Retention Of Tumor Suppressor Proteins Research Articles

Efficacy of Selinexor in Relapsed/Refractory Multiple Myeloma (RRMM) Patients with del17p and Other High-Risk Abnormalities (A Retrospective Single-Center Study).

Selinexor (Seli) is a first-in-class, oral selective inhibitor of the nuclear export protein, exportin-1 (XPO1). Seli exhibits its antitumor effect through the blockage of XPO1, which increases nuclear retention of tumor suppressor proteins (TSPs), including p53, thereby limiting the translation of oncogenes, triggering cell cycle arrest and the death of malignant cells. Multiple Myeloma (MM) patients with del17p are deficient in TP53 and have a particularly poor prognosis. Given its unique mechanism of action, we investigated whether Seli has increased efficacy in RRMM patients with del17p compared to other high-risk cytogenetics (OHRC). This is an IRB-approved observational study of RRMM patients with high-risk cytogenetics (del17p, t (4;14), t (14;16) or gain 1q) or standard-risk cytogenetics treated at the Levine Cancer Institute (LCI) with a Seli-based regimen between January 2019 and December 2022. Time-to-event endpoints (PFS, OS) were evaluated using Kaplan-Meier (KM) methods. Log-rank tests compared time-to-event endpoints between cohorts [del17p vs. OHRC vs. standard risk]. We identified 40 RRMM patients with high-risk cytogenetics, including 16 patients with del17p and 24 patients with OHRC, as well as 20 with standard-risk cytogenetics. The median age was 62.5 vs. 69 vs. 65.5 years (del17p group vs. OHRC vs. standard risk). The median prior line of therapies was five (range: 3-16) with similar rates of prior autologous stem cell transplant in all arms (68.8% vs. 62.5% vs. 70.0%). The most frequently used regimens were Seli-Pomalidomide-dexamethasone(dex) or Seli-Carfilzomib-dex (Seli-Kd) in the del17p group and Seli-Kd in the OHRC and standard-risk groups. The median time to start the Seli-based regimen after initial MM diagnosis was 5.6 years for the del17p group, 4.1 years in OHRC, and 4.8 years in the standard-risk group. The median follow-up time after the start of the Seli-based regimen was 10.5 months (mos) in the del17p group, 8.4 mos in OHRC, and 10.3 mos in the standard-risk group. In the del17p group, 50% had an objective response, 41.7% in the OHRC, and 35% in the standard-risk group (p = 0.71). Depth of response was also similar across the arms (12.5% vs. 12.5% vs. 10.0% VGPR p = 0.99). The median OS was 10.9 mos in the del17p group, 10.3 mos in the OHRC, and 10.3 mos in the standard-risk group (p = 0.92). The median OS was 15.5 mos for patients who received Seli as a bridging therapy versus 9 mos for Seli use for other reasons rather than as a bridge. Overall, Seli-based regimens showed promising responses even in this heavily pretreated population. Our analysis suggests that Seli-based regimens lead to similar outcomes among RRMM patients with del17p, OHRC, and standard-risk cytogenetics. This contrasts with previously reported outcomes using combinations of novel therapies in this population, where the del17p patients often have a poorer prognosis. Interestingly, our data suggest that Seli is a particularly effective bridging modality for patients preparing for CAR-T cell therapies in our population. Further investigation into this population is warranted, including in earlier lines of therapy, in hopes of seeing a more durable response.

A Phase 2b Study of Selinexor in Combination with Carfilzomib, Daratumumab, or Pomalidomide in Patients with Multiple Myeloma Relapsing on Current Therapy

Background: Selinexor is an oral, selective inhibitor of nuclear export compound that blocks exportin 1 (XPO1), leading to retention of tumor suppressor proteins and apoptosis in malignant cells. Preclinical studies suggest synergistic activity when selinexor is combined with other treatments for multiple myeloma (MM). We hypothesize the addition of selinexor can recapture response to patients relapsing on their current carfilzomib (CFZ), pomalidomide (POM) or daratumumab (DARA)-based regimens. Methods: This is an ongoing phase 2b, open-label, multicenter investigator-initiated study to evaluate the safety and efficacy of selinexor plus CFZ, POM, or DARA. MM patients with documented disease progression or refractory disease while on current treatment with any CFZ-containing regimen (Arm 1), any POM-containing regimen (Arm 2), or any DARA-containing regimen (Arm 3) were eligible for this study. Patients were assigned to the respective arm only if their most recent line of therapy contained the specific drug of interest. Arm 1 consists of selinexor 80 mg orally on D1, 8, 15; CFZ 56 mg/m 2 IV on D1, 8, 15; and dexamethasone (DEX) 40 mg or 20 mg if ≥75 years old on D1, 8, 15, 22. Arm 2 consists of selinexor 60 mg orally on D1, 8, 15; POM 4 mg orally on D1-21; and DEX 40 mg or 20 mg if ≥75 years old on D1, 8, 15, 22. Arm 3 consists of selinexor 100 mg orally on D1, 8, 15, 22; DARA 16 mg/kg IV or 1,800 mg SQ dosing as per standard of care; and DEX 40 mg or 20 mg if ≥75 years old on D1, 8, 15, 22. The primary endpoint is overall response rate (ORR), which includes patients who experience partial response (PR), very good partial response (VGPR), complete response (CR), or stringent CR (sCR), based on International Myeloma Working Group response criteria (Kumar 2016). Secondary endpoints include duration of response (DOR), clinical benefit rate (CBR = sCR + CR + VGPR + PR + stable disease [SD]), progression-free survival (PFS), and treatment-emergent adverse events (TEAEs). Results: At data cutoff (July 14, 2023), there were 23 patients enrolled, including 20 patients on trial, 2 screen failures, and 1 in screening. Of the 20 patients on trial, 1 patient was enrolled onto Arm 1, 9 patients onto Arm 2, and 10 patients onto Arm 3. The median age was 66 years (range 52-83), 30% with ISS stage III MM, 75% with high-risk cytogenetics, 90% refractory to the last LOT, 50% triple refractory, and 35% quadruple refractory. Fifteen unique patients had high-risk cytogenetics, which included 9 patients with 1q21 duplication, 7 with t(4;14), 2 with del(17p)/monosomy 17, 4 with TP53 mutation, and 1 with complex karyotype. In 18 patients evaluable for response, the ORR was 33% (6/18; PR, 6 [33%]) and the CBR was 89% (16/18; PR, 6 [33%]; SD, 10 [56%]); 2 patients withdrew consent before response evaluation. For Arm 2, the ORR was 29% (2/7; PR, 2 [29%]) and the CBR was 86% (6/7; PR, 2 [29%]; SD, 4 [57%]). For Arm 3, the ORR was 44% (4/9; PR, 4 [44%]) and the CBR was 100% (9/9; PR, 4 [44%]; SD, 5 [56%]). In the overall cohort, median DOR was 9.46 months (95% CI, 5.1-12.3), and median PFS was 5.98 months (95% CI, 3.9-NR) (Figure 1). There were 18/20 patients who experienced any grade TEAEs (Table 1). No treatment-related deaths occurred. The most common grade 3 TEAEs were neutropenia (25%), thrombocytopenia (15%), pneumonia (10%), and hyperglycemia (10%). One patient experienced a grade 4 TEAE of neutropenia. There were 3 unique patients who experienced SAEs related to study treatment, which included pneumonia (n=2), chest pain (n=1), sepsis (n=1), and lactic acidosis (n=1). Seventeen patients discontinued treatment due to disease progression (n=12) and withdrawal of consent (n=5). Two patients are deceased: one patient died during long-term follow-up unrelated to treatment of disease; and one patient was hospitalized for dehydration, anorexia, and hyponatremia after C1D8 of KCD then experienced progressive cytopenias upon discharge likely due to disease progression. Conclusions: Preliminary results suggest selinexor plus CFZ, POM, or DARA is safe and restores sensitivity to therapy in relapsed/refractory MM patients. In this population of heavily pretreated MM patients relapsing on their most recent line of therapy, the ORR of 33% and CBR 89% highlights the clinical potential and generally manageable side effect profile of selinexor as an add-on drug for patients with relapsed/refractory MM in combination with CFZ, POM, and DARA. Clinical trial information: NCT04661137.

A phase IIb study of selinexor in combination with carfilzomib, daratumumab, or pomalidomide in patients with multiple myeloma relapsing on current therapy.

e20042 Background: Selinexor is an oral, selective inhibitor of nuclear export compound that blocks exportin 1 (XPO1), leading to retention of tumor suppressor proteins and apoptosis in malignant cells. Preclinical studies suggest synergistic activity when selinexor is combined with other treatments for MM. We hypothesize the addition of selinexor can recapture response to patients relapsing or refractory on carfilzomib (CFZ), pomalidomide (POM) or daratumumab (DARA)-based regimens. Methods: This is an ongoing phase IIb, open-label, multicenter study to evaluate the safety and efficacy of selinexor plus CFZ, POM, or DARA in patients with MM. Patients were enrolled to each arm only if their disease was refractory to the specific drug of interest (CFZ for Arm 1, POM for Arm 2, and DARA for Arm 3). Arm 1 (n=1) consists of selinexor 80 mg orally on D1, 8, 15; CFZ 56 mg/m2 IV on D1, 8, 15; and dexamethasone (DEX) on D1, 8, 15, 22. Arm 2 (n=7) consists of selinexor 60 mg orally on D1, 8, 15; POM 4 mg orally on D1-21; and DEX on D1, 8, 15, 22. Arm 3 (n=7) consists of selinexor 100 mg orally on D1, 8, 15, 22; DARA 16 mg/kg IV or 1,800 mg SQ on D1, 8, 15, 22 for C1-2; D1 and 15 for C3-6; D1 for ≥C7; and DEX on D1, 8, 15, 22. The primary endpoint is ORR and secondary endpoints include PFS, OS, MRD negativity and TEAEs. Results: At data cutoff (Feb 6 2023), 15 patients were enrolled with a median age of 64 years (range 60-83), 27% with ISS stage III MM, 73% with high-risk cytogenetics, 93% refractory to the last LOT, 47% triple refractory, and 33% quadruple refractory (Table). The ORR was 33% (4/12; 4 PR) and the CBR was 92% (11/12; 7 SD); 3 patients are pending. The most common grade 3-4 TEAEs (n=34) were neutropenia (32%), hypophosphatemia (9%), pneumonia (6%), hyperglycemia (6%), and fatigue (6%). Ten patients discontinued treatment due to PD (n=5), withdrawal of consent (n=2), or TEAEs (n=3; fall, diarrhea, and RSV pneumonia), of whom 1 patient died while hospitalized for diarrhea and weakness. Conclusions: Preliminary results show selinexor plus CFZ, POM, or DARA is safe and restores sensitivity to therapy in relapsed/refractory MM patients. In this population of heavily pretreated MM patients, the ORR (33%) and CBR (92%) observed are higher compared to those seen with single-agent selinexor (ORR 4% and CBR 21%) (Chen C et al, Blood 2018). Clinical trial information: NCT04661137 . [Table: see text]

Abstract 4195: Evaluation of the anti-myeloma effects of high and metronomic selinexor alone and in combination with duvelisib in vitro and in vivo

Abstract Introduction: Selinexor (Karyopharm Therapeutics) is an FDA-approved oral selective inhibitor of nuclear export (SINE) compound. It specifically blocks XPO1 enforcing the nuclear retention of tumor suppressor proteins restoring their anti-cancer functions. It also attenuates the phosphoinositide-3 kinase (PI3K)-AKT pathway by activating PTEN. Although activation of PI3K signaling is often detected in hematological malignancies, there are no data regarding the combination of selinexor with the PI3K inhibitor duvelisib for MM treatment. Thus, we evaluated the anti-myeloma effects of selinexor in combination with duvelisib in vitro and also studied alternative schedules of selinexor with dexamethasone in vivo using our MM xenograft models LAGκ-2. Methods: The human MM cell line U266 was obtained from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow (BM) aspirates. Cell viability was quantified using the MTS cell proliferation assay. For the in vivo studies, the human myeloma xenograft LAGκ-2 was surgically implanted into SCID mice. Selinexor was administered via oral gavage at 2.5 mg/kg 5x per week, 5 mg/kg 4x per week on consecutive days, or 10 mg/kg 2x per week with one day between treatments. Dexamethasone was administered via intraperitoneal injection at 4 mg/kg 2x per week except the vehicle control. Results and Discussion: MTS assays showed selinexor induced concentration dependent inhibition of cell viability/proliferation in the MM cell line MM1s at 0.1, 0.4 and 1.6 μM. Selinexor alone at 0.2 μM reduced cell proliferation of primary MM tumor cells obtained from BM mononuclear cells (MCs) from a patient with progressive disease. We next examined cell viability in the presence of selinexor and duvelisib. MM1s or fresh BM mononuclear cells were incubated with a fixed concentration of selinexor (0.1μM) with increasing concentrations of duvelisib (1.5 μM -25 μM) for 48 h. The results showed that duvelisib enhanced the cytotoxic effects of selinexor in a concentration dependent manner. We also evaluated dosing and scheduling of selinexor and dexamethasone in vivo using SCID mice bearing LAGκ-2 MM xenografts. When selinexor was combined with dexamethasone, lower more frequent dosing showed greater inhibition of tumor growth than mid-range and higher less frequent dosing of this SINE. This study illustrates that the combination of selinexor and duvelisib showed enhanced cytotoxic effects in vitro compared with single agent treatment, and we will further evaluate the anti-MM effects of selinexor in combination with duvelisib in vivo using our human MM xenograft models. These studies suggest that the combination of a SINE and PI3K inhibitor may be effective for MM treatment. Citation Format: Haiming Chen, Stacy Behare, Mingjie Li, Marissa Goldwater, Ava Hekmati, Jasmin Cao, Rocky Levin, Ning Xu, James Berenson. Evaluation of the anti-myeloma effects of high and metronomic selinexor alone and in combination with duvelisib in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4195.

Targeting Nuclear Exporter Protein XPO1/CRM1 in Gastric Cancer.

Gastric cancer remains an unmet clinical problem in urgent need of newer and effective treatments. Here we show that the nuclear export protein, Exportin 1 (XPO1, chromosome region maintenance 1 or CRM1), is a promising molecular target in gastric cancer. We demonstrate significant overexpression of XPO1 in a cohort of histologically diverse gastric cancer patients with primary and metastatic disease. XPO1 RNA interference suppressed gastric cancer cell growth. Anti-tumor activity was observed with specific inhibitor of nuclear export (SINE) compounds (selinexor/XPOVIO), second-generation compound KPT-8602/eltanexor, KPT-185 and +ve control Leptomycin B in three distinct gastric cancer cell lines. SINE compounds inhibited gastric cancer cell proliferation, disrupted spheroid formation, induced apoptosis and halted cell cycle progression at the G1/S phase. Anti-tumor activity was concurrent with nuclear retention of tumor suppressor proteins and inhibition of colony formation. In combination studies, SINE compounds enhanced the efficacy of nab-paclitaxel in vitro and in vivo. More significantly, using non-coding RNA sequencing studies, we demonstrate for the first time that SINE compounds can alter the expression of non-coding RNAs (microRNAs and piwiRNAs). SINE treatment caused statistically significant downregulation of oncogenic miR-33b-3p in two distinct cell lines. These studies demonstrate the therapeutic significance of XPO1 in gastric cancer that warrants further clinical investigation.

PL3.5 Efficacy and safety of selinexor in recurrent glioblastoma

Abstract BACKGROUND New treatment modalities are needed for recurrent glioblastoma (rGBM). Selinexor is a novel, oral selective inhibitor of nuclear export which forces nuclear retention of tumor suppressor proteins including p53 and p27, leading to apoptosis. We previously reported interim results showing tolerability, preliminary efficacy, and blood-brain barrier penetration in a surgical cohort (N=8). We now report updated results following completion of accrual to non-surgical cohorts (N=68). MATERIALS AND METHODS This is an open-label, multicenter, phase 2 study of selinexor monotherapy. Patients (pts) not undergoing surgery for measurable rGBM per response assessment neuro-oncology criteria (RANO) were enrolled in one of 3 arms encompassing different dosing schedules of selinexor (50 mg/m2 [~ 85 mg] BIW, 60 mg BIW, and 80 mg QW). Treatment was continuous, although cycles were defined as 28 days and response was assessed every other cycle by MRI. Prior treatment with radiotherapy and temozolomide was required and prior bevacizumab was exclusionary. The primary endpoint was 6-month progression free survival (6mPFS) rate, calculated by the Kaplan-Meier method. RESULTS 76 pts were enrolled; 24, 14 and 30 pts on doses of ~85 mg BIW, 60 mg BIW, and 80 mg QW, respectively. Median age was 56 years (range 21–78). Median number of prior treatments was 2 (range 1–7) At the end of the 6 cycles, 30.2% pts on 80 mg QW were free from progression. The 6mPFS rate on 80 mg QW was 18.9%. Best RANO-defined responses (assessed locally) among 26 evaluable pts on 80 mg QW included 1 complete response, 2 partial responses, 7 stable disease, and 16 with progressive disease. Complete and partial responses were durable: the complete and a partial responder remain on selinexor for 393 and 1093 days respectively, as of the cut-off date. Median duration of response was 10.8 months. The most common related adverse events (all grades) in pts on ~85 mg BIW/60 mg BIW/80 mg QW were nausea (42%/64%/63%), leukopenia (38%/7%/43%), fatigue (71%/71%/47%), neutropenia (29%/14%/33%), decreased appetite (46%/71%/27%), and thrombocytopenia (67%/29%/23%). CONCLUSION Selinexor demonstrated efficacy, with durable responses and disease stabilization in rGBM. Based on the favorable efficacy and safety profile, selinexor at a dose of 80 mg QW is recommended for further development in rGBM.

Efficacy and safety of selinexor in recurrent glioblastoma.

2005 Background: New treatment modalities are needed for recurrent glioblastoma (rGBM). Selinexor (SEL) is a novel, oral selective inhibitor of nuclear export which forces nuclear retention of tumor suppressor proteins including p53 and p27, leading to apoptosis. We previously reported interim results showing tolerability, preliminary efficacy, and blood-brain barrier penetration in a surgical cohort (N = 8). We now report updated results following completion of accrual to non-surgical cohorts (N = 68). Methods: This is an open-label, multicenter, phase 2 study of SEL monotherapy. Patients (pts) not undergoing surgery for measurable rGBM (per RANO) were enrolled in one of 3 arms encompassing different dosing schedules. Treatment was continuous, although cycles were defined as 28 days and response was assessed every other cycle by MRI. Prior treatment with radiotherapy and temozolomide was required and prior bevacizumab was exclusionary. The primary endpoint was 6-month progression free survival (6mPFS) rate, calculated by the Kaplan Meier method. Results: A total of 76 pts were enrolled. Median age was 56 years (range 21-78). Median number of prior treatments was 2 (range 1-7). At the end of the 6 cycles, 30.2% patients on 80 mg QW were free from progression. The 6mPFS rate on 80 mg QW was 15.1%. Best RANO-defined responses (assessed locally) among 26 evaluable pts on 80 mg QW included 1 complete response, 2 partial responses, 7 stable disease, and 16 with progressive disease. Median duration of response was 10.8 months. The most common related adverse events in pts on ~85 mg BIW/60 mg BIW/80 mg QW were nausea (42%/64%/60%), leukopenia (38%/7%/43%), fatigue (71%/71%/43%), neutropenia (29%/14%/33%), decreased appetite (46%/71%/27%), and thrombocytopenia (67%/29%/23%). Conclusions: SEL demonstrated efficacy, with durable responses and disease stabilization in rGBM. Based on the favorable efficacy and safety profile, SEL at a dose of 80 mg QW is recommended for further development in rGBM. Clinical trial information: NCT01986348. [Table: see text]

Selinexor plus low-dose bortezomib and dexamethasone for patients with relapsed or refractory multiple myeloma

AbstractSelinexor is an oral inhibitor of the nuclear export protein exportin 1. Preclinical studies demonstrated synergistic antimyeloma activity between selinexor and proteasome inhibitors (PI) through suppression of NF-κB signaling and nuclear retention of tumor suppressor proteins. We tested selinexor in combination with low-dose bortezomib and dexamethasone (SVd) for the treatment of relapsed or refractory multiple myeloma (MM). The primary objectives of this study were to determine the safety profile, overall response rate (ORR), and a recommended phase 2 dose (RP2D) of SVd. We enrolled 42 patients to receive selinexor (60, 80, or 100 mg orally) plus bortezomib (1.3 mg/m2 subcutaneously) and dexamethasone (20 mg orally) once or twice weekly in 21- or 35-day cycles. Patients had a median of 3 (range 1-11) prior lines of therapy, and 50% were refractory to a PI. Treatment-related grade 3 or 4 adverse events reported in ≥10% of patients were thrombocytopenia (45%), neutropenia (24%), fatigue (14%), and anemia (12%). Incidence (4 patients, 10%) and grade (≤2) of peripheral neuropathy were low. The ORR for the entire population was 63%: 84% ORR for PI nonrefractory and 43% for PI-refractory patients. The median progression-free survival for all patients was 9.0 months; 17.8 months for PI nonrefractory, and 6.1 months for PI refractory. SVd treatment produced high response rates in patients with relapsed or refractory MM, including borezomib-refractory MM, with no unexpected side effects. The RP2D is selinexor (100 mg once weekly), bortezomib (1.3 mg/m2 once weekly for 4 weeks), and dexamethasone (40 mg once weekly) per 35-day cycle. This trial was registered at www.clinicaltrials.gov as #NCT02343042.

Abstract 4646: Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer

Abstract Introduction: Selinexor (sel) is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports include major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. We have previously shown that HER2+ breast cancer (BC) cells are highly sensitive to sel (SKBR3 and MCF7 IC50 0.21 and 0.073 μM, respectively). Here we tested the hypothesis that inhibition of HER2 signaling with trastuzumab (tras) synergizes with sel as both mediators (PI3K-AKT and Ras-Raf-MEK pathways) and effectors (FOXO3A, MDM2) of the HER2 pathway are among direct cargos of XPO1. Methods: The effects of sel and tras alone or in combination on cell viability were tested on MCF7 and BT474 HER2+ BC cells using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, BT474 cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of sel and tras alone or in combination and with the therapeutic dose of sel. Tumor Growth (TG) was monitored for 60 days. Xenografts were harvested and analyzed by immunohistochemestry (IHC). Results: Sel-tras combination was highly effective in-vitro and in-vivo. In MTT assays, both sel and tras demonstrated low IC50 values (nanomolar) and when combined, they synergistically inhibit proliferation. In-vivo, the combination resulted in significant survival benefit and enhanced TG inhibition of 88% compared to the monotherapy groups 57% (sel) and 25% (tras). Interestingly, an increase in pro-survival cytoplasmic FOXO3A protein was observed in the tras-treated group however, in the combination group, FOXO3A, which is an XPO1cargo, was restricted to the nucleus where it can trigger apoptosis. In addition, a synergistic increase in P27 protein, the G1 phase arrest regulator, was observed in the sel-tras-treated group. While in the monotherapy groups p-ERK was not modulated, in the combination group an increase in cytoplasmic p-ERK was observed, suggesting an affect on its downstream regulators of apoptosis. Indeed, apoptosis was evident in the combination samples by increased IHC staining to Cleaved caspase3 and Apoptag. In addition, an increase in PARP protein was observed in the tras-treated group however, in the combination group, PARP was dramatically reduced. We have previously shown that selinxor modulates DNA Damage Response (DDR) proteins suggesting that one mechanism by which the combination enhances cancer cell death is by accumulation of DNA damage that cannot be resolved. Conclusion: Sel-tras combination is a novel candidate therapy to HER2+ BC modulating DDR and apoptosis. It synergizes to induce survival benefit and TG inhibition. These data provide rational support for study of sel-tras combination in clinical trials. Citation Format: Sivan Elloul, Hua 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 trastuzumab in a mouse model of breast cancer. [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 4646.

Abstract 2219: Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows synergistic anti-tumor activity when combined with PD-1 blockade in a mouse model of colon cancer

Abstract Introduction: Selinexor is an oral, first-in-class SINE compound that specifically binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports over 200 cargos, including major tumor suppressor proteins (TSPs), leading to their functional inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. Interestingly, XPO1 also mediates the export of NFAT1c, STAT1 and STAT3, which have been implicated in regulation of the inhibitory T-cell receptor PD-1 (NFAT1c, STAT3) and its ligand, PD-L1 (STAT1). Therefore, we hypothesized that selinexor treatment will result in up-regulation of PD-1 and PD-L1, making tumor cells more amenable to immunotherapy. Methods: The effect of selinexor on PD-1 and PD-L1 gene expression was tested in human normal donor leukocytes and in tumor cells [HCT-116(colon), PC-3 (prostate) and MDA-MB-468 (breast)], respectively by quantitative PCR. A Colon26 syngeneic mouse model of CRC was generated and animals were assigned to the following treatment groups: (i) vehicle, (ii) selinexor at sub-therapeutic dose of 5 mg/kg (M/W/F), (iii) Anti PD-1 (BioXCell), 100ug biwk and (iv) selinexor + anti-PD-1 combination. Tumor growth, weight loss and signs of toxicity were monitored for 45 days. Xenografts were harvested, RNA and DNA were collected and tumors were analyzed microscopically and by immunohistochemestry (IHC). Results: In-vitro studies showed that selinexor induces PD-1 gene expression by ∼2-fold in normal donor leukocytes as early as 4 hrs after treatment initiation and that this change is sustained for at least 24 hrs. A similar effect was measured on PD-L1 gene expression induction in HCT-116, PC-3 and MDA-MB-468 tumor cells. In-vivo, the combination was well tolerated and no weight loss or signs of toxicity were evident. Selinexor-anti-PD-1 combination therapy exhibited a synergistic effect on percent Tumor Growth Inhibition (%TGI) were the selinexor-treated group and the anti-PD-1-treated group alone were very similar with mean%TGI measuring 27% and 24% respectively, and the combination group measuring a mean%TGI of 67%. Importantly, 4-out-of-10 mice in the combination group did not have detectable tumors at day 22 and these mice were also tumor free at the end of the study, at day 45. IHC analysis showed overall enhanced effect of the combination treatment on induction of apoptosis and reduced proliferation as well as effect on specific T-cell checkpoint regulatory molecules. Conclusion: Selinexor induces PD-1 and PD-L1 gene expression, sensitizing tumor cells to immunotherapy. Selinexor synergizes with anti-PD-1 to inhibit tumor cell proliferation and to induce apoptosis in-vivo. These data provide rational support for study of selinexor/ anti-PD-1 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. Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows synergistic anti-tumor activity when combined with PD-1 blockade in a mouse model of colon cancer. [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 2219.

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.

Safety, Efficacy, and Determination of the Recommended Phase 2 Dose for the Oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330)

Safety, Efficacy, and Determination of the Recommended Phase 2 Dose for the Oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330)

Abstract LB-255: Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows enhanced antitumor activity in combination with the PARP inhibitor, olaparib, in models of triple-negative breast cancer

Abstract Background: Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound currently in Phase I and II clinical trails for the treatment of hematological and solid malignancies. Selinexor blocks the key nuclear export protein XPO1 to force nuclear retention of tumor suppressor proteins (TSPs), including p53, BRCA1/2, pRB and FOXO3A. Olaparib is an FDA approved therapy for BRCA1/2 mutated ovarian cancer, which inhibits Poly-ADP-Ribose Polymerase (PARP) and prevents DNA damage repair. Furthermore, olaparib is being evaluated for the treatment of Triple Negative Breast Cancer (TNBC). We hypothesized that selinexor would restore genomic surveillance through nuclear accumulation of wild type BRCA1 and therefore combination treatment with olaparib would prevent DNA damage repair to amplify cancer cell death. Methods: The effects of selinexor alone or in combination with olaparib were tested on a panel of 7 TNBC cell lines using MTT and soft-agar colony formation assays in parallel with FACS analysis. In vivo efficacy of single-agent or combination therapy was evaluated using an MDA-MB-468 (BRCA1 wild type, TNBC) xenograft model. Combination index (CI) values were determined using the CompuSyn software and treatment was considered synergistic when CI<1. Results: The median IC50 values for selinexor and olaparib were 1.88 μM (range: 0.27 μM to >10 μM) and 92.6 μM (range: 17.5 μM to >300 μM), respectively. Combination treatment led to synergistic inhibition of proliferation in the 7 TNBC cell lines evaluated. The median CI tested on the panel of cell lines was 0.68 (ranging from 0.4 to 0.96). FACS analysis revealed an additive effect of the selinexor and olaparib combination on S-phase inhibition and G2 arrest in BRCA1 mutated and wild type cells. Furthermore, AnnexinV/PI staining showed an additive effect on TNBC cell apoptosis regardless of BRCA1 mutational status. In the MDA-MB-468 xenograft model, 75% tumor growth inhibition (TGI) was observed in the combination group by day 22 compared to 55% and 35% TGI for single-agent selinexor and olaparib, respectively. Conclusion: Selinexor and olaparib in combination act synergistically to induce apoptosis in TNBC cells and amplify anti-tumor effects in a TNBC xenograft model. These data provide a rationale supporting the study of selinexor/olaparib combination in clinical trials. Citation Format: Helene Marijon, Sigal Gery, Sivan Elloul, Sharon Y. Friedlander, TJ Unger, Robert Carlson, Sharon Shacham, Michael Kauffman, Harold P. Koeffler. Selinexor, a selective inhibitor of nuclear export (SINE) compound, shows enhanced antitumor activity in combination with the PARP inhibitor, olaparib, in models of triple-negative breast cancer. [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-255. doi:10.1158/1538-7445.AM2015-LB-255

Piperlongumine is a novel nuclear export inhibitor with potent anticancer activity

Piperlongumine is a natural compound recently identified to be toxic selectively to tumor cells in vitro and in vivo. However, the molecular mechanism underlying its anti-tumor action still remains unclear. In this report, we describe another novel mechanism by which piperlongumine mediates its anti-tumor effects. We found that piperlongumine is a novel nuclear export inhibitor. Piperlongumine could induce nuclear retention of tumor suppressor proteins and inhibit the interactions between CRM1 and these proteins. Piperlongumine could directly bind to the conserved Cys528 of CRM1 but not to a Cys528 mutant peptide. More importantly, cancer cells expressing mutant CRM1 (C528S) are resistant to piperlongumine, demonstrating the nuclear export inhibition via direct interaction with Cys528 of CRM1. The inhibition of nuclear export by piperlongumine may account for its therapeutic properties in cancer diseases. Our findings provide a good starting point for development of novel CRM1 inhibitors.

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.

Abstract B198: Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC).

Abstract Selinexor (KPT-330) is a novel small molecule inhibitor of 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 IκB, resulting in selective death of cancer cells. Preclinical studies have shown that oral Selinexor is well tolerated, even with prolonged (4-8 month) administration. Human Phase 1 clinical studies with Selinexor in hematological and solid cancer patients are ongoing (Clinicaltrials.gov NCT01607892 and NCT01607905). In this study we evaluated the mechanisms involved in Selinexor-induced growth inhibition of NSCLC in vitro and in vivo. Methods: A panel of NSCLC cells with various genetic backgrounds were treated with Selinexor and tested in proliferation (MTT), clonogenic survival, and flow cytometric assays. The cellular localization of TSPs were evaluated by immunofluorescence (IF). Western blotting was used to study levels of cell cycle regulating proteins and quantify markers of cell survival. Functional inhibition of NF-κB was measured using a transcription factor assay kit. A SCID mouse xenograft model of NSCLC, adenocarcinoma A549 (wt p53, NF-κB IC50 of 1.19 uM), was used to evaluate Selinexor effects in vivo and tumors were evaluated by histology and immunohistochemistry (IHC). Results: Selinexor induced cell cycle arrest, inhibited tumor cell growth and clonogenic formation and induced apoptosis in lung cancer cell lines regardless of genetic background (IC50 25-700 nM). Treated normal human dermal fibroblasts entered into cell cycle arrest but resisted apoptosis induction (IC50 4.0 uM). IF of the TSPs p53, p21, IκB, E2F4, and survivin demonstrated strong nuclear localization after 4 to 24 hour treatment with Selinexor followed by apoptosis as determined by cleavage of PARP, caspases 3 and 8 by immunoblot analysis. Functional evaluation of NF-kB in A549 and H1299 revealed transcriptional repression with IC50 values similar to MTT assays. Finally, A549 xenografts treated with Selinexor (10 - 20 mg/kg QoDx3) showed marked tumor suppression when compared to vehicle and cisplatin-treated mice (growth inhibition was 78% at 20 mg/kg, p&amp;lt;0.0001). Histological and IHC analysis of treated tumors indicated increased nuclear accumulation of p53, p21, FOXO1, survivin, NF-κB and IκB, reduced cell proliferation, and tumor cell death. Conclusions: In NSCLC, Selinexor forces nuclear retention of TSPs, inhibits NF-κB transcriptional activity, inhibits tumor growth and induces cell death regardless of p53 status. Selinexor thus has therapeutic potential for the treatment of NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B198. Citation Format: William Senapedis, Marsha Crochiere, Tami Rashal, Trinayan Kashyap, Boris Klebanov, Jean-Richard Saint-Martin, Ori Kalid, Sharon Shechter, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Eran Shacham, Sharon Tamir, Dilara McCauley, Erkan Baloglu, Michael Kauffman, Sharon Shacham, Yosef Landesman. Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC). [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 B198.

Abstract 2075: Combination tTerapy KPT-SINE (selective inhibitors of nuclear export) with radiotherapy have additive effects in non-small cell lung cancer (NSCLC) cells in vitro and in vivo.

Abstract Radiation therapy is an established therapeutic option for lung cancer, but its use is limited due to side effects. Combining radiosensitizers with RT is a way to augment the efficacy of RT. Chromosome Region Maintenance Protein 1/Exportin 1 (CRM1/XPO1) is a key nuclear export protein, the cargoes of which are Tumor Suppressor Proteins (TSPs) such as p53, FOXO, PTEN, pRB and I-κB. Small molecule SINE block CRM1-dependent nuclear export and force the nuclear retention of TSPs, potentially sensitizing cells to RT. Suggested mechanisms include: 1) Induction of “genome fidelity review” by forced nuclear retention of TSPs; 2) Prevention of single stranded DNA break repair, and thereby reduction of resistance of cancer cells to RT. Methods: IC50 of KPT-SINE e.g. KPT-185 and clinical compound KPT-330, were determined in MTT assays using human NSCLC p53 wild type (wt) cancer cell lines H1299 and A549. Clonogenic assays in the SINE sensitive H1299 (IC50 100nM) and SINE-resistant A549 (IC50 1.7μM) lines were conducted in the presence or absence of escalating doses of radiation. An in vivo tumor xenograft model using the A-549 cell line in SCID mice was also conducted with KPT-330. Results: KPT-185 demonstrated inhibitory effects on the clonogenicity of two NSCLC cell lines in a dose dependent fashion. We showed that, while RT alone treatment caused coloines growing curve reduced to less than &amp;lt;40% of colonies in both cell lines. Combination of both agents (RT and SINE at IC25 concentrations) demonstrated an additive effect for A549 cell line, and a dose-related effect for H1299 cell line. In vivo, we demonstrated significant reductions in tumor growth, where treatment with KPT-330 caused a &amp;gt;75% tumor volume reduction when compared to vehicle, and a &amp;gt;65% reduction when compared to Cisplatin. In this model, KPT-330 was fairly well-tolerated, with minimal body weight loss by animals over the course of the study. Conclusions: These studies showed that KPT-SINE significantly inhibited the growth of two p53 wt NSCLC lines by MTT assay. Similar inhibition was observed in clonogenic assays with cells (A549) resistant to SINE. We described the additive effects of SINE and RT on NSCLC cell lines, suggesting that SINEs can act as radiosensitizers. We further demonstrated that SINEs are efficacious in vivo in the A549 xenograft model, and display superior efficacy when compared to Cisplatin. SINE compounds represent a promising novel therapy against NSCLC both as stand-alone agents and in combination with RT. These data support further development of SINE-based therapies for NSCLC. Further work on in vivo studies of KPT-SINE in combination with RT will be reported. Citation Format: Tami Rashal, Dilara McCauley, Maya Ilouze, Nir Raphael, Inessa Solomonik, Yaccov Lawrence, Ronen Shavit, Sharon Shacham, Michael Kauffman, Nir Peled. Combination tTerapy KPT-SINE (selective inhibitors of nuclear export) with radiotherapy have additive effects in non-small cell lung cancer (NSCLC) cells in vitro and in vivo. [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 2075. doi:10.1158/1538-7445.AM2013-2075

Abstract 4336: Selective inhibitors of nuclear export (SINE) display single agent efficacy against gastric (NCI-N87) and colon (HCT-116) xenografts.

Abstract 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 &amp;lt;1 μM, but did not affect non-malignant human intestinal epithelial cells. The inhibition of CRM1 nuclear export with SINE resulted with the accumulation of TSPs p53, p21, p27 and FOXO in the nucleus. Immunoblot and qPCR studies showed increased expression of stress-related genes, reduced expression of proliferation markers, and evidence of cell death. SINE therapy was well-tolerated in mice. In the HCT-116 (p53wt, K-ras mutant) xenograft model, oral SINE treatment inhibited tumor growth by &amp;gt;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 &amp;gt;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 3775: Pharmacokinetic (PK) / pharmacodynamic (PD) and efficacy relationship of selective inhibitors of nuclear export (KPT-SINE)

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. 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

CRM1 Is Highly Expressed in Myeloma Plasma Cells and Its Inhibition by KPT-SINE Induces Cytotoxicity by Increasing p53 in the Nucleus of Multiple Myeloma (MM) Cells

Abstract 1852 Background:CRM1 (XPO1, exportin) is a nuclear export protein which controls the nuclear-cytoplasmic localization of multiple tumor suppressor proteins and cell proliferation pathways including p53, p21, PI3K/AKT/FOXO, Wnt/ß-catenin/APC, topoisomerase II, and NF-κB/I-κB. Transport of nuclear proteins to the cytoplasm can render them ineffective as tumor suppressors or as targets for chemotherapy. Small molecule, selective inhibitors of nuclear export (SINE) that block CRM1-dependent nuclear export can force the nuclear retention of tumor suppressor proteins, thus rendering cancer cells more susceptible to apoptosis and responsive to other chemotherapy. In this study we evaluated CRM1 as a potential target in MM and the effect of SINE on the activity of established anti-myeloma agents currently in use in treatment of MM. KPT-276 is the lead CRM1 inhibitor being investigated which will be submitted for IND in 2012. Methods:To evaluate expression of CRM1, bone marrow aspirates from MM patients and tonsil tissue from normal patients were enriched for plasma cells (PC) and proteins from cell lysates were separated by SDS-PAGE followed by immunoblotting with CRM1 antibodies. In functional experiments, isolated fresh MM PCs from patients, and NCI-H929, MM1.S, MM1.R and RPMI-8226 cell lines were cultured in RPMI-1640 with 10–15% serum. Cells were treated for 24–72 hrs with CRM1 inhibitors KPT-SINE compounds with or without bortezomib and dexamethasone and were analyzed for cytotoxicity by MTT assay. Drug concentrations for combination experiments were chosen to be at or below IC50 for each individual drug. Apoptosis induction in primary MM cells and cell lines was studied by Annexin V labeling and flow cytometry. Cell lysates from primary MM PCs and cell lines were prepared after treatment with KPT-SINE and were used to determine the expression of p53 and CRM1. Results:Primary MM plasma cells derived from naïve, previously untreated patients show 4–20 fold higher CRM1 protein expression, compared to normal peripheral blood mononuclear cells (PBMCs) and normal tonsilar PCs. Dose response analysis of KPT-SINE compounds in myeloma cell lines showed potent activity with IC50s in the range of 10–100nM. The lead compound KPT-276 had an IC50 of <100 nM in NCI-H929, MM1.S, MM1.R and RPMI-8226 cells. Functional studies in MM patient plasma cells showed that in vitro inhibition of CRM1 with related SINEs KPT-185, −225 or −276 increase apoptosis induction as measured by Annexin V assay. In addition, the inhibition of CRM1 with KPT-SINE results in a dose-dependent increase in levels of nuclear as well as total p53 in MM patient plasma cells within 48 hrs. When combined with proteasome inhibitors like bortezomib and/or dexamethasone, KPT-SINE compounds potently increase the cellular cytotoxicity of these drugs in MM cell lines. Mechanism of activity of drug combinations is under investigation in MM cell lines and MM patient plasma cells. Conclusions:MM plasma cells express CRM1 that is functionally active and therefore is a valid target in the treatment of myeloma. Moreover, higher expression of CRM1 in malignant plasma cells compared to normal PBMCs and normal PCs suggests possibility of therapeutic index. Early mechanistic studies indicate that CRM1 inhibition can lead to an increased expression of p53 (and other tumor suppressors) and its nuclear localization in myeloma cells and therefore might serve as a mechanism for the activity of CRM1 inhibitors in MM. Potentiation of cytotoxicity of bortezomib and dexamethasone by KPT-SINE suggests that these drugs might be useful in treating MM refractory to currently used agents and provide rationale for combining inhibitors of nuclear transport with other drugs. Disclosures:Off Label Use: KPT-SINE family of drugs are not approved for the treatment of multiple myeloma. These drugs have a novel mechanism and are in pre-clinical development for the treatment of several malignancies. McCauley:Karyopharm Therapeutic Inc.: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Jakubowiak:Exelixis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.