Abstract 1831: Selective inhibitors of nuclear export (SINE) induce multiple tumor suppressor proteins (TSP) activity and show single agent antitumor effect and synergy with Bcl-2 antagonist in non-small cell lung cancer
Authors: McCauley Dilara , Rashal Tami , Shacham Sharon , Kauffman Michael , Landesman Yossi , St-Martin Jean , Kashyap Trinayan , Senapedis William , Williams Jennifer L. , Nir Raphael , Illuz Maya , Peled Nir 
Published: August 2012

Abstract
Neoplastic cells must inactivate their tumor suppressor proteins (TSP) in order to perpetuate their growth. Oncogene and growth factor driven nuclear export of TSPs is an increasingly recognized mechanism for TSP inactivation. We have developed orally active, small molecule SINE that irreversibly block the major nuclear export protein CRM1 (exportin 1, XPO1) and selectively induce the death of cancer cells. Forced nuclear accumulation of TSPs is believed to initiate a “genome survey” leading to the death of cancer cells, whereas normal cells undergo transient, reversible cell cycle arrest. We show that the small molecule SINE CRM1 inhibitor KPT-185 potently kills many types of tumor cells in vitro (IC50 20-500nM, IC80 ≤ 1µM at 72 hours) including ∼75% of NSCLC lines with diverse genetic signatures (EGFR wt & mut, p53 wt & mut, K-ras wt & mut). In contrast, ∼25% of NSCLC lines with similar genotypes show cytotoxicity IC50s ≤ 1.5µM and IC80 is not reached. Treatment of the SINE-resistant A549 NSCLC (adenocarcinoma, EGFR wt, p53 wt, K-ras mut, KPT-185 IC50 = 1.75µM) leads to nuclear localization of p53, p21, FOXO, E2F4, IkB, underphosphorylated pRb and other CRM1 cargoes at concentrations similar to that of sensitive NSCLC cell lines, but apoptosis is not readily induced. To test whether the resistance to CRM1 inhibition-mediated cytotoxicity is due to activation of anti-apoptotic (or inactivation of pro-apoptotic) BCL-2 family proteins, we combined SINE with ABT-737, a small-molecule BCL-2 inhibitor currently in Phase 2 clinical trials, and examined the molecular mechanisms leading to tumor cell death. We show that combination of low doses (500nM, the IC20) of KPT-185 with non-cytotoxic doses of the Bcl-2 inhibitor (≥ 100µM) induces synergistic cytotoxicity. Moreover, the antitumor effects of the combination therapy are manifested within 48 hours, whereas significant cytotoxicity by higher doses of SINE alone requires at least 72 hours. Similar results have been obtained with another SINE resistant cell line, HCC-2935 (EGFR mut, p53 mut, K-ras mut). We noted that in the resistant A549 line, levels of the pro-apoptotic BCL2 member, Bax, were reduced by ∼50% with SINE treatment, but that addition of the BCL2 inhibitor reversed this effect and induced significant synergistic cytotoxicity. The sensitive NSCLC cell line H-226 (EGFR wt, p53wt, K-ras wt; IC50 12nM) showed nuclear localization of CRM1 cargoes when treated with KPT-185 (100nM), but only modest additional killing by the addition of the Bcl-2 inhibitor. We conclude that mechanism of resistance to SINE mediated cytotoxicity can be overcome by antagonism of Bcl-2. In vivo studies using A-549 xenografts comparing the effects of KPT-SINE and ABT-737 alone or in combination are ongoing and will be reported at the meeting.
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 1831. doi:1538-7445.AM2012-1831