Abstract 1825: Novel small molecule CRM-1 inhibitor for Non Hodgkin's Lymphoma

Abstract

Abstract Recent evidence demonstrates that Non Hodgkin's Lymphoma (NHL) tumors have elevated expression of the XPO1 gene. XPO1 codes for the nuclear exporter protein CRM-1 that controls localization of critical tumor suppressors including p53 family members. For apoptosis and cell cycle regulation, p53/p73 nuclear localization and DNA binding are highly critical making CRM-1 an attractive therapeutic target for NHL; a disease that carries >90% wild type/functional p53 and only rare mutations in p73. Earlier strategies to develop CRM-1 targeted agents such as Leptomycin B failed in the clinic due to off target toxicity. As a significant advancement to the field, we have identified novel small molecule inhibitors of CRM-1 (KPTs) that bind irreversibly and lock tumor suppressors (including p53 and p73) in cancer cell nucleus leading to apoptosis selectively in tumor cells. The KPTs show minimal toxicity to normal tissues, and possess clinically acceptable pharmacokinetic parameters. Here we demonstrate for the first time that KPTs can induce apoptosis in resistant NHL cell lines and corresponding xenograft models. The most potent CRM-1 inhibitor (KPT-185 and not its inactive analog KPT-Tran) induced growth inhibition, cell cycle arrest and apoptosis in a panel of NHL cell lines with a median IC50 ∼25 nM. The drug does not have growth inhibitory or apoptotic effects against normal peripheral lymphocytes (IC50 ∼20 µM). Fluorescent microscopy, western blot ad co-immunoprecipitation analyses demonstrated that KPT-185 treatment resulted in nuclear localization as well as dissociation of CRM-1-p53 in wt-p53 and CRM-1-p73 in mut-p53 cell lines. Additionally, we observed KPT-185 mediated activation of p21 and Bax that are known downstream executioners of p53/p73 mediated cell cycle control and apoptosis, respectively. Most significantly, knockdown of p53 in wt-p53 WSU-FSCCL and p73 in mut-p53 WSU-DLCL2 abrogated the apoptotic potential of KPT-185, confirming that these were indeed p53/p73 dependent apoptotic events. KPT-185 showed a substantial enhancement in apoptosis when combined with genotoxic p53/p73 re-activating regimen CHOP. Using WSU-DLCL2 SCID models, we further show that oral administration of related CRM-1 inhibitor KPT-276 (75 and 150 mg/Kg p.o) resulted in 65 and 70% tumor reduction, respectively and subcutaneous injections of KPT-251 (25 and 75 mg/Kg) resulted in 70 and 74% suppression of tumor growth with no observed toxicity to the host. Remnant tumor tissue analysis (histology and protein markers) fell in line with our in vitro results with clear activation of p73 pathway. Additionally, Co-immunoprecipitation studies showed dissociation of CRM-1-p73 interaction in KPT treated animal tumors. Our study verifies CRM-1 as a potential therapeutic target in NHL irrespective of the functional status of p53. These results build a strong case for the clinical use of our novel CRM-1 inhibitors either as single agent or in combination with CHOP. 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 1825. doi:1538-7445.AM2012-1825