regulated by Sel-Dex combination. It has been shown by others that silencing GILZ expression decreased the therapeutic effects of dexamethasone in multiple myeloma.Conclusions: Selinexor increased the nuclear retention of dexamethasone-activated-GR. The increased GR transcriptional activity induces expression of genes across different pathways leading to inhibition of cell proliferation and increase cancer cell death. We are currently testing the function of several of these genes in the context of Sel-Dex combination. The current finding supports the the previously reported anti cancer activity of Sel-Dex combination in patients with heavily pretreated relapsed/refractory multiple myeloma. DisclosuresKashyap:Pharma: Employment. Klebanov:Karyopharm Therapeutics Inc: Employment. Lee:Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm Therapeutics: Employment.

Phase I Study of Selinexor, a Selective Inhibitor of Nuclear Export, in Combination with Fludarabine and Cytarabine in Pediatric Patients with Relapsed or Refractory AML

Background and Purpose: Although novel approaches to the treatment of acute myeloid leukemia (AML) are urgently needed, the heterogeneity of AML and the paucity of known actionable targets indicate that there is an important need for broadly active treatment approaches that are not limited to specific genetic subtypes. Selinexor, an oral selective inhibitor of nuclear export, inhibits XPO1 and causes differentiation and apoptosis of a variety of AML subtypes while sparing normal hematopoiesis. We therefore undertook a pilot study to determine the safety and to explore the efficacy of selinexor in combination with fludarabine and cytarabine in pediatric patients with relapsed leukemia.Patients and Methods: Twelve children and adolescents with relapsed or refractory AML (n=10) or mixed phenotype leukemia (n=2) have been enrolled on the study to date. Four patients previously received only chemotherapy, whereas eight had undergone at least one prior stem cell transplant. High-risk features included t(6;9), t(6;12), t(4;11), -7 (2 cases), and megakaryoblastic leukemia. Selinexor, initially at 30 mg/m2/dose, was given orally on days 1, 3, 8, 10, 22, and 24 and escalated according to a rolling-6 design. Fludarabine (30 mg/m2) and cytarabine (2 g/m2) were administered on days 15-19.Results: Among 11 patients who completed at least one cycle and were evaluable for toxicity and response, 3 were treated at dose level 1 (30 mg/m2), 3 at dose level 2 (40 mg/m2), 4 at dose level 3 (55 mg/m2), and 1 at dose level 4 (70 mg/m2). No dose-limiting toxicities were observed at any dose level. The most common Grade 3 non-hematologic toxicity related to selinexor was hyponatremia, which was observed in 10 patients and easily corrected in all cases. As expected, the combination of selinexor plus fludarabine and cytarabine resulted in Grade 3 neutropenia and thrombocytopenia in all patients. Mean pharmacokinetic parameters indicate that plasma exposure is generally dose proportional across selinexor doses, with no apparent accumulation. Plasma exposure in pediatric patients is similar to adult patients treated in phase I trials. Inhibition of XPO1 was assessed by qRT-PCR of XPO1, which is upregulated at the RNA level in response to XPO1 protein inactivation (PDn). Six of the first seven patients enrolled on the trial demonstrated at least 2-fold induction of XPO1 that persisted for at least 48 hours (see figure), indicating prolonged inhibition of the protein by selinexor. The overall response rate in this group of heavily pretreated, relapsed, and refractory patients was 55%. Five patients achieved complete remission (4 with complete count recovery) and 1 had a partial response. Eight of the 11 patients underwent subsequent stem cell transplantation. A trend toward stronger and persistent XPO1 inhibition (measured by PDn) was observed among patients who achieved CR compared to those who did not.Conclusion: Selinexor, given in combination with fludarabine and cytarabine, is tolerable in pediatric patients with relapsed leukemia. Selinexor pharmacokinetic parameters are generally dose proportional and are similar to those seen in adult patients. Most patients demonstrate XPO1 target inhibition. Response rates are encouraging and will be further explored in the Phase II portion of this trial. [Display omitted] DisclosuresKaufman:Karyopharm Therapeutics Inc: Employment. Klebanov:Karyopharm Therapeutics Inc: Employment. Ellis:Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm Therapeutics: Employment. Youssoufian:Karyopharm Therapeutics Inc: Employment. Rashal:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm: Employment, Equity Ownership.

Selinexor, a selective inhibitor of nuclear export (SINE), shows enhanced activity in combination with PD-1/PD-L1 blockade in syngeneic murine models of colon cancer and melanoma

Exportin-1 (XPO1) is a nuclear export protein with >220 cargo proteins, including tumor suppressors and cell cycle modulators. Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >900 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. Selinexor blocks nuclear export of NFAT1c, STAT1 and STAT3, which are implicated in regulating the inhibitory T cell receptor PD-1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and that combination treatment with anti-PD-1 or anti-PD-L1 would thereby enhance the ability of selinexor to elicit antitumor activity. Selinexor increased PD-1 gene expression by ~2-fold in normal lymphocytes and induced PD-L1 gene expression in tumor cell lines. Mice bearing syngeneic colon tumors (colon26) treated with selinexor and anti-PD-1 for 2 weeks demonstrated a significant reduction in tumor growth rate (P < 0.05), while monotherapy with either agent had no significant effect on tumor growth. Similar results were obtained in mice bearing syngeneic B16F10 melanoma tumors, whereby combined treatment with selinexor + anti-PD-1 was superior to either single agent alone (p < 0.034). Combined therapy of mice bearing B16F10 tumors with selinexor and anti-PD-L1 was similarly effective, with significantly smaller tumors at the study endpoint (p < 0.001). No weight loss or signs of toxicity were evident in any in vivo study. Immunophenotypic analysis by flow cytometry revealed that selinexor alone or in combination with anti-PD-1/anti-PD-L1 significantly increased the percentage of splenic NK cells (p≤0.050), while selinexor ± anti-PD-L1 significantly increased the percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p < 0.001). These data indicate that the efficacy of selinexor may be enhanced by disrupting the pre-existing PD-1/PD-L1 signaling in effector cells (T and NK cells). Altogether, these data suggest that the efficacy of selinexor in combination with anti-PD-1 or anti PD L1 in mouse syngeneic tumor models may be due to both disrupting immunosuppressive PD-1/PD-L1 signaling and increasing the frequency of potentially tumor reactive NK cells and Th1 T cells. This provides a rational basis for this treatment combination as a novel therapeutic approach for advanced cancer.

Abstract 1756: Preclinical activity in non-Hodgkin's lymphoma of Selinexor, a selective inhibitor of nuclear export (SINE), is enhanced through combination with standard-of-care therapies

Abstract The nuclear export protein Exportin 1 (XPO1) is overexpressed in diffuse large B-cell lymphoma (DLBCL), follicular small cell cleaved Lymphoma (FSCCL) and a wide variety of other cancers, which often correlates with poor prognosis. Selinexor is an oral SINE currently in Phase 1/2 clinical testing, which targets XPO1 to induce apoptosis across a broad spectrum of tumor types. This broad action is primarily due to forced nuclear retention and reactivation of tumor suppressor proteins (TSPs), resulting in selective tumor cell death. We have previously reported anti-lymphoma activity of selinexor in murine models of DLBCL, in spontaneous canine aggressive lymphomas and in preliminary results from B-cell lymphoma patients enrolled in a Phase 1 trial of hematological malignancies. One promising finding thus far is that efficacy of selinexor appears to be independent of the presence of the mutations in MYC and BCL2/6 found in “double-hit” DLBCL, which is typically resistant to standard treatments. Here we report combination studies involving Selinexor-Dexamethasone and Selinexor-mTOR inhibitor everolimus in Non Hodgkin's lymphoma relative to front-line standard of care. Selinexor was found to be potently cytotoxic across a broad panel of B-cell lymphoma cell lines. Furthermore, combination of selinexor with dexamethasone (Dex) or the mTOR inhibitor everolimus resulted in synergistic cytotoxicity in the WSU-DLCL2 and WSU-FSCCL cell lines. The superior cytotoxicity observed in the combination was consistent with enhanced nuclear retention of different TSPs. In WSU-DLCL2 xenografts, analogs of selinexor KPT-251 and KPT-276 showed comparable efficacy to R-CHOP and in a model of disseminated follicular lymphoma using WSU-FSCCL cells, selinexor had similar efficacy to rituximab for extending survival. The combination of selinexor and Dex or selinexor and everolimus in both xenografts are ongoing. B-cell lymphoma cells in culture are highly sensitive to selinexor. Furthermore, combination of selinexor with Dex or everolimus is synergistically toxic to DLBCL cells. This synergism is consistent with the mechanism of action of these drugs in that both selinexor and Dex antagonize NF-κB and mTOR is an XPO1 cargo protein. The comparable in vivo activity of selinexor and related SINE compounds relative standard of care is consistent with the observations of objective responses in B-cell lymphoma patients in Phase I clinical testing. Ongoing preclinical work is designed to extend comparisons of selinexor with other standard of care and broaden the characterization of combination therapies in an effort to provide a rational basis for studying specific combinations in B-cell lymphoma clinical trials. Citation Format: Asfar S. Azmi, Amro Aboukameel, Robert O. Carlson, Sivan Elloul, Sharon Shacham, Michael Kauffman, Ran Frenkel, Ramzi M. Mohammad. Preclinical activity in non-Hodgkin's lymphoma of Selinexor, a selective inhibitor of nuclear export (SINE), is enhanced through combination with standard-of-care therapies. [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 1756. doi:10.1158/1538-7445.AM2015-1756

Abstract 2074: Selective inhibitor of nuclear export (SINE) compounds show synergistic anti-tumor activity in combination with dexamethasone in multiple myeloma

Abstract Background: The primary nuclear export protein, Exportin 1 (XPO1/CRM1), is overexpressed in most cancers, which is frequently correlated with poor prognosis. SINE compounds are small-molecule bioavailable drugs that bind covalently to XPO1, which leads to nuclear retention of major tumor suppressor proteins, such as p53, FOXO and IκB, resulting in selective cancer cell death. Selinexor is the most advanced SINE with &amp;gt;500 hematological and solid tumor cancer patients treated to date in Phase I/II clinical trials. Glucocorticoid Receptor (GR) is an XPO1 cargo and dexamethasone (Dex) acts through GR agonism and inhibition of NF-κB activity. The combination of selinexor with Dex seems likely to have enhanced anti-tumor potency and this hypothesis was tested in multiple myeloma models. Methods: Total RNA and whole protein cell lysates from multiple myeloma cell line (MM1S) treated with selinexor with or without dexamethasone were analyzed by quantitative PCR and by immunoblots. Localization of GR was evaluated by immunofluorescence. GR and NF-κB transcriptional activity was analyzed using ELISA assays. MM1S xenograft model in NOD-SCID mice were treated with selinexor with or without dexamethasone to evaluate the effect on tumor growth. Results: In MM.1S cells, we found Dex, but not selinexor, induced phosphorylation of GR resulting in GR nuclear localization. Selinexor prevented nuclear export of phosphorylated GR, leading to the synergistic induction of GR-dependent transcriptional activity. RNA levels of GR regulated genes such as MNK2 were induced by this combination treatment. Interestingly, between the two MNK2 isomers, the combination treatment increased the expression of MNK2α, which is a tumor suppressor protein but not the MNK2β isoform. NF-κB transcriptional activity was inhibited additively by this treatment in MM1S cells. The combination treatment of suboptimal doses of selinexor (5 mg/kg; 60% TGI) and Dex (1 mg/kg; 47% TGI) led to nearly complete growth suppression (91% TGI) in the MM1.S multiple myeloma xenografts. Conclusions: We confirmed the hypothesized synergistic anti-tumor activity for the combination of selinexor with dexamethasone and provided evidence that this synergy is mediated through enhanced GR transcriptional activity, inhibition NF-κB and upregulation of the tumor suppressor MNK2α. This work provides a rationale basis for conducting clinical trials of combinations of selinexor with dexamethasone against cancers known to be sensitive to dexamethasone, such as multiple myeloma. Promising anti tumor activity for the combination of selinexor and dexamethasone in patients with relapsed/refractory MM was reported in the ongoing Phase 1 study (NCT01607892). Citation Format: Yosef Landesman, Trinayan Kashyap, Boris Klebanov, Sivan Elloul, Marsha Crochiere, Sharon Friedlander, William Senapedis, Robert Carlson, Michael Kauffman, Sharon Shacham. Selective inhibitor of nuclear export (SINE) compounds show synergistic anti-tumor activity in combination with dexamethasone in 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 2074. doi:10.1158/1538-7445.AM2015-2074

Abstract 5472: Selinexor, a selective inhibitor of nuclear export (SINE), acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death

Abstract Background: The primary nuclear export protein, Exportin 1 (XPO1/CRM1) is overexpressed in most cancers, which frequently correlates with poor prognosis. SINE compounds are a family of small-molecule bioavailable drugs that bind covalently to XPO1 leading to nuclear retention of major tumor suppressor proteins such as p53 and IκB, resulting in selective tumor cell death. Selinexor is the most advanced SINE with &amp;gt;500 hematological and solid tumor cancer patients treated to date in Phase I/II clinical trials. Here we investigated the role of NF-κB transcriptional deactivation in SINE induced anti tumor activity by nuclear retention of IκB. We report the results of combining selinexor or related SINE compounds with proteasome inhibitors, for which inhibition of NF-κB has also been shown to be an important mechanism for cancer cell killing. Methods: Whole protein cell lysates from solid and hematological cancer cell lines treated with selinexor with or without proteasome inhibitors were analyzed by immunoblots. IκB localization was evaluated by immunofluorescence. Cytotoxic effects of SINE compounds were evaluated in the presence or absence of proteasome inhibitors. NF-κB transcriptional activity was analyzed using an ELISA assay. H929 multiple myeloma xenografts in NOD-SCID mice were treated with selinexor alone or in combination with carfilzomib to determine effects on tumor growth. Results: NF-κB transcriptional activity was upregulated in SINE resistant sarcoma cell lines HT1080R (IC50 = 2.4 μM versus HT1080 parental IC50 = 0.03 μM) and ASPS-KY (IC50&amp;gt;10μM). In those resistant lines, combination of the proteasome inhibitors bortezomib or carfilzomib with SINE compound showed dramatic synergistic cytotoxic potency of more than 10 folds. Also, the combination of bortezomib with selinexor led to synergistic nuclear retention of IκB. Furthermore, IκB silencing by siRNA led to reduced selinexor cytotoxic potency, increasing IC50 from 58 nM to 830 nM (9-fold) in IM-9 multiple myeloma cells and from 27 nM to 1.9 μM (66-fold) in U2OS osteosarcoma cells. Finally, the combination of selinexor with carfilzomib was synergistic in reducing tumor growth in the H929 multiple myeloma xenograft model. Conclusion: Inhibition of NF-κB transcriptional activity through forced nuclear retention of IκB appears to be an important mechanism in the selective tumor cell cytotoxicity of selinexor and related SINE compounds. Furthermore, the combination of selinexor and proteasome inhibitors, which are also known to act at least in part through inhibition of NF-κB, leads to synergistic activity in vitro and in vivo, suggesting that such combinations may provide clinically more effective than the single agents. A Phase 1 trial to study the safety and efficacy of selinexor in combination with carfilzomib in multiple myeloma is ongoing (NCT02199665). Citation Format: Yosef Landesman, Trinayan Kashyap, Marsha Crochiere, Boris Klebanov, Sharon Friedlander, William Senapedis, Robert Carlson, Michael Kauffman, Sharon Shacham. Selinexor, a selective inhibitor of nuclear export (SINE), acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death. [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 5472. doi:10.1158/1538-7445.AM2015-5472

Abstract 501: Inhibition of exportin 1 (XPO1) by selinexor (KPT-330) synergistically suppresses growth of neuroblastoma in combination with doxorubicin or bromodomain inhibition

Abstract Background: Neuroblastoma is a childhood malignancy of the peripheral nervous system, and most patients with high-risk disease are not cured. Exportin 1 (XPO1; Chromosome Region Maintenance 1 Protein Homolog, CRM1) is the sole nuclear exporter of TP53, FOXO1, RB1, CDKN1A, and other critical tumor suppressor proteins (TSPs) and is abundantly expressed in high-risk neuroblastoma. In addition, these major tumor suppressor proteins are rarely mutated in neuroblastoma, and thus inhibition of XPO1 is a rational anti-neuroblastoma strategy. The orally bioavailable drug selinexor (KPT-330) is a selective inhibitor of nuclear export that forms a slowly reversible covalent bond to XPO1 and inhibits XPO1-mediated nuclear export. We previously showed that selinexor showed cytotoxicity with sensitivity independent of MYCN amplification status resulting in decreased Myc family protein levels and activation of apoptotic pathways. In addition, flank xenograft models and mouse models of metastatic disease showed significant tumor growth inhibition across multiple neuroblastoma cell lines. Methods: A time course microarray experiment was performed to discover temporal effects across four neuroblastoma cell lines following exposure to selinexor. Models were selected at the extremes of drug sensitivity and expression profiles were generated after 8 and 24 hours of selinexor exposure. Results: The intersection of linear and K-clustering methods revealed genes that followed a consecutively increasing or decreasing profile with respect to time. There were 4 genes in common to all cell lines, 29 genes across 3 cell lines, and 88 between the two most sensitive cell lines. Based off these observations, MAST3, GPRIN1, SMACRD2, and SENP5 were further explored in a larger cohort of neuroblastoma cell lines. In addition, gene set enrichment analysis revealed a doxorubicin-sensitivity expression profile in more resistant lines after exposure to selinexor. The combination of selinexor and doxorubicin resulted in synergistic increases in cell death. Chromatin remodeling genes were significantly disrupted by selinexor across all models, and the combination of selinexor and the bromodomain inhibitor JQ1 resulted in decreased MYCN protein levels and also showed evidence of synergy in vitro. Conclusions: Expression profiling of neuroblastoma cell lines following selinexor exposure has uncovered candidate biomarkers of activity and sensitivity. The potent synergy with other chemotherapeutic agents provides evidence for rational drug combinations. Selinexor is currently in pediatric phase 1 trials and has the potential to be rapidly translated into a novel therapeutic approach for children with neuroblastoma. Citation Format: Pietro J. Ranieri, Rebecca Trillo, Yosef Landesman, William Senapedis, Dilara McCauley, Sharon Shacham, Michael Kauffman, John M. Maris, Edward F. Attiyeh. Inhibition of exportin 1 (XPO1) by selinexor (KPT-330) synergistically suppresses growth of neuroblastoma in combination with doxorubicin or bromodomain inhibition. [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 501. doi:10.1158/1538-7445.AM2015-501

Abstract LB-062: XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas

Abstract Mutation and constitutive expression of MYC and BCL2 and/or BCL6 (a.k.a. double and triple-hit lymphomas) defines subsets of diffuse large B-cell lymphoma (DLBCL) pts with particularly poor outcome. Almost 60% of pts with BCL2 and MYC translocations die within six months of diagnosis due to chemorefractory disease, a prognosis that cannot be overcome with intense chemotherapy. A further hindrance to patient survival is that these double and triple-hit lymphomas are frequently found in the elderly who have limited tolerability to aggressive chemotherapeutic regimens. To identify chemoresistant double and triple-hit DLBCL, we screened 38 DLBCL cell lines for mutations in BCL2, MYC and BCL6 by FISH and response to chemotherapy administration in vitro. We found 3 triple-hit and 2 double-hit DLBCL cell lines. Previous work in our laboratory showed that the nuclear export protein, XPO1, is a critical regulator of MYC, BCL2 and BCL6 mRNA transport in DLBCL. We therefore analyzed XPO1 amplification (by PCR) and expression (by immunoblot) in these cell lines and found that all 5 cells expressed XPO1 at higher levels than centroblasts and that, in at least 2 of them, this was result of gene amplification. Inhibition of XPO1 with selinexor, a Selective Inhibitor or Nuclear Export (SINE) compound, increased nuclear localization of MYC, BCL2 and BCL6 transcripts and consequently decreased their protein expression. Moreover, selinexor administration induced chemosensitization in doxorubicin-resistant DLBCL cells through decreasing DNA damage repair mechanisms as demonstrated by comet assays. To determine the potential extent of DLBCL patients that could benefit from such a treatment, we first analyzed 110 DLBCL patient samples by immunohistochemistry and found that XPO1 expression was increased in 85 cases compared to normal tonsils. Within the cohort analyzed, 6 patients had double or triple hit lymphoma (by FISH) and all overexpressed XPO1. Since this population has higher incidence of chemorefractory disease, we decided to develop a pre-clinical model of this disease using primary patient samples. We developed two patient-derived xenograft (PDX) models representing a double and a triple-hit lymphoma. In both cases XPO1 was also amplified. We compared the molecular and pathological characteristics of 12 generations of PDXs with the original pt sample, and found that protein expression and mutations of these genes were stable. We therefore administered selinexor alone, CHOP alone (cyclophosphamide, vincristine, doxorubicin and dexamethasone) and the combination of selinexor with CHOP to these PDXs. We found that selinexor significantly decreased tumor growth, compared to vehicle or CHOP alone. Moreover, combinatorial treatment indicated that selinexor was able to revert the chemorefractoriness of these DLBCLs, without inducing toxicity by clinical chemistry or pathologic examination. Analysis of selinexor vs. vehicle treated PDXs indicated a decrease in protein levels of MYC, BCL6, BCL2 as well as DNA damage and checkpoint regulators such as CHK1. In summary, selinexor has potent anti-proliferative effects in double/triple-hit DLBCL and can be safely combined with CHOP to enhance cancer cell death. These data present a new therapeutic approach for pts with double/triple hit lymphomas, and provide rational support for the study of selinexor/CHOP combination in clinical trials. Citation Format: Rossella Marullo, ShaoNing Yang, Tami Rashal, Yosef Landesman, Robert Carlson, Sharon Shacham, Leandro C. Cerchietti. XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas. [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 LB-062. doi:10.1158/1538-7445.AM2015-LB-062

Abstract 4490: Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo

Abstract The primary nuclear export protein, Exportin 1 (XPO1/CRM1), is overexpressed in most cancers and this overexpression is frequently correlated with poor prognosis. Selective Inhibitors of Nuclear Export (SINE) compounds are a family of small-molecule bioavailable drugs that bind covalently to XPO1 to inhibit nuclear export. This results in nuclear retention of major tumor suppressor proteins, such as p53, pRB, FOXO3A and other critical proteins in cancer biology, which leads to selective cancer cell death. Selinexor is the most advanced SINE with &amp;gt;500 hematological and solid tumor cancer patients treated to date in a number of Phase I/II clinical trials. Blocking XPO1 function also blocks nuclear transport of key DNA damage repair (DDR) proteins and we therefore hypothesized that combination of selinexor with radiation therapy (RT) would lead to synergistic anti-tumor activity. Radiation-chemotherapy combination is an established therapeutic option for patients with medically inoperable non-small cell lung cancer (NSCLC), but its use is limited due to tolerability and adverse effects. Moreover, this treatment strategy typically has short-term benefits and the patients relapse with progressive disease. Here we report on studies designed to test whether selinexor could interfere with DDR and synergize with radiation to enhance anti-tumor potency relative to either treatment alone. Cell cycle analysis and clonogenic assays were conducted in the presence or absence of selinexor in combination with escalating doses of RT on the radiation-sensitive and radiation-insensitive H1299 and A549 cell lines, respectively. Interestingly, Selinexor-treatment induced G1 cell cycle arrest in the p53-deficient cell line H1299 and a prominent G2 cell cycle arrest in p53-wild type A549 cells 24 hrs post treatment. Furthermore, clonogenic assays revealed that selinexor + RT treatment act synergistically to produce a dose-dependent growth inhibition in both cell lines. In vivo combination treatment in the A549 NSCLC xenograft model with low doses of selinexor and RT displayed a strong synergistic effect for reduction of tumor volume. In addition, microscopic and immunohistochemical analysis of the resected tumors showed an overall reduction in tumor cell numbers, increased fibrosis and induction of apoptosis in the selinexor-RT treated tumors compared to controls. Finally, mechanistic studies revealed that selinexor does not induce DNA damage like RT, but downregulates CHK1, RAD51 and other DDR proteins. Together, our results suggest that selinexor treatment sensitizes cells to RT by preventing single-stranded DNA break repair via downregulation of DDR protein expression. In contrast, selinexor induces cell cycle arrest at G1 in p53 deficient H1299 cells, allowing DNA damage accumulation and induction of apoptosis. These results provide a rationale basis for combining selinexor with RT in clinical trials studies. Citation Format: Tami Rashal, Sivan Elloul, Marsha Crochiere, Trinayan Kashyap, William Senapedis, Ryan George, Sharon Friedlander, Maya Ilouze, Yosef Landesman, Robert Carlson, Nir Peled, Michael Kauffman, Sharon Shacham, Yaacov Lawrence. Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo. [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 4490. doi:10.1158/1538-7445.AM2015-4490

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia

AbstractDespite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

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.

Effects Of Inhibition Of XPO1/CRM1-Dependent Nuclear Export By Selinexor (KPT-330), Alone and In Combination With Carfilzomib (CFZ), On Apoptosis and Autophagy In Multiple Myeloma (MM)

IntroductionExportin 1 (XPO1/CRM1) is the sole transporter of most tumor suppressor proteins (TSP) from the nucleus to the cytoplasm. Small molecule selective inhibitors of nuclear export (SINE) block XPO1-mediated nuclear export, leading to nuclear retention of TSP, inducing cancer cell death and sensitizing cancer cells to other cytotoxic drugs. Although the cytotoxic and apoptotic effects of SINE on different cancer cells have now been established, the mechanism of cell death is still not fully understood. Recently, autophagy emerged as a possible cell death pathway and alternate to the ubiquitin-proteasome pathway (UPP) by which excess and/or dysfunctional proteins and organelles are degraded and recycled. MM cells require basal autophagy for survival and caspase 10 protease activity is required to limit autophagic cell death. The possibility that autophagy may be involved in the mechanism of action of SINE is supported by observations that knockdown of XPO1 can promote autophagy and that cytoplasmic p53 can repress autophagy. In this study, we evaluated the contribution of autophagy to the effects of Selinexor (KPT-330), a SINE currently in two phase I clinical trials, on MM cell cytotoxicity. Because proteasome inhibition can also induce autophagy, we hypothesized that the combination of Selinexor and CFZ, a next generation irreversible proteasome inhibitor approved for treatment of MM, may synergistically augment cytotoxicity in MM cells. MethodsPlasma cells (PC) were purified from consented MM patient bone marrow aspirates using EasySep (Stem Cell Technology). PC purity (>80%) was determined by Wright-Giemsa staining of cytospins. Human myeloma cell lines (HMCL) NCI-H929, RPMI-8226, MM1.S and MM1.R were cultured in RPMI1640 with 10% FBS. IC50 values were determined using GraphPad Prism. Drug combination efficacy was determined using CalcuSyn (Biosoft). Combination index (CI) values <1.0 indicate synergy. Transcription factor profiling plate array II was from Signosis (Sunnyvale, CA). Otherwise, our studies used standard cellular and molecular biology techniques. ResultsSelinexor caused significant cytotoxicity in HMCL (IC50 10-100 nM), induced cell cycle arrest in G1, and promoted apoptotic cell death typified by caspase activation, DNA fragmentation, and Annexin V binding. Importantly, purified PC from newly-diagnosed MM patients were also sensitive to the cytotoxic effects of Selinexor.HMCL treated with KPT-330 also exhibited nuclear retention of p53. To gain insight into more global effects of SINE treatment, we assayed DNA binding activity of approx. 100 different transcription factors. TSP, such as Rb, p53, and EGR-1, exhibited increased activity, whereas proto-oncogenes, including NF-κB, Myc, and Myb, were inactivated in response to KPT-330. Western blot analysis of inactivated targets showed nearly total loss of protein. Unlike XPO1, which is degraded via the UPP upon treatment with SINE, loss of Myc and NF-κB subunits, including RelA, RelB, and p52, could not be reversed by proteasome inhibition. Instead, we found that Selinexor treatment induced markers of autophagy, including LC3B induction and processing, and promoted loss of caspase 10, which is associated with autophagic cell death in MM.Importantly, we have determined that the combination of Selinexor and CFZ results in synergistic cell death (CI 0.2-0.6) characterized by enhanced induction of both apoptosis and autophagy in HMCL. The effects of Selinexor and CFZ were also evaluated in NOD-SCID mice inoculated subcutaneously with NCI-H929 cells. Mice were treated 3 times weekly per oswith Selinexor (5 or 10 mg/kg) alone and in combination with twice-weekly IV-administered CFZ (1.5 or 3 mg/kg). After 16 days of treatment, high doses of either CFZ or Selinexor alone moderately inhibit tumor growth. Treatment with the combination of CFZ and Selinexor at all dose levels was more effective than single agent treatment, and high dose combination treatment completely impaired xenograft tumor growth with good tolerability. ConclusionOur studies are the first to suggest that Selinexor-induced cell death correlates with both apoptosis and autophagy, and that both cell death pathways are enhanced in response to combined treatment with CFZ. Overall, our pre-clinical study provides strong rationale for evaluation of Selinexor in combination with CFZ for the treatment of MM. Disclosures:McCauley:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Jakubowiak:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.