Abstract 1815: CRM-1 as a potential therapeutic target in pancreatic cancer

Abstract CRM-1 exportin protein encoded by the XPO-1 gene maintains cancer cells through nuclear export signal (NES)-dependent protein exclusion of tumor suppressor proteins (TSPs). High CRM-1 expression has been correlated with poor prognosis in pancreatic cancer making it an attractive therapeutic target. Numerous attempts have been made in the past to develop specific CRM-1 inhibitors; however, earlier inhibitors such as Leptomycin B failed in the clinic due to acute off target toxicity. Using structure based drug design; we have identified new class of selective inhibitors of nuclear export (SINE) that irreversibly inhibits CRM1 and lock TSPs in cancer cell nucleus leading to cancer cell selective apoptosis. The inhibitors have excellent pharmacokinetic parameters and minimal toxicity to normal tissue. Here, we tested SINEs against pancreatic cancer, a disease that urgently needs novel and effective therapies. CRM-1 inhibitors [(KPT-185 (most potent analog), KPT-127, KPT-205, KPT-227 as well as Leptomycin B (as positive control) and not its inactive analog KPT-Tran (as negative control)] induced growth inhibition (MTT) and apoptosis (Annexin V and Histone DNA ELISA) in five cell lines (IC50 ∼150 nM). KPT-SINEs did not have any effect on normal HPDE cells (IC50>5 µM). Mechanistically, KPT-185 mediated apoptosis was found to be through activation, phosphorylation and nuclear localization of a TSP prostate apoptosis response 4 (PAR-4). PAR-4 has earlier been established by our laboratory as a therapeutic target in pancreatic cancer. Co-immunoprecipitation studies demonstrated that KPT-185 dissociated CRM-1-PAR-4 and CRM-1-p-PAR-4 interaction. Further, PAR-4 siRNA knockdown abrogated KPT-SINEs ability to induce growth inhibition or apoptosis. In normal cells PAR-4 localization or phosphorylation was not observed and this was associated with low basal expression of PAR-4 and its phosphorylation inducer protein kinase A protein. Role of other TSPs such as p53 and p27 was also explored, however, our investigations directly point to a PAR-4 specific mechanism. KPT-251 (structurally very similar to KPT-185 with improved pharmacokinetics properties) was well tolerated in SCID mice upto 150 mg/kg, p.o. Oral administration of analog KPT-251 at 25 and 150 mg/kg daily for two weeks reduced pancreatic tumors significantly (p<0.01). Histological examination of remnant tumors showed enhanced staining of PAR-4 and reduced Ki67. Tumor tissue lysates were probed for protein expression using western blotting that revealed activation of PAR-4 signaling and co-immunoprecipitation assay demonstrated dissociation of CRM-1-PAR-4 in tumors as well. In conclusion, discovery of our novel non-toxic KPTs can significantly impact the advancement of the field of CRM-1 inhibitors and could become effective anti-cancer therapeutics for pancreatic cancer as well as other solid tumors. 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 1815. doi:1538-7445.AM2012-1815