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Purpose Selinexor, a first-in-class, oral, selective exportin 1 (XPO1) inhibitor, induces apoptosis in cancer cells through nuclear retention of tumor suppressor proteins and the glucocorticoid receptor, along with inhibition of translation of oncoprotein mRNAs. We studied selinexor in combination with low-dose dexamethasone in patients with multiple myeloma refractory to the most active available agents. Patients and Methods This phase II trial evaluated selinexor 80 mg and dexamethasone 20 mg, both orally and twice weekly, in patients with myeloma refractory to bortezomib, carfilzomib, lenalidomide, and pomalidomide (quad-refractory disease), with a subset also refractory to an anti-CD38 antibody (penta-refractory disease). The primary end point was overall response rate (ORR). Results Of 79 patients, 48 had quad-refractory and 31 had penta-refractory myeloma. Patients had received a median of seven prior regimens. The ORR was 21% and was similar for patients with quad-refractory (21%) and penta-refractory (20%) disease. Among patients with high-risk cytogenetics, including t(4;14), t(14;16), and del(17p), the ORR was 35% (six of 17 patients). The median duration of response was 5 months, and 65% of responding patients were alive at 12 months. The most common grade ≥ 3 adverse events were thrombocytopenia (59%), anemia (28%), neutropenia (23%), hyponatremia (22%), leukopenia (15%), and fatigue (15%). Dose interruptions for adverse events occurred in 41 patients (52%), dose reductions occurred in 29 patients (37%), and treatment discontinuation occurred in 14 patients (18%). Conclusion The combination of selinexor and dexamethasone has an ORR of 21% in patients with heavily pretreated, refractory myeloma with limited therapeutic options.

Selinexor, a selective inhibitor of nuclear export compound that blocks exportin 1 (XPO1) and forces nuclear accumulation and activation of tumor suppressor proteins, inhibits nuclear factor κB, and reduces oncoprotein messenger RNA translation, is a potential novel treatment for myeloma that is refractory to current therapeutic options. We administered oral selinexor (80 mg) plus dexamethasone (20 mg) twice weekly to patients with myeloma who had previous exposure to bortezomib, carfilzomib, lenalidomide, pomalidomide, daratumumab, and an alkylating agent and had disease refractory to at least one proteasome inhibitor, one immunomodulatory agent, and daratumumab (triple-class refractory). The primary end point was overall response, defined as a partial response or better, with response assessed by an independent review committee. Clinical benefit, defined as a minimal response or better, was a secondary end point. A total of 122 patients in the United States and Europe were included in the modified intention-to-treat population (primary analysis), and 123 were included in the safety population. The median age was 65 years, and the median number of previous regimens was 7; a total of 53% of the patients had high-risk cytogenetic abnormalities. A partial response or better was observed in 26% of patients (95% confidence interval, 19 to 35), including two stringent complete responses; 39% of patients had a minimal response or better. The median duration of response was 4.4 months, median progression-free survival was 3.7 months, and median overall survival was 8.6 months. Fatigue, nausea, and decreased appetite were common and were typically grade 1 or 2 (grade 3 events were noted in up to 25% of patients, and no grade 4 events were reported). Thrombocytopenia occurred in 73% of the patients (grade 3 in 25% and grade 4 in 33%). Thrombocytopenia led to bleeding events of grade 3 or higher in 6 patients. Selinexor-dexamethasone resulted in objective treatment responses in patients with myeloma refractory to currently available therapies. (Funded by Karyopharm Therapeutics; STORM ClinicalTrials.gov number, NCT02336815.).

Purpose We evaluated the pharmacokinetics (PKs), pharmacodynamics, safety, and efficacy of selinexor, an oral selective inhibitor of nuclear export compound, in patients with advanced soft tissue or bone sarcoma with progressive disease. Patients and Methods Fifty-four patients were treated with oral selinexor twice per week (on days 1 and 3) at one of three doses (30 mg/m2, 50 mg/m2, or flat dose of 60 mg) either continuously or on a schedule of 3 weeks on, 1 week off. PK analysis was performed under fasting and fed states (low v high fat content) and using various formulations of selinexor (tablet, capsule, or suspension). Tumor biopsies before and during treatment were evaluated for pharmacodynamic changes. Results The most commonly reported drug-related adverse events (grade 1 or 2) were nausea, vomiting, anorexia, and fatigue, which were well managed with supportive care. Commonly reported grade 3 or 4 toxicities were fatigue, thrombocytopenia, anemia, lymphopenia, and leukopenia. Selinexor was significantly better tolerated when administered as a flat dose on an intermittent schedule. PK analysis of selinexor revealed a clinically insignificant increase (approximately 15% to 20%) in drug exposure when taken with food. Immunohistochemical analysis of paired tumor biopsies revealed increased nuclear accumulation of tumor suppressor proteins, decreased cell proliferation, increased apoptosis, and stromal deposition. Of the 52 patients evaluable for response, none experienced an objective response by RECIST (version 1.1); however, 17 (33%) showed durable (≥ 4 months) stable disease, including seven (47%) of 15 evaluable patients with dedifferentiated liposarcoma. Conclusion Selinexor was well tolerated at a 60-mg flat dose on a 3-weeks-on, 1-week-off schedule. There was no clinically meaningful impact of food on PKs. Preliminary evidence of anticancer activity in sarcoma was demonstrated.

Purpose This trial evaluated the safety, pharmacokinetics, pharmacodynamics, and efficacy of selinexor (KPT-330), a novel, oral small-molecule inhibitor of exportin 1 (XPO1/CRM1), and determined the recommended phase II dose. Patients and Methods In total, 189 patients with advanced solid tumors received selinexor (3 to 85 mg/m2) in 21- or 28-day cycles. Pre- and post-treatment levels of XPO1 mRNA in patient-derived leukocytes were determined by reverse transcriptase quantitative polymerase chain reaction, and tumor biopsies were examined by immunohistochemistry for changes in markers consistent with XPO1 inhibition. Antitumor response was assessed according Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 guidelines. Results The most common treatment-related adverse events included fatigue (70%), nausea (70%), anorexia (66%), and vomiting (49%), which were generally grade 1 or 2. Most commonly reported grade 3 or 4 toxicities were thrombocytopenia (16%), fatigue (15%), and hyponatremia (13%). Clinically significant major organ or cumulative toxicities were rare. The maximum-tolerated dose was defined at 65 mg/m2 using a twice-a-week (days 1 and 3) dosing schedule. The recommended phase II dose of 35 mg/m2 given twice a week was chosen based on better patient tolerability and no demonstrable improvement in radiologic response or disease stabilization compared with higher doses. Pharmacokinetics were dose proportional, with no evidence of drug accumulation. Dose-dependent elevations in XPO1 mRNA in leukocytes were demonstrated up to a dose level of 28 mg/m2 before plateauing, and paired tumor biopsies showed nuclear accumulation of key tumor-suppressor proteins, reduction of cell proliferation, and induction of apoptosis. Among 157 patients evaluable for response, one complete and six partial responses were observed (n = 7, 4%), with 27 patients (17%) achieving stable disease for ≥ 4 months. Conclusion Selinexor is a novel and safe therapeutic with broad antitumor activity. Further interrogation into this class of therapy is warranted.

BACKGROUND: There is no consensus regarding resection of the primary tumour with few or absent symptoms in patients with synchronous unresectable metastatic colorectal cancer (CRC). A potential benefit of resection of the primary tumour is to prevent complications of the primary tumour in later stages of the disease. We here propose a randomized trial in order to demonstrate that resection of the primary tumour improves overall survival.METHODS/DESIGN: The CAIRO4 study is a multicentre, randomized, phase III study of the Dutch Colorectal Cancer Group (DCCG). Patients with synchronous unresectable metastases of CRC and few or absent symptoms of the primary tumour are randomized 1:1 between systemic therapy only, and resection of the primary tumour followed by systemic therapy. Systemic therapy will consist of fluoropyrimidine-based chemotherapy in combination with bevacizumab. The primary objective of this study is to determine the clinical benefit in terms of overall survival of initial resection of the primary tumour. Secondary endpoints include progression free survival, surgical morbidity, quality of life and the number of patients requiring resection of the primary tumour in the control arm.DISCUSSION: The CAIRO4 study is a multicentre, randomized, phase III study that will assess the benefit of resection of the primary tumour in patients with synchronous metastatic CRC.TRIAL REGISTRATION: The CAIRO4 study is registered at clinicaltrials.gov (NCT01606098).

The key nuclear export protein CRM1/XPO1 may represent a promising novel therapeutic target in human multiple myeloma (MM). Here we showed that chromosome region maintenance 1 (CRM1) is highly expressed in patients with MM, plasma cell leukemia cells and increased in patient cells resistant to bortezomib treatment. CRM1 expression also correlates with increased lytic bone and shorter survival. Importantly, CRM1 knockdown inhibits MM cell viability. Novel, oral, irreversible selective inhibitors of nuclear export (SINEs) targeting CRM1 (KPT-185, KPT-330) induce cytotoxicity against MM cells (ED50

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p

Resistance to BRAF inhibitor therapy places priority on developing BRAF inhibitor-based combinations that will overcome de novo resistance and prevent the emergence of acquired mechanisms of resistance. The CRM1 receptor mediates the nuclear export of critical proteins required for melanoma proliferation, survival, and drug resistance. We hypothesize that by inhibiting CRM1-mediated nuclear export, we will alter the function of these proteins resulting in decreased melanoma viability and enhanced BRAF inhibitor antitumoral effects. To test our hypothesis, selective inhibitors of nuclear export (SINE) analogs KPT-185, KPT-251, KPT-276, and KPT-330 were used to induce CRM1 inhibition. Analogs PLX-4720 and PLX-4032 were used as BRAF inhibitors. Compounds were tested in xenograft and in vitro melanoma models. In vitro, we found CRM1 inhibition decreases melanoma cell proliferation independent of BRAF mutation status and synergistically enhances the effects of BRAF inhibition on BRAF-mutant melanoma by promoting cell-cycle arrest and apoptosis. In melanoma xenograft models, CRM1 inhibition reduces tumor growth independent of BRAF or NRAS status and induces complete regression of BRAF V600E tumors when combined with BRAF inhibition. Mechanistic studies show that CRM1 inhibition was associated with p53 stabilization and retinoblastoma protein (pRb) and survivin modulation. Furthermore, we found that BRAF inhibition abrogates extracellular signal–regulated kinase phosphorylation associated with CRM1 inhibition, which may contribute to the synergy of the combination. In conclusion, CRM1 inhibition impairs melanoma survival in both BRAF-mutant and wild-type melanoma. The combination of CRM1 and BRAF inhibition synergizes and induces melanoma regression in BRAF-mutant melanoma. Mol Cancer Ther; 12(7); 1171–9. ©2013 AACR.

2044 Background: Patients (pts) with recurrent GBM have few treatment options and a poor prognosis. Selinexor is an oral inhibitor of XPO1 mediated nuclear export resulting in nuclear retention of ...

10569 Background: Sarcomas are a heterogeneous group of malignancies with diverse genetic abnormalities. Selinexor is a first-in-class, oral, inhibitor of XPO1, (nuclear exportin protein 1) with potent anti-tumor activity in multiple sarcoma cell lines and in murine liposarcoma xenografts. Here we report results from a Phase 1b dose expansion trial of selinexor in sarcoma patients (NCT01896505). Methods: Patients (pts) with advanced, refractory sarcomas with radiographic progression received oral selinexor at 50 mg/m2 twice weekly per 28 day cycle. Pharmacokinetics (PK, n = 12) was assessed in the fasted and fed state. Pharmacodynamic studies were performed on fresh tumor biopsies. Response was evaluated every 2 cycles (RECIST 1.1). Results: 36 pts (14 M / 22 F, ECOG 0/1: 19/17, median age 57.5 years [range 18–86], median lines of previous treatments: 3 [range 1–9]). Disease subtypes include liposarcoma (LPS; N = 12), leiomyosarcoma (LMS; N = 8) and other sarcomas (N = 16). Grade 3 drug related adverse ev...

SINE (Selective Inhibitors of Nuclear Export) block the activity of XPO1/CRM1, 1 of 7 nuclear export proteins in cells, forcing the nuclear retention of key tumor suppressor proteins, leading to selective apoptosis of tumor cells. The purpose of these studies was to evaluate the in vitro activity of SINE against canine tumor cell lines and investigate the biological activity of verdinexor (KPT-335) in dogs with spontaneous cancers as proof of principle for human clinical studies. Several different canine tumor cell lines including those derived from non-Hodgkin Lymphoma (NHL) exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE; NHL cells were particularly sensitive with IC50 2-42 nM. A Phase I clinical trial of verdinexor was performed in dogs with cancer with an emphasis on NHL given in vitro activity demonstrated against the tumor cell lines. The MTD was 1.75 mg/kg twice per week although biological activity was observed at 1 mg/kg. Clinical benefit including Partial Response (PR) and Stable Disease (SD) for at least 4 weeks was observed in 9/14 dogs with NHL with a median time to progression of 66 days (range 35-256). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg verdinexor on a Monday/Wednesday/Friday (MWF) regimen; clinical benefit (PR+SD) was observed in 4/6 dogs with a median time to progression of 83 days (range 35-354). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care and dose modulation. A validated health related Quality of Life (QOL) form used to assess dogs during treatment demonstrated that the overall quality of life did not decrease in dogs in this study supporting the notion that clinical toxicities associated with verdinexor are generally well tolerated. Based on these findings, a Phase IIb study was performed in 58 dogs with either newly diagnosed or relapsed NHL. Drug was administered initially at 1.5 mg/kg MWF, but this dosing regimen was changed to 1.25 mg/kg M/Th due to the high rate of anorexia and weight loss on the MWF regimen; dose escalation was permitted to 1.5 mg/kg on the M/Th regimen.. The objective response rate was 29% (1 CR, 16 PR) with an additional 25 dogs experiencing SD for a minimum of 4 weeks, resulting in a of 72% disease control rate. While the median time to progression was approximately 6 weeks, 19 dogs (32%) remained on study drug for more than 8 weeks. Laboratory abnormalities were minimal. Together, these data provide robust evidence that the novel orally bioavailable XPO1 inhibitor vrdinexor exhibits single agent biological activity in a spontaneous large animal model of human NHL. Furthermore, verdinexor was well tolerated even in the absence of supportive care, suggesting that SINE compounds could exhibit good long-term tolerability in people. Citation Format: Cheryl A. London, Luis Feo Bernabe, Sandra Barnard, William Kisseberth, Antonella Borgatti, Michael Henson, Heather Wilson-Robles, Kiersten Jensen, Daisuke Ito, Jaime Modiano, Misty Bear, Michael Pennell, Jean-Richard Saint-Martin, Dilara McCauley, Michael Kauffman, Sharon Shacham. Evaluation of the novel, orally bioavailable selective inhibitor of nuclear export (SINE) KPT-335 (verdinexor) in spontaneous canine cancer: Results of phase I and phase II clinical trials. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3809. doi:10.1158/1538-7445.AM2014-3809

Aim: Increased XPO1 expression has been linked to progression of OvCa. Nearly all tumor suppressor proteins (TSPs) are transported out of the nucleus exclusively by XPO1 and thereby inactivated. The XPO1 inhibitor, Selinexor, forces the nuclear retention and activation of >10 TSPs resulting in OvCa cell death. Methods: SINE induced TSP nuclear localization and induction of apoptosis were tested in OvCa cell lines and patient-derived cells. Combination with cisplatin was assessed in vitro & in patient-derived xenograft models (30 mg/m2 po, 3 times/week). An on-going Phase 1 (KCP-330-002, NCT01607905) in pts with solid tumors, oral selinexor (8 -10 doses/4-week cycle) was administered to pts with heavily pretreated OvCa that were progressing on study entry. Response was evaluated every 2 cycles (RECIST 1.1). Results: SINE potently induced cell death in platinum-sensitive and -resistant OvCa cell lines (IC50s Conclusions: Selinexor treatment provides synergistic activity with cisplatin in preclinical models. In pts, selinexor shows preliminary single agent antitumor activity against heavily pretreated platinum resistant/refractory OvCa. A single agent phase 2 study is ongoing (NCT02025985) & combination studies are planned. Disclosure: All authors have declared no conflicts of interest.

5522 Background: Increased XPO1 expression has been linked to progression of OvCa and is an independent poor prognostic for survival. Most tumor suppressor proteins (TSP) are transported out of the...

SINE (Selective Inhibitors of Nuclear Export) are novel small molecule drugs in phase I clinical trials for advanced cancers. SINEs inhibit nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB, resulting in selective death of cancer cells. Resistant cells were created by treating the sensitive fibrosarcoma cell line HT1080 with increasing concentrations of SINE over 10 months. Gene chip analysis of parental-sensitive and drug-resistant cells treated with SINE demonstrated activation of distinct pathways that mediate either cell death or survival. In addition, SINE resistance was overcome by drug combination with proteasome inhibition. Methods: Resistant cells were generated with selection in increasing concentrations of SINE. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. FACS and immunoblots were used to measure effects on cell cycle, protein expression and cell death. RNA from nai[[Unable to Display Character: ̈]]ve and drug treated sensitive/resistant cells was analyzed by Affymetrix microarrays and qPCR. A drug combination study was performed to evaluate whether resistance to SINE could be overcome with proteasome inhibition. Results: Treatment of SINE-sensitive fibrosarcoma cell line HT1080 (IC50 = 13.6nM) with gradually increasing concentrations of SINE for 10 months resulted in > 100 fold decrease in sensitivity to SINE cytotoxicity (IC50 = 1.7μM). Resistant cells displayed prolonged cell cycle (∼72 vs 24 hrs) compared to parental cells. Resistant cells did not show increased MDR1 and MRP1 activity, suggesting that resistance to SINE was not mediated by induction of the multidrug resistance mechanism. Sequencing of XPO-1 from the SINE resistant cells revealed no mutations in the SINE / cargo binding pocket including the reactive Cys528. Upon exposure to SINE, resistant cells had reduced nuclear accumulation of p53, p21, FOXO1A, IkB, p27, and PP2A proteins compared with SINE-sensitive cells. SINE treatment of both sensitive and resistant cells resulted in pRB de-phosphorylation and induction of p53 and its downstream target p21. In addition, SINE treatment reduced the levels of the anti-apoptotic protein Mcl-1, and induced the apoptotic markers Caspase 3 and PARP cleavage. Microarray analysis revealed changes in cell survival and cell death pathways, which were confirmed by qPCR. In spite of the changes, resistant cells continue to show synergistic death effects induced by SINE in combination with proteasome inhibition. Conclusions: The extensive selection time (10 months) needed to achieve drug resistance suggests that generation of resistance may be difficult and that drug response may be prolonged. However such a resistance would be overcome by drug combination treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A188. Citation Format: Marsha L. Crochiere, Trinayan Kashyap, Jean-Richard Saint-Martin, Sharon Shechter, Ori Kalid, Eran Shacham, William Senapedis, Boris Klebanov, Sharon Tamir, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Erkan Baloglu, Dilara McCauley, Michael Kauffman, Sharon Shacham, Yosef Landesman. SINE resistant fibrosarcoma cells reveal changes in profile of gene expression, but continue to be sensitive to combination treatment by proteasome inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A188.

2505 Background: In cancers, the majority of tumor suppressor proteins (TSP) are transported out of the nucleus exclusively by Exportin 1 (XPO1/CRM1), rendering these TSPs non-functional. KPT-330 is a potent inhibitor of XPO1, and forces the nuclear retention and activation of > 10 TSPs resulting in tumor cell death in vitro, in murine preclinical models and in dogs with spontaneous lymphomas. Methods: KPT-330 was administered orally for 10 doses in a 28-day cycle. Detailed pharmacokinetic (PK) and pharmacodynamic (PDn) analyses and serial tumor biopsies were performed. Response evaluation was done every 2 cycles (RECIST 1.1). All pts entering the study had documented progressive disease. Results: 23 pts (10 males; median age 62 yrs; ECOG PS 0/1: 5/18) received KPT-330 across 6 dose levels (3 to 30 mg/m2). There has been no dose limiting toxicity. Nine drug related grade 3/4 adverse events (AEs) post cycle 1 were reported in 6 pts (neutropenia, thrombocytopenia, hyponatremia, increased ALT, fatigue, vomiting [n=2], nausea [n=2]). The most common grade 1/2 AEs were nausea (78%), fatigue (74%) and anorexia (74%). PK analysis demonstrated a fairly proportional increase in Cmax and AUC with increasing dose, with no accumulation and without affecting half-life or clearance of KPT-330. At 30 mg/m2, AUC0-last. (4375 ng*h/mL) was comparable to the anti tumor exposure observed in mice and dogs. Tmax (~3 hrs) and T1/2 (6-7 hrs) were consistent across doses. Significant increase (2-20x) in XPO1 mRNA levels (PDn marker) in circulating leukocytes was observed at all doses, with higher doses demonstrating higher levels of XPO1 mRNA induction. Analysis of tumor biopsies confirmed nuclear localization of TSPs (e.g. p53, FOXO3A, IκB) and apoptosis of cancer cells following KPT-330 administration. RECIST response was evaluable in 13 pts. Stable disease (SD) was noted in 9 pts, with 3 (colon, endocervical & endometrial stromal tumors) remaining with SD at 6+ months (dose levels 3 & 6 mg/m2), as well as one minor response (colon). Conclusions: KPT-330 treatment is generally well tolerated, with favorable PK and PDn properties. Preliminary signals of clinical antitumor activity were observed. Clinical trial information: NCT01607905.

The key nuclear export protein CRM1 (chromosomal region maintenance 1, Exportin 1, XPO1) may directly contribute to the pathophysiology of human multiple myeloma (MM). Here, we characterized the role of CRM1 in MM biology and defined the efficacy and molecular mechanisms of novel oral, irreversible, selective inhibitors of nuclear export (SINEs) targeting CRM1 against MM. CRM1 is significantly elevated in patient MM vs. normal plasma cells at transcript and protein levels. CRM1 downregulation by shCRM1 lentiviruses inhibited cell growth and survival of MM cells (p< 0.01). Importantly, SINEs (KPT-185, KPT-251, KPT-276, and KPT-330) blocked proliferation and decreased survival of MM cell lines and patient MM cells (LD50 2-log differences). SINEs potently enhanced nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins, including rapid induction of p53 and IκB (as early as 2h after drug treatment) followed by FOXO1A, FOXO3A, p27, and PP2A in MM cells. Transcripts of p53 and its downstream targets (p21, PUMA, BAX) were induced by KPT-185, thereby inducing strong growth arrest and apoptosis. KPT-185 decreased MM oncogenes (c-myc, c-maf), anti-apoptosis molecules Mcl-1 and Bcl-xL; increased pro-apoptotic protein BAX; as well as inhibited pIκBα. Inhibition of pIκBα further correlated with KPT-185-blocked NFκB p65 DNA-binding activity in MM cells with or without A Proliferation-Inducing Ligand (APRIL) stimulation. KPT-185 further downregulated CRM1 protein, which was blocked by bortezomib; concurrently, KPT-185 (or KPT-330) upregulated CRM1 mRNA in MM cells. Cleavage of caspase 3 and PARP was markedly increased in MM1R cells treated with KPT-185 and bortezomib vs. either drug alone, confirming enhanced cytotoxicity by combination of these agents. Combined dex with KPT-185 (or KPT-276) induced synergistic cytotoxicity against MM cells (combination indices < 1). Moreover, KPT-185 and KPT-330 impaired osteoclastogenesis and bone resorption via blockade of RANKL-induced NFκB activation and NFATc1 in OC precursor cells, without impacting osteoblasts and BMSCs. Finally, SINEs (KPT-251 and KPT-276) suppressed MM cell growth (p< 0.01), diminished MM cell-induced osteolysis, and prolonged survival of SCID mice with diffuse human MM bone lesions. Together, these results identify CRM1 as a promising novel target in MM, strongly supporting clinical development of SINE CRM1 inhibitors to inhibit both MM cell growth and related bone disease. The oral SINE KPT-330 is ongoing in MM and other hematological malignancies. Citation Format: Yu-Tzu Tai, Yosef Landesman, Chirag Acharya, Yolanda Calle, Mike Zhong, Michele Cea, Daniel Tannenbaum, Antonia Cagnetta, Michaela Reagan, Aditya Munshi, William Senapedis, Jean-Richard Saint-Martin, Trinayan Kashyap, Sharon Shacham, Michael Kauffman, Yumei Gu, Lizi Wu, Steve Schey, Irene Ghobrial, Andrew Kung, Nikhil Munshi, Paul Richardson, Kenneth Anderson, Haoqiang Ying. The nuclear export protein CRM1 (XPO1) regulates multiple myeloma cell growth, osteoclastogenesis, and myeloma-induced osteolysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2142. doi:10.1158/1538-7445.AM2013-2142

Abstract SINE are novel small molecules in human phase I clinical trials for advanced cancers. SINEs inhibit nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, PTEN, pRB and IKB, leading to selective death of cancer cells. Resistant cells were created by treating the sensitive fibrosarcoma cell line HT1080 with increasing concentrations of SINE over 12 months. Gene chip analysis of parental-sensitive and drug-resistant cells that were treated with SINE resulted in activation of distinct pathways that mediate mechanisms of response and drug resistance. Methods Resistant cells were generated by selection in increasing concentrations of SINE. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. FACS, qPCR and immunoblots were used to measure effects on cell cycle, gene expression and cell death. RNA from naïve and drug treated sensitive/resistant cells was analyzed by Affymetrix microarrays. Results Treatment of SINE-sensitive fibrosarcoma cell line HT1080 (IC50 = 13.6nM) with gradually increasing concentrations of SINE for ∼1 year resulted with > 100 fold decrease in sensitivity to SINE cytotoxicity (IC50 = 1.7μM). Resistant cells displayed prolonged cell cycle (∼72 vs 24hrs) compared to parental cells. Resistant cells did not show increased PgP activity, suggesting that resistance to SINE was not the result of the induction of the multidrug resistance mechanism. Sequencing of CRM1 from the SINE resistant cells revealed no mutations in the SINE / cargo binding pocket including the reactive Cys528. Upon exposure to SINE, resistant cells had reduced nuclear accumulation of p53, p21, FOXO1A, IKB and p27 compared with SINE-sensitive cells. Interestingly, in SINE-sensitive cells, but not in resistant cells, SINE treatment resulted in potent pRB dephosphorylation (growth suppressive form of pRB), nuclear localization and the activation of p53 as measured by the induction of p53-downstream targets p21 and MIC1 (GDF15). In addition, SINE reduced levels of the anti-apoptotic protein Mcl-1, and induced the apoptotic markers Caspase 3 and PARP cleavage in sensitive but not in SINE resistant cells. Genes involved in cell adhesion, cytoskeleton formation, tight junctions, vesicle transport, cell cycle, and apoptosis were identified as key pathways correlating with drug response and resistance. Conclusions This study identified key pathways and genes that are likely mediating response and resistance to SINE antagonists. The extensive selection time (∼1 year) needed to achieve drug resistance suggests that generation of resistance may be difficult and that drug response may be prolonged. Citation Format: Yosef Landesman, Sharon Shechter, Jean-Richard Saint-Martin, Trinayan Kashyap, Marsha Crochiere, William Senapedis, Paul Ippolitti, Boris Klebanov, Sharon Tamir, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Ori Kalid, Erkan Baloglu, Dilara McCauley, Michael Kauffman, Sharon Shacham. Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitors of Nuclear Export (SINE). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 875. doi:10.1158/1538-7445.AM2013-875

Inhibition of nuclear export can promote re-activation of tumor suppressor proteins (TSPs) by restoring them to the nucleus. CRM1 (chromosome region maintenance 1, XPO1) is the exclusive exporter of many TSPs. We hypothesized that CRM1 inhibition can be used as a therapeutic target in melanoma. The growth inhibitory and pro-apoptotic effects of KPT-185, KPT-276 and KPT-330, potent, small molecule, selective inhibitors of nuclear export (SINEs) were evaluated in a panel of human metastatic melanoma cell lines using an MTS assay and Annexin V/PI staining, respectively. The weak CRM1 inhibitor, KPT-185 trans-isomer, and DMSO vehicle were controls in all assays. CRM1 protein was expressed in melanoma cell lines regardless of molecular profile (BRAF, NRAS, TP53) as determined by immunoblot. SINEs inhibited cell growth in a concentration-dependent manner and induced apoptosis at nanomolar concentrations (range=139.7nM - 2.41μM). Both BRAF wild type (WT) and BRAF V600E mutant lines were sensitive to apoptosis by SINE, but BRAF V600E lines had an 8.8-fold lower IC50s as compared to BRAF (WT) lines (p=0.018). The cytostatic and pro-apoptotic effects of CRM1 inhibition were associated with nuclear accumulation of p53, and p21 induction in A375 and CHL-1 melanoma cell lines with functional p53 at time points prior to apoptosis, while pERK accumulated in melanoma cells regardless of p53 status. In pharmacokinetic studies in mice, KPT-276 and KPT-330 showed >50% oral bioavailability with Cmax >5μM. Mice bearing either A375 (BRAF V600E) or CHL-1 (BRAF WT) melanoma xenografts were given KPT-276 or KPT-330 SINE by oral gavage. A375 xenografts (p Citation Format: Jennifer Yang, Matthew A. Bill, Gregory S. Young, Yosef Landesman, Sharon Shacham, Michael Kauffman, William Senapedis, Trinayan Kashyap, Jean-Richard Saint-Martin, Gregory Lesinski. Novel small molecule CRM1 inhibitors induce nuclear accumulation of p53, phosphorylated ERK and apoptosis in human melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3443. doi:10.1158/1538-7445.AM2013-3443

Abstract Chromosomal Maintenance Protein 1/Exportin 1 (CRM1/XPO1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of Tumor Suppressor Proteins (TSPs) such as p53, FOXO, PTEN, pRB and I-κB. SINE are a novel class of compounds currently in clinical development for a variety of cancers. SINE irreversibly block CRM1-dependent nuclear export and force the nuclear retention of TSPs, inducing apoptosis in cancer cells. Here we report in vitro activity of SINE on a broad range of cancer cell lines and in vivo efficacy results in NCI-N87 gastric and HCT-116 colorectal carcinoma murine tumor xenograft models. Methods MTT cytotoxicity assays were used to determine IC50s of KPT-SINE on various cell lines. The effects of SINE on the cell cycle and gene expression were investigated by FACS and quantitative RT/PCR respectively. Inhibition of CRM1 nuclear export was determined by immunofluorescence of CRM1 cargos. The effects of SINE treatment on tumor growth in vivo were assessed by measuring tumor volumes and by imaging with FluoroThymidine Positron Emission Tomography (FLT-PET). In tumors, gene expression was analyzed and immunohistochemistry performed to detect TSP, proliferation and apoptosis markers. Results SINE showed potent cytotoxicity on the majority of the cell lines tested with IC50 values 80%. Treated tumor samples showed very low levels of Ki67 and increased TUNEL staining consistent with induction of cell cycle arrest and apoptosis in SINE-treated groups. Also, marked upregulation of the TSPs p53 and p21 was observed, indicating that SINE induced nuclear localization of these proteins in situ. In the NCI-N87 gastric carcinoma xenograft model, oral SINE treatment inhibited tumor growth >75% compared with vehicle- treated animals. Immunofluorescence analysis with biomarkers of proliferation, along with FLT-PET results, indicated significant inhibition of proliferation in tumors. Histological analysis of the tumors confirmed in vitro observations of cytotoxic effects on tumors, induction of apoptosis and the replacement of cancer cells with fibrotic tissue. Conclusions These studies demonstrate potent in vitro and in vivo efficacy of single agent SINE CRM1 inhibitors against NCI-N87 and HCT-116 xenografts. These data further support the development of SINE-based therapies for gastric and colorectal cancers. Citation Format: Dilara McCauley, Trinayan Kashyap, Mwanasha Hamuza, Yosef Landesman, Paul Ippolitti, Sharon Shechter, Jean Richard Saint-Martin, Marsha Crochiere, William Senapedis, Boris Klebanov, Sharon Tamir, Diego Del Alamo, Erkan Baloglu, Giulio Draetta, Michael Kauffman, Sharon Shacham. Selective inhibitors of nuclear export (SINE) display single agent efficacy against gastric (NCI-N87) and colon (HCT-116) xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4336. doi:10.1158/1538-7445.AM2013-4336

Abstract 326 The key nuclear export protein CRM1 (chromosome region maintenance 1, Exportin 1, XPO1) may directly contribute to the pathophysiology of human multiple myeloma (MM). Here, we characterized the role of CRM1 in MM biology and defined molecular mechanisms whereby novel oral, irreversible, selective inhibitors of nuclear export (SINE) targeting CRM1 mediate anti-MM activity. CRM1 gene expression is increased with disease progression, since it is significantly elevated in active MM and plasma cell leukemia (PCL) vs. normal/MGUS/SMM patients (p< 0.02). CRM1 downregulation by shCRM1 lentiviruses significantly decreases MM cell viability regardless of drug sensitivity and p53 status. Importantly, SINE (KPT-185, KPT-251, KPT-276, and KPT-330) specifically blocked proliferation and decreased survival of MM cell lines (n=14) and patient MM cells (n=17) (LD50 < 200 nM), cultured alone and with bone marrow stromal cells (BMSCs) or osteoclasts. Caspases 3, 8, and 9 were not induced by any SINE in BMSCs derived from MM patients, cultured either alone or with MM cells, under conditions inducing marked apoptosis of MM cells (>2-log differences). These agents potently enhanced nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins p53, IκB, FOXO1A, FOXO3A, p27, and PP2A in MM cells. Transcripts of p53 and its downstream targets p21, PUMA, BAX were also induced by KPT-185, thereby inducing strong growth arrest and apoptosis. KPT-185 decreased MM oncogenes (c-myc, c-maf), anti-apoptosis molecules Mcl-1 and BCL-xL; increased pro-apoptotic protein BAX; as well as inhibited HSP70 and pIkBa. KPT-185 further blocked baseline and APRIL-induced NFkB p65 DNA-binding activity in MM cells. It triggered proteasome-dependent reduction of CRM1 protein; concurrently, KPT-185 and KPT-330 upregulated CRM1 mRNA. Furthermore, KPT-185 induced a number of tumor suppressing, regulatory, apoptotic and anti-inflammatory genes, i.e., p53, p21, PUMA, BAX, CHOP, C10orf10, MIC1, IκBα in MM1S cells in a dose-dependent manner, regardless of the presence of BMSCs. Cleavage of caspase 3 and PARP was markedly increased in MM1R cells treated with KPT-185 and bortezomib vs. either drug alone, validating that the combination of these agents triggered stronger cytotoxicity against MM cells. Combined treatment with dex and KPT-185 (or KPT-276) induced synergistic cytotoxicity against MM cells. Moreover, KPT-185 and KPT-330 impaired osteoclastogenesis and bone resorption via blockade of RANKL-induced NFκB activation in osteoclast precursor cells, without impacting osteoblasts and BMSCs (Abstract#48190). Importantly, SINEs (KPT-251 and KPT-276) suppressed MM cell growth (p< 0.01), diminished MM cell-induced osteolysis, and prolonged survival of SCID mice with diffuse human MM bone lesions (p=0.0004). Together, these results identify CRM1 as a promising novel target in MM, strongly supporting clinical development of SINE CRM1 antagonists to inhibit both MM cell growth and related bone disease. Disclosures: Landesman: Karyopharm Therapeutics Inc: Employment. Senapedis:Karyopharm Therapeutics Inc: Employment. Saint-Martin:Karyopharm Therapeutics Inc: Employment. Kashyap:Karyopharm Therapeutics Inc: Employment. Ying:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy. Richardson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

e13549 Background: Inhibition of nuclear export can promote re-activation of tumor suppressor pathways. CRM1 (chromosomal regional maintenance 1) or XPO1 (exportin 1) is the major protein that mediates nuclear export. We hypothesized that CRM1 mediated nuclear export represents a novel therapeutic target that can be manipulated to inhibit melanoma cell survival. Methods: The growth inhibitory and pro-apoptotic effects of KPT-185, KPT-276 and KPT-330, small molecules selective inhibitor of nuclear export (SINE) were evaluated in human melanoma cell lines using an MTT assay and Annexin V/PI staining, respectively. Fluorescence microscopy and immunoblots were used to assess nuclear accumulation of tumor suppressor proteins. The trans-isomer of KPT-185 and DMSO (vehicle) were used as a negative controls in all assays. The pharmacokinetic (PK) profile of all compounds was evaluated in mice. Results: CRM1 protein was highly expressed in human melanoma cell lines with diverse molecular profiles (i.e., B-Raf, NRAS, p53). KPT-SINE inhibited melanoma cell growth in a concentration-dependent manner and induced apoptosis at nanomolar concentrations. Importantly, there was no evidence that B-Raf V600 mutational status influenced melanoma cell response to these agents. Nuclear accumulation and/or induction of p53, p21, FOXO3a, STAT1 and BAD, and reduction of MCL-1 occurred in melanoma cells at time points prior to apoptosis as shown by increase in cleaved PARP and caspase 3 levels. PK studies were conducted in mice following oral administration of 10 mg/kg, to guide drug selection for our ongoing efficacy studies in murine melanoma models. KPT-185 showed limited bioavailability and systemic exposure, while KPT-276 and KPT-330 showed >50% bioavailability reaching Cmax >5µM. Conclusions: This study represents the first report of CRM1 inhibition in melanoma. These data indicate that the novel SINE compounds can effectively inhibit CRM1-mediated nuclear export and induce apoptosis in melanoma cells. KPT-330 is currently under development as orally bioavailable, small molecule inhibitors for a human clinical trial.

Chromosomal Maintenance Protein 1/Exportin 1 (CRM1/XPO1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of Tumor Suppressor Proteins (TSPs) such as p53, FOXO, PTEN, pRB and I-κB. Small molecule, KPT-SINE that block CRM1-dependent nuclear export can force the nuclear retention of TSPs, thus render cancer cells more susceptible to apoptosis and more responsive to other chemotherapies. KPT-SINE display significant in vitro and in vivo activity over a broad range of tumor cell lines and xenografts. To optimize the design of future clinical trials, we developed a pharmacodynamics assay and tested it in cell lines, leukocytes in vitro and leukocytes from animal models. Methods: We used quantitative PCR and immunoblotting to study patterns of gene expression of CRM1, Macrophage Inhibitory Cytokine 1 (MIC1), p53 and p21 in human, mouse, rat and monkey leukocytes. The selection and the validation of the four PD markers included 1. In vitro studies where expression of PD markers was determined in tumor cell lines. 2. In vitro studies where expression of PD markers was determined in purified leukocytes. 3. In vivo studies where expression of PD markers was determined in leukocytes isolated from mice, rats and monkeys that were treated with KPT-SINE in pharmacokinetic studies. Drug levels were determined in the plasma of all animals. Results: KPT-SINE treatment at 75 mg/kg QDX5 each week for 4 weeks inhibited tumor growth by more than 80% in Molt-4, Z-138, Hep3b and U87 xenograft models. In separate PK and TK studies, expression levels of the four PD markers were investigated and were found to correlate well with KPT-SINE exposure and duration. In addition, we found that the full induction of those markers was p53-dependent. However, we also observed partial p53-independent induction of CRM1 and MIC1. PK analysis in treated rats and monkeys indicated that KPT-SINE plasma concentrations above 250 nM were sufficient to induce significant changes in the mRNA expression levels of the PD markers. Those concentrations were higher than the individual cytotoxicity indexes (EC50), of each of the above cancer cell lines, that we measured in vitro by cell proliferation assay. Conclusions: The results from our studies suggest that KPT-SINE display potent in vivo efficacy in various xenograft models. We were able to identify PD markers that correlated well with in vitro and in vivo levels of KPT-SINE. Further studies investigating CRM1, p53, p21 and MIC1 as biomarkers of target inhibition and response to KPT-SINE treatment are on-going. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3775. doi:1538-7445.AM2012-3775

Background Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330. Methods Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays. Results Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in > 100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental and resistant cells, the extent of response was more robust in the parental cells. Conclusions These results suggest that SINE resistance is conferred by alterations in signaling pathways downstream of XPO1 inhibition. Modulation of these pathways could potentially overcome the resistance to nuclear export inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1790-z) contains supplementary material, which is available to authorized users.

10587 Background: Sarcomas are heterogeneous diseases with multiple genetic abnormalities. XPO1 inhibition can restore the activity of multiple tumor suppressor proteins (TSP) including p53, Rb, an...