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)
Authors: Senapedis William , Landesman Yossi , St-Martin Jean , Kashyap Trinayan , Plamondon Louis , Sandanayaka Vincent , Shechter Sharon , Froim Doriana , McCauley Dilara , Nir Raphael , Williams Jennifer L. , Kauffman Michael , Shacham Sharon 
Published: August 2012

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