Localization Of Tumor Suppressor Proteins Research Articles

Abstract B147: Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models

Abstract Background: Resistance to topo-I inhibitors is of significant importance in treating patients with metastatic CRC (mCRC). Selinexor and eltanexor are selective inhibitors of nuclear export, by binding to exportin 1 (XPO1, the primary nucleic export receptor, and is overexpressed in CRC). Eltanexor is an investigational XPO1 inhibitor, with similar mechanism and potency but improved tolerability in animal models. XPO1 inhibition results in nuclear localization of tumor suppressor proteins, oncoprotein mRNAs, and topoisomerases, and decreases DNA damage repair (DDR) response. Irinotecan is a topo-I inhibitor which induces DNA damage and is commonly used in mCRC. We hypothesized that XPO1 inhibition may improve response to topo-I inhibitor in mCRC by impairing the DDR response. Methods: The synergism of XPO1 inhibition (selinexor or eltanexor) with SN38 was assessed in CRC cell lines by CellTiter-Glo 2.0 and quantitated using Bliss Additivity Model using Synergy Finder 2.0. Each condition was plated either concurrent (both drugs for 72 hours) or sequential (SN38 for 24 hours then removed, followed by XPO1 inhibitor for 48 hours). Nude mice were implanted with one microsatellite stable CRC PDX model in the flanks followed by treatment with vehicle, XPO1 inhibitor, irinotecan (15 mg/kg intraperitoneal weekly), or combination. Tumor growth was measured by specific growth rate (SGR), within an IACUC-approved protocol. Tumor tissue was collected at day 7 for molecular analysis. Tissues were lysed and protein was run on 4%-12% SDS-PAGE Gel. p21 Waf/Cip and P-Histone H2A.X were used to determine DNA damage by western blot. P-Histone H2AX (pH2AX) was evaluated by immunofluorescence using primary conjugated P-Histone H2A.X antibody and DAPI for nuclear counter stain. Slides were imaged using Olympus iX83 microscope and analysis was performed using Olympus CellSens software. Results: In CRC cell lines, concurrent combination eltanexor and SN38 did not synergistically decrease viability but increased pH2AX compared to SN38 alone (~30-fold). However, sequential SN38 followed by eltanexor synergistically decreased viability at multiple dose levels; evaluation of pH2AX is ongoing. In PDX model CRC254, eltanexor (10 mg/kg and reduced to 7.5 mg/kg on day 8 to improve tolerability, orally 5 days per week) with irinotecan decreased SGR compared to irinotecan alone (SGR=0.007 vs 0.026, respectively, p=0.0003) and compared to eltanexor alone (SGR=0.029, p <0.0001). Combination treatment increased pH2AX and increased p21 expression compared to either single-agent treatment. Similar SGR results were observed with selinexor (5 mg/kg orally twice weekly). Conclusions: XPO1 inhibition and topo-I inhibition decreased CRC tumor growth in PDX models, presumably by increasing DNA double strand breaks. Cell line data suggests sequential dosing of topo-I inhibition followed by XPO1 inhibition may be effective. This work supports continued development of this combination in mCRC. Citation Format: Robert W Lentz, Adrian Dominguez, Stacey Bagby, Cameron Binns, Alexis Leal, Sunnie Kim, Sarah Lindsey Davis, Christopher H Lieu, Wells A Messersmith, Todd M Pitts. Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B147.

Selinexor and Other Selective Inhibitors of Nuclear Export (SINEs)—A Novel Approach to Target Hematologic Malignancies and Solid Tumors

Exportin 1 (XPO1) is a crucial molecule of nucleocytoplasmic transport. Among others, it exports molecules important for oncogenesis from the nucleus to the cytoplasm. The expression of XPO1 is increased in numerous malignancies, which contributes to the abnormal localization of tumor suppressor proteins in the cytoplasm and subsequent cell cycle dysregulation. Selective inhibitors of nuclear export (SINEs) are novel anticancer agents that target XPO1, arrest tumor suppressor proteins in the nucleus, and induce apoptosis in cancer cells. Selinexor, a first-in-class SINE, has already been approved for the treatment of relapsed/refractory multiple myeloma and relapsed/refractory diffuse large B cell lymphoma not otherwise specified. It has also been proven effective in relapsed/refractory and previously untreated acute myeloid leukemia patients. In addition, numerous studies have yielded promising results in other malignancies of the hematopoietic system and solid tumors. However, future clinical use of selinexor and other SINEs may be hampered by their significant toxicity.

Abstract 2383: Mir-7974: An oncogenic miRNA that perpetuates gastric cancer

Abstract The global incidence of gastric cancer exceeds 1 million people per year and has a 5-year survival rate of 31%. Current chemotherapies are not effective because of genetic and epigenetic heterogeneity and late stage detection. Previously, we showed that exportin 1 (XPO1) is a therapeutic target in gastric cancer, as it facilitates the nuclear export of various cancer related tumor suppressor proteins (TSPs) through direct interaction with their nuclear export sequences (NES). XPO1 is upregulated in a variety of tumors including gastric cancer, leading to increased cytoplasmic localization of TSPs supporting malignant transformation and cancer cell proliferation, Knockdown of XPO1 leads to anti-tumor effects and pharmacologic targeting of XPO1 is achievable with selective inhibitors of nuclear export (SINE) compounds. In 2019 and 2020, the first generation XPO1 inhibitor KPT-330 was FDA approved for the use in penta-refractory multiple myeloma and non-Hodgkin's Lymphoma. Besides protein, mRNA and non-coding RNAs are also transported across the nuclear membrane. Non-coding RNA transport is generally attributed to another nuclear export protein, exportin 5 (XPO5), which plays a critical role in microRNA (miRNA) biogenesis. Nevertheless, there is emerging evidence supporting the role of XPO1 in non-coding RNA transport. In order to elucidate the role of XPO1 in non-coding RNA biology, we performed small RNA sequencing on gastric cancer cell lines treated with the XPO1 inhibitor selinexor. Our results revealed statistically significant changes in miR-7974 and miR-129-1-3p. These two miRs are recognized to influence protein modifications and cell survival. Administration of a miR-7974 mimic and a miR-129-1-3p inhibitor reduced proliferation of gastric cancer cells. Next, we evaluated the miR target genes of interest. At the mRNA level, we found upregulation of RasGEF Domain Family Member 1a (RASGEF1A), downregulation of DNA damage-regulated autophagy modulator 1 (DRAM1), downregulation of TIMP2 and downregulation of Cyclin G2 protein encoding gene CCNG2. Western Blots confirmed inhibition of AKT, RAS and Wnt signaling. Our results also showed that miR-7974 overexpression correlated with cellular XPO1 levels through the enhancement of XPO1 and its cargoes RCC1 and RANBP2. Finally inhibition of XPO1 by SINE compounds reversed the observed oncogenic phenotypes caused by these microRNAs. Our results unveil a novel interaction network between XPO1 and small non-coding RNAs and show that XPO1 induced small-noncoding microRNAs influence gastric cancer proliferation, and can be reversed by SINE compounds. This implies that modulation of microRNAs might be explored as a biomarker for XPO1 inhibitor therapy and warrants further investigation. Citation Format: Rachel E. Sexton, Mohammad Najeeb Al-Hallak, Bayan Al-Share, Yiwei Li, Amro Aboukameel, Yosef Landesman, Trinayan Kashyap, Steve Kim, Ramzi M. Mohammad, Philip A. Philip, Asfar S. Azmi. Mir-7974: An oncogenic miRNA that perpetuates gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2383.

Immunohistochemical detection of p53, PTEN, Rb, and p16 in canine osteosarcoma using tissue microarray.

Although inactivating mutations of tumor suppressor genes are well described in cell lines of canine osteosarcoma (OS), expression of tumor suppressor proteins in spontaneous disease is poorly characterized. We determined the immunohistochemical expression of p53, PTEN, Rb, and p16 in a large cohort of dogs with OS. Formalin-fixed, paraffin-embedded samples of canine OS were analyzed retrospectively. Primary tumor samples from 145 dogs, collected between 2003 and 2008, were evaluated by tissue microarray. Streptavidin-biotin complex immunohistochemistry was performed using monoclonal antibodies for Rb and PTEN and polyclonal antibodies for p16 and p53. The average age of dogs was 7.6 y, and 118 of 145 (81%) were purebred. Most commonly represented purebreds were Greyhound (23%), Rottweiler (11%), and Labrador Retriever (10%). Immunohistochemical detection of p53, PTEN, Rb, and p16 was 81%, 61%, 66%, and 66%, respectively. The staining pattern for p16 was primarily cytoplasmic; the predominant pattern for PTEN, Rb, and p53 was cytoplasmic and nuclear. Exclusively cytoplasmic staining was noted in 19% of samples positive for p53 and 8% of samples positive for Rb. Kaplan-Meier curves showed that protein expression was not associated with significant differences in overall survival ( p > 0.191). We documented heterogeneity in both immunostaining and subcellular localization of tumor suppressor proteins, providing further characterization of canine OS.

Abstract 329: Selinexor or KPT-8602 mediated XPO1 inhibition synergizes with dexamethasone to repress convergent pathways in the mTORC1 signaling network and drive cell death in multiple myeloma

Abstract Background: Dexamethasone (DEX), a synthetic glucocorticoid (GC), is administered to nearly all multiple myeloma (MM) patients as a single agent and in combination with other chemotherapies or targeted agents. DEX and other GCs bind to glucocorticoid receptors (GR) in the cytoplasm, induce nuclear translocation and regulate GR-dependent gene expression networks in the nucleus. Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and KPT-8602) exhibit potent anti-tumor activity in MM especially when combined with DEX. SINE compounds enhance nuclear localization of tumor suppressor proteins (TSPs) through inhibition of the nuclear export protein, XPO1. We discovered that inhibition of the mechanistic Target of Rapamycin Complex 1 (mTORC1) pathway is a primary driver of the combination effect. Here we further elucidate the molecular mechanism of action of the SINE-DEX synergy in MM. Methods: GR+ MM.1S and GRnull MM.1R MM cell lines were treated with SINE compounds and/or DEX for 24 hours. Whole cell lysates were subjected to SDS-PAGE and western blot analysis. Gene expression and GR transcriptional activity was analyzed using qPCR and ELISA, respectively. Cell viability was examined using the Celltiter-Fluor assay. Results: We found that MM cell lines treated with SINE compounds (selinexor or KPT-8602) have increased basal GR protein levels. Consistent with these results, the SINE-DEX combination shows enhanced GR transcriptional activity. Several GR-DEX target genes are known to inhibit the GTPase, Ras homolog enriched in brain (RHEB), which is required for mTORC1 activation. We discovered that the SINE-DEX combination not only reduces RHEB protein but also induces the RHEB inhibitory pathways containing REDD1 and the KLF15-BCAT2 axis. Although SINE compound-mediated inhibition of mTORC1 (i.e. reduced phosphorylation of S6K1 and 4E-BP1) is GR independent, SINE-DEX inhibition is more robust in GR+ MM.1S cell line when compared to the GRnull MM.1R cells. The combination resulted in the selinexor IC50 in MM.1S cells shifting from 40 nM to 11 nM in the presence of low dose DEX. As expected, DEX did not modulate the SINE compounds IC50s in MM.1R cells. Conclusion: We show that SINE compound inhibition of MM cell viability is enhanced with DEX. Our results indicate that this combinatorial effect is due to convergent suppression of mTORC1 signaling by GR targets. The findings provide mechanism of action data around the SINE-DEX combination in MM with suggestive biomarkers (REDD1, KLF15, BCAT2 and GR) that may predict best response to the combination. Therefore, these data may translate directly to the current clinical development of SINE compounds. Citation Format: Christian Argueta, Trinayan Kashyap, Boris Klebanov, Hua Chang, Sharon Friedlander, Erkan Baloglu, Yosef Landesman, Margaret Lee, Humphrey Gardner, Sharon Shacham, William Senapedis. Selinexor or KPT-8602 mediated XPO1 inhibition synergizes with dexamethasone to repress convergent pathways in the mTORC1 signaling network and drive cell death in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 329. doi:10.1158/1538-7445.AM2017-329

Abstract B38: Clinical translation of nuclear export inhibitor in metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease in urgent need of newer therapeutic modalities. PDAC tumors are very heterogeneous and carry alterations in many critical pathways rendering the design of therapy against a single pathway unrealistic. The disease requires a broad form of therapy that can target the activation of multiple tumor suppressor proteins (TSPs) simultaneously. We have shown that over-expression of the nuclear exporter protein CRM1 in PDAC leads to functional inactivation of TSPs (such as FOXO3a, p27, Par-4 and p53) through their mislocalization to the cytoplasmic compartment. We clearly demonstrated that targeted inhibition of CRM1 by CRISPR/Cas9 validated Specific Inhibitor of Nuclear Export (SINE) KPT-330/Selinexor can restore the function of multiple TSPs leading to PDAC cell death and tumor inhibition in orthotopic models. More significantly, our findings show that SINE synergize with gemcitabine (GEM) and nab-paclitaxel leading to enhanced PDAC growth inhibition, apoptosis, and spheroid disintegration of PDAC derived cancer stem cells (CSCs) that undergo epithelial-to-mesenchymal transition (EMT). The observed synergy was due in part to enhanced nuclear localization of TSPs and suppression of CSC markers alongside reversal of EMT markers. RNA from quadruplet combination treatment samples were subjected to Agilent HT12 microarrays and differentially expressed genes (p<0.01) were subjected to pathway analysis. Our computational analysis demonstrated reversal of immune suppressive networks and suppression of CSC sustaining networks. Most importantly we observed activation of fibroblast specific cell death pathways highlighting an as of yet unexplored role of CRM1 in sustaining fibroblast growth that is recognized to support high desmoplastic reaction (DR) in PDAC. Our multi-model pre-clinical work has led to the approval of a Phase Ib/II clinical study involving GEM-nab-paclitaxel-Selinexor for metastatic PDAC (NCT02178436). Citation Format: Asfar S. Azmi, Irfana Muqbil, Amro Aboukameel, Michael Kauffman, Sharon Shacham, Ramzi M. Mohammad, Philip A. Philip.{Authors}. Clinical translation of nuclear export inhibitor in metastatic pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B38.

CRM1 inhibitor S109 suppresses cell proliferation and induces cell cycle arrest in renal cancer cells.

Abnormal localization of tumor suppressor proteins is a common feature of renal cancer. Nuclear export of these tumor suppressor proteins is mediated by chromosome region maintenance-1 (CRM1). Here, we investigated the antitumor eff ects of a novel reversible inhibitor of CRM1 on renal cancer cells. We found that S109 inhibits the CRM1-mediated nuclear export of RanBP1 and reduces protein levels of CRM1. Furthermore, the inhibitory eff ect of S109 on CRM1 is reversible. Our data demonstrated that S109 signifi cantly inhibits proliferation and colony formation of renal cancer cells. Cell cycle assay showed that S109 induced G1-phase arrest, followed by the reduction of Cyclin D1 and increased expression of p53 and p21. We also found that S109 induces nuclear accumulation of tumor suppressor proteins, Foxo1 and p27. Most importantly, mutation of CRM1 at Cys528 position abolished the eff ects of S109. Taken together, our results indicate that CRM1 is a therapeutic target in renal cancer and the novel reversible CRM1 inhibitor S109 can act as a promising candidate for renal cancer therapy.

Abstract 1696: Selective nuclear export inhibitor kpt330 enhances the antitumor activity of gemcitabine in human pancreatic cancer

Abstract Background: Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine is currently the standard chemotherapeutic agent for advanced pancreatic cancer, but has limited efficacy due to chemoresistance and dose escalation toxicity. Therefore an effective and safer therapy is warranted against this deadly malignancy. Selective nuclear export inhibitor KPT-330 inhibits the exportin 1 (CRM1) leading to nuclear localization of tumor suppressor proteins. The aim of this study was to evaluate the combination of KPT-330 and gemcitabine on anchorage-dependent and indepencent metastatic tumor cell growth, pro and antiapoptotic protein expression in vitro and in vivo orthotopic mouse model of metastatic human pancreatic cancer. Methods: Human pancreatic cancer cell MiaPaCa-2 and human metastatic pancreatic cancer cell L3.6pl were treated with different concentrations of KPT-330 (0.1-10 μM), and gemcitabine (0.1-10 μM) alone and in combination and anchorage-dependent growth recorded using MTT assay for 72 h. The effect of KPT330 and gemcitabine alone and in combination on anchorage independent growth were performed using soft agar colony formation assay. Human metastatic pancreatic cancer cells L3.6pl with luciferase were injected orthotopically into pancreas of female athymic nude mice and were treated with 1) Vehicle (PBS 1 ml/kg, ip, 2/week and PVP/PF68 1ml/kg, po, 3/week), 2) KPT330 (20 mg/kg, po, 3/week), 3) gemcitabine (100 mg/kg, ip, 2/week) and 4) KPT330 (10 mg/kg, po, 3/week + gemcitabine (50 mg/kg, ip, 2/week) for 4 weeks. The tumor volume and tumor weights were recorded after 4 weeks of treatment. The CRM1, p53, proapoptotic Bax and antiapoptotic survivin protein expressions in cells and tumor tissues were determined by Western blotting. Results: KPT-330 and gemcitabine alone significantly inhibited the anchorage-dependent growth in a concentration dependent manner. The combination of the two drugs inhibited the growth synergistically and almost completely inhibited the malignant transformation of anchorage independent cell growth. KPT-330 and gemcitabine alone decreased tumor volume and tumor weight compared to vehicle (p<0.05) but when combined together significantly decreased tumor size and weight (P<0.001). KPT-330 and gemcitabine alone and in combination induced apoptosis (Bax expression). KPT-330 but not gemcitabine depleted CRM1 expression whereas no significant change was observed in mutant p53 expression with any treatment. KPT330 and gemcitabine alone moderately depleted survivin expression compared to vehicle but when combined together, they markedly depleted survivin expression. Conclusion: KPT-330 potentiates antitumor activity of gemcitabine through inhibition of metastatic pancreatic tumor growth, depletion of the antiapoptotic protein and induction of apoptosis in human metastatic pancreatic cancer. Citation Format: Sabiha Kazim, Mokenge P. Malafa, Kazim Husain, Michael Kauffman, Shacham Sharon, Amit Mahipal. Selective nuclear export inhibitor kpt330 enhances the antitumor activity of gemcitabine in human pancreatic cancer. [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 1696. doi:10.1158/1538-7445.AM2014-1696

758O - Selinexor (Kpt-330), an Oral, Selective Inhibitor of Nuclear Export (Sine) Shows Anti-Prostate Cancer (Prca) Activity Preclinically & Disease Control in Patients (Pts) with Chemotherapy Refractory, Castrate-Resistant Prostate Cancer (Crpc)

ABSTRACT Aim: Exportin 1 (XPO1) is a nuclear export protein whose inhibition leads to the nuclear accumulation of tumor suppressor proteins (TSPs). XPO1 is overexpressed in many tumors including PrCa. Selinexor is an oral SINE that activates ≥10 TSPs with potent activity against various cancers including CRPC. Methods: Selinexor mediated nuclear localization of TSP & apoptosis induction was evaluated in CRPC cell lines & in pt derived tumors in vitro & xenografts (po 10 mg/kg QoDx3). Oral selinexor was given at 8 doses (twice weekly) 28-day cycle as part of an ongoing Phase 1 study in advanced solid tumors (KCP-330-002, NCT01607905). Response evaluation was every 2 cycles (CT & bone scans). Pts had progressing, chemotherapy refractory CRPC on study entry. Results: Selinexor induced nuclear accumulation of p53, p21, FOXO3a, & p27 in PrCa cells as well as nuclear retention of AR in androgen depleted PrCa cells. Oral selinexor showed strong (∼100%) inhibition of tumor growth in pt derived CRPC xenografts. 11 pts with CRPC (median 65 years; ECOG PS 0/1: 5/6; median prior regimens: 5.5 (range 2-10) received selinexor across 3 dose levels (35-65 mg/m2) & 8 were evaluable for response. All pts received taxanes & 5 pts either abiraterone and/or enzalutamide prior to selinexor. Grade 4 toxicities include leukopenia in 1 pt. The most common AEs (Grade 1/2/3) include: fatigue (18%/27%/18%), nausea (27%/18%/0%), anorexia (18%/27%/0%) & thrombocytopenia (9%/18%/9%). Supportive care with dexamethasone (9 pts, 8-24 mgs/weekly or equivalent), appetite stimulants & anti-emetics improved constitutional symptoms. Pharmacokinetics & pharmacodynamics showed dose-dependent increases in Cmax/AUC0-inf & in XPO1 mRNA. PSA & pain reductions were observed, 7 of 8 evaluable pts achieved prolonged SD (114-300+ days). 1 pt progressed during treatment. Conclusions: Selinexor shows potent activity in preclinical models of CRPC with activation of TSPs & retention of nuclear AR. Single agent oral selinexor induced long-term disease control in pts with chemotherapy-resistant CRPC. Future studies in pts with CRPC are planned in 2014. Disclosure: All authors have declared no conflicts of interest.

Clinical Activity of the Oral Selective Inhibitor of Nuclear Export (Sine) Selinexor (Kpt-330) in Patients with Head & Neck Squamos Cell Carcinoma (Hn-Scc)

ABSTRACT Aim: The nuclear export of most tumor suppressor proteins (TSPs) inactivates their anti-cancer functions. Exportin 1 (XPO1) is the exclusive nuclear exporter of nearly all TSPs. Multiple TSP pathways are altered in HN-SCC including p53, CDKN2A, pRB and others. Selinexor (KPT-330) is a novel, orally bioavailable, selective inhibitor of nuclear export (SINE) that blocks XPO1, leading to the nuclear accumulation and re-activation of TSPs. Selinexor has shown potent in vitro and in vivo activity in numerous models of solid tumors with squamous histology. Methods: Oral selinexor was given at 8-10 doses (2-3 times/week)/28-day cycle as part of an ongoing Phase 1 study in advanced solid tumors (KCP-330-002, NCT01607905). Response evaluation was done every 2 cycles (RECIST 1.1). Over 130 patients (pts) were enrolled, including 17 pts with heavily pretreated HN-SCC with objectively progressive disease (PD) on study entry. Results: Seventeen pts with HN-SCC (14 M, 3 F; median age 56 yrs; ECOG PS 0/1: 4/13; median prior regimens: 2.6 (range 1-4)) received selinexor across 6 dose levels (23 to 65 mg/m2). Grade 4 AEs included thrombocytopenia without bleeding in 3 pts. The most common AEs (Grade 1/2/3) include: fatigue (12%/41%/6%), nausea (41%/6%/6%), anorexia (12%/41%/0%), diarrhea (35%/12%/0%), weight loss (18%/24%/0%) and thrombocytopenia (18%/0%/12%). Supportive care with glucocorticoids, appetite stimulants and anti-emetics improved constitutional symptoms. Pharmacokinetics and pharmacodynamics showed dose-dependent increases in Cmax/AUC0-inf and in XPO1 mRNA. Tumor biopsies showed nuclear localization of TSPs (p53, FOXO3A, IkB) and apoptosis induction. Fourteen pts with PD on study entry were evaluable for efficacy. Nine patients (64%) had stable disease and 5 pts (36%) had progressive disease. 5 pts have remained on therapy for ≥4 months (4-13). Conclusions: Oral selinexor is generally well tolerated and can be administered over prolonged periods. Durable single agent disease control was observed in heavily pretreated HN-SCC pts. The recommended dose is 65 mg/m2 twice weekly and a Phase 2 study in patients with HN-SCC is expected to begin in mid 2014. Disclosure: All authors have declared no conflicts of interest.

Safety and antitumor activity of selinexor (KPT-330), a first-in-class, oral XPO1 selective inhibitor of nuclear export: A phase I study expanded with colon cancer cohort.

482 Background: Tumor suppressor proteins (TSPs) are inactivated by their export from the nucleus by Exportin1 (XPO1/CRM1). The oral selective inhibitor of nuclear export selinexor (KPT-330)restores the nuclear localization and function of TSPs and shows a broad anti-tumor activity in animal models. Here we report on the treatment of a subset of patients (pts) with metastatic colorectal cancer (CRC) in a phase I trial of selinexor. Methods: Objectives were to determine the recommended phase 2 dose, evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD), and tumor response (RECIST 1.1) of selinexor administered in two different schedules with 8 or 10 doses in a 28-day cycle. An expansion cohort of 15 pts with CRC was planned. Results: Twenty-seven pts with CRC were treated across eight doses (3-40 mg/m2) including 15 pts in the expansion cohort (30-35 mg/m2). Median age was 61 yrs and median number of prior regimens was 4. MTD of the 10-dose schedule was 30 mg/m2. MTD for the 8-dose schedule has not been reached. Thirteen pts experienced drug related gr 3-4 adverse events (AEs) including fatigue (n = 6), hyponatremia (n = 5), thrombocytopenia (n = 3), anorexia (n = 3), dehydration (n = 2), anemia (n = 2), hyperglycemia (n = 1), pulmonary embolism (n = 1), and hypotension (n = 1). The most common gr 1-2 drug related AEs were nausea (74 %), anorexia (67 %), fatigue (67 %), vomiting (59 %), dysgeusia (52 %), and weight loss (48 %). Cmax increased with dose to ≈ 1.4 µM with T½ of 4.0 – 7.4 hrs. Significant increases (2-20x) in PD marker XPO1 mRNA in circulating leukocytes were observed at all doses. Analysis of tumor biopsies confirmed nuclear localization of TSPs and induction of apoptosis following selinexor. CEA decreased in 4 of 12 pts. One pt had a partial response at 23 mg/m2, 6 patients had stable disease ≥8 weeks, and 3 patients had stable disease ≥24 weeks in 25 evaluable pts Conclusions: Preliminary signals of antitumor activity in CRC pts were observed. Selinexor is generally well tolerated and prolonged drug exposure is feasible. Selinexor induces Exportin in leucocytes and apoptosis in tumor biopsies with restoration of the nuclear location of TSPs. Clinical trial information: NCT01607905.

Phase I Trial of Selinexor (KPT-330), A First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE) In Patients (pts) With Advanced Acute Myelogenous Leukemia (AML)

Background AML cells overexpress the nuclear exporter, Exportin 1 (XPO1/CRM1) and higher XPO1 levels correlate with poor outcome. The novel selective inhibitor of nuclear transport (SINE), Selinexor, antagonizes XPO1 and shows potent cytotoxicity for AML and ALL cells in vitro , independent of genotype. Mechanistic studies show that SINE induces nuclear localization and activation of multiple tumor suppressor proteins (TSPs), leading to rapid apoptosis of AML cells. Methods Relapsed and refractory AML patients (pts) were dosed with oral Selinexor (8-10 doses / 4-week cycle) in one arm of a Phase 1 trial in advanced hematological malignancies (NCT 01607892). Detailed pharmacokinetic (PK) and pharmacodynamic (PDn) analyses were done on serial marrow biopsies. Response evaluation was performed every cycle. Results Sixteen AML pts with median age 71yrs; ECOG PS 0/1: 2/14; median number of prior regimens: 2 [range 0-7; Two patients had not received treatment specifically for AML, but had received hypomethylating agents and other therapy for myelodysplastic syndrome (MDS) prior to transformation to AML, and enrollment on the trial], received KPT-330 across 2 dose levels (16.8 to 23 mg/m2 (with additional cohorts ongoing). There has been no dose limiting toxicity. Four patients experienced drug-related grade 3/4 non-hematological Adverse Events (AEs) including hypotension (n=1), AST increased (n=1), hypokalemia (n=1), nausea (n=1), headache (n=1), fatigue (n=1)). The most common grade 1/2 toxicities were: nausea (9/17; 53%), anorexia (8/17pts; 47%), vomiting (6/17; 35%), fatigue (5/17; 29%), weight loss (5/17; 29%) and diarrhea (3/17; 18%). These adverse events were manageable with supportive care. There were no clinically significant cumulative toxicities or major organ dysfunction. PK analysis demonstrated a proportional increase in Cmax and AUC with increasing dose with no accumulation. Elimination half life was (6-7 hours) with rapid clearance of Selinexor. Of 14 pts who were evaluable for response, complete response (CR) with full hematological recovery was achieved in 2 patients (14%), and CR without hematological recovery (CRi) in 2 patients (14%). Four (29%) of the remaining patients have had stable disease for > 30 days, and 6 (43%) have had progressive disease. Conclusions Oral Selinexor treatment is generally well tolerated and has a favorable pharmacokinetic profile. Remissions have been observed at doses below the MTD, and dose escalation is ongoing with the current cohort being dosed at 40 mg/m2 twice weekly. Disclosures: Garzon: Karyopharm : Research Funding. Yee: Karyopharm : Research Funding. Baz: Karyopharm : Research Funding. Kuruvilla: Karyopharm : Research Funding. Shacham: Karyopharm : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Rashal: Karyopharm : Employment, Equity Ownership. Yau: Ozmosis Research Inc: Employment. McCauley: Karyopharm : Employment, Equity Ownership, Patents & Royalties. Saint-Martin: Karyopharm : Employment, Equity Ownership. McCartney: Karyopharm : Employment, Equity Ownership. Landesman: Karyopharm : Employment, Equity Ownership, Patents & Royalties. Klebanov: Karyopharm : Employment, Equity Ownership. Pond: Ozmosis Research: Consultancy. Oza: Ozmosis Research: Employment. Kauffman: Karyopharm : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Mirza: Karyopharm : Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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<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.

CRM1 expression in pancreatic carcinoma.

e15115 Background: Increased expression of chromosomal region maintenance (CRM1) protein, also known as exportin 1, has been described in several human cancers. It is an important regulator of subcellular localization of tumor suppressor proteins and growth regulatory proteins. Direct inhibition of CRM1 blocks cell proliferation and induces apoptosis leading to the current evaluation of CRM1 inhibitors as anticancer agents in early phase clinical trials. There is a paucity of data regarding the prevalence of CRM1 expression in pancreatic cancer. Methods: We analyzed the expression of CRM1 by immunohistochemistry (IHC) in pancreatic tissue microarray (TMA) samples (malignant and non-malignant) obtained from patients who underwent potentially curative resection for pancreatic ductal adenocarcinoma. CRM1 antibody (Santa Cruz Biotechnology) was used for immunostaining the formalin fixed paraffin embedded core sections in the TMA samples. The intensity of staining and percentage of cells stained were graded on a scale of 0 to 3, with 3 being highest. The final IHC score was obtained using the product of immunostain intensity and percentage of cells stained (Range: 0-9). Low and high CRM1 expression was considered if the IHC score was 0 to 4 and 6 to 9 respectively. Results: Seventy nonmalignant and 91 pancreatic carcinoma samples were evaluated in this study. The median IHC score was 6 (range: 0-9) and 3 (range: 1-9) in malignant and nonmalignant pancreatic tissue samples respectively (P<0.0001). High CRM1 expression was found in 11% (8/70) of normal tissue samples and 69% (63/91) of tumor samples (P<0.0001). There were 48 paired samples of pancreatic cancer tissue and normal tissue obtained from same patient. Among these patients, 33 (69%) patients had higher CRM1 expression, 7 (15%) patients had similar expression and 8 (17%) patients had lower expression in malignant tissue sample as compared to their adjacent normal tissues. Conclusions: Higher CRM1 expression occurs frequently in pancreatic cancer as compared to nonmalignant pancreatic tissue, reinforcing its putative tumor oncogenic activity, and raising the value of targeting it for pancreatic cancer therapy.

First-in-class, first-in-human phase I trial of KPT-330, a selective inhibitor of nuclear export (SINE) in patients (pts) with advanced solid tumors.

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.

Selective inhibitors of nuclear export for the treatment of non-Hodgkin's lymphomas

The nuclear export protein chromosome maintenance region 1, found to be elevated in non-Hodgkin's lymphomas, controls localization of critical tumor suppressor proteins. Nuclear localization of tumor suppressor proteins is necessary for their cell surveillance function. However, their nuclear exclusion by chromosome maintenance region 1 renders them ineffective making this nuclear transporter an attractive therapeutic target. We have identified selective inhibitors of nuclear export that lock tumor suppressor proteins in the cell nucleus leading to apoptosis of lymphoid but not normal cells. Our inhibitors induce tumor suppressor protein nuclear retention-dependent growth inhibition and apoptosis in a panel of non-Hodgkin's lymphoma cell lines. Western blot of nuclear protein fraction and confocal microscopy analysis demonstrated retention of major tumor suppressor proteins in the cell nucleus. Co-immunoprecipitation studies showed disruption of the tumor suppressor protein-chromosome maintenance region 1 interaction. Small inhibitor RNA knockdown of two major tumor suppressor proteins, p53 in wild-type protein-53 and protein 73 in mutant-protein-53, abrogated inhibitor activity. Oral administration of related inhibitor at 75 and 150 mg/kg resulted in 65 and 70% tumor reduction, respectively and subcutaneous injections of inhibitor (25 and 75 mg/kg) resulted in 70 and 74% suppression of non-Hodgkin's lymphoma tumor growth with no toxicity; residual tumors showed activation of the protein 73 pathway. Our study verifies chromosome maintenance region 1 as a therapeutic target in non-Hodgkin's lymphoma, indicating that this nuclear export protein warrants further clinical investigations.

Abstract 2943: KPT-SINE (Selective Inhibitors of Nuclear Export) induce apoptosis in colon cancer cells in-vitro and in-vivo through nuclear localization of Tumor Suppressor Proteins (TSPs)

Abstract Chromosome Region Maintenance 1/Exportin-1 (CRM1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of TSPs and negative regulators of cell proliferation. Through CRM1 inhibition, nuclear localization of these proteins restores cell cycle checkpoints and genome surveying functions culminating in apoptosis of tumor cells. Conversely, CRM1 inhibition of normal cells induces reversible cell cycle arrest. We are developing novel small molecules, KPT-SINE, which irreversibly bind and inhibit CRM1 nuclear export killing a broad range of tumor cell lines and xenografts. We used human colon cancer cells HCT116 to track the molecular and cellular events that follow KPT-SINE treatment and ultimately lead to cancer cell death. Methods: Using our proprietary in silico hierarchical structure-based discovery platform, we designed and tested novel SINE in a cell-based microscopy assay to confirm CRM1-mediated nuclear export inhibition. Cytotoxic IC50s of KPT-SINE were determined on a panel of cancer cell lines. KPT-SINE – KPT-185, -251 and -276 were selected for additional testing based on their potency and pharmacokinetics. These include; 1. Nuclear localization of TSPs, 2. Effects on cell cycle and viability, 3. Effects on TSP mRNA and protein expression. Additionally, we transiently transfected mutant CRM1-C528S into cells and treated with KPT-SINE to show binding to Cysteine 528. We tested the effects of KPT-276 in an in vivo HCT116 xenograft model. Results: KPT-276, a potent inhibitor in the cell-based microscopy assay (EC50 = 130 nM), was a robust inhibitor of HCT116 proliferation (IC50 = 400 nM). KPT-SINE inhibition of CRM1 started within 30 minutes and reached a maximum in 8 hrs. Additionally, KPT-SINE effects were blocked by transient transfection of CRM1-C528S, confirming KPT-SINE - Cysteine 528 interaction. Washout experiments demonstrated that 4 hrs of KPT-SINE incubation sustained 24 hrs of CRM1 inhibition. We also identified nuclear accumulation of p53, FOXO3a, pRb, APC, IαB, p27, PTEN, and p21, which was followed by cell cycle arrest and cell death. Although KPT-276 treatment had less effect on cell cycle and cytotoxicity assays of p53null HCT116 cells, the p53 wildtype and null HCT116 cells showed similar kill curves after 8 days of treatment. Mouse xenografts treated with 75 mg/kg QDX5 of KPT-276 each week for 4 weeks inhibited tumor growth by more than 70%. Further analysis of the tumor molecular markers from KPT-276 treated animals will be reported. Conclusions: In this study we described the correlative effects of KPT-SINE from the accumulation of nuclear TSPs through to cancer cell death in mouse models. These results suggest that KPT-SINE display potent in vivo efficacy in the xenograft model of human colon cancer. 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 2943. doi:1538-7445.AM2012-2943