Abstract 4646: Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer
Authors: Elloul Sivan , Hua Chang Hua , Klebanov Boris , Kashyap Trinayan , Werman Maxwell , Lee Margaret , Landesman Yosef , Shacham Sharon , Kauffman Michael , Friedlander Sharon Y. 
Published: July 2016

Abstract
Introduction: Selinexor (sel) is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports include major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. We have previously shown that HER2+ breast cancer (BC) cells are highly sensitive to sel (SKBR3 and MCF7 IC50 0.21 and 0.073 μM, respectively). Here we tested the hypothesis that inhibition of HER2 signaling with trastuzumab (tras) synergizes with sel as both mediators (PI3K-AKT and Ras-Raf-MEK pathways) and effectors (FOXO3A, MDM2) of the HER2 pathway are among direct cargos of XPO1.
Methods: The effects of sel and tras alone or in combination on cell viability were tested on MCF7 and BT474 HER2+ BC cells using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, BT474 cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of sel and tras alone or in combination and with the therapeutic dose of sel. Tumor Growth (TG) was monitored for 60 days. Xenografts were harvested and analyzed by immunohistochemestry (IHC).
Results: Sel-tras combination was highly effective in-vitro and in-vivo. In MTT assays, both sel and tras demonstrated low IC50 values (nanomolar) and when combined, they synergistically inhibit proliferation. In-vivo, the combination resulted in significant survival benefit and enhanced TG inhibition of 88% compared to the monotherapy groups 57% (sel) and 25% (tras). Interestingly, an increase in pro-survival cytoplasmic FOXO3A protein was observed in the tras-treated group however, in the combination group, FOXO3A, which is an XPO1cargo, was restricted to the nucleus where it can trigger apoptosis. In addition, a synergistic increase in P27 protein, the G1 phase arrest regulator, was observed in the sel-tras-treated group. While in the monotherapy groups p-ERK was not modulated, in the combination group an increase in cytoplasmic p-ERK was observed, suggesting an affect on its downstream regulators of apoptosis. Indeed, apoptosis was evident in the combination samples by increased IHC staining to Cleaved caspase3 and Apoptag. In addition, an increase in PARP protein was observed in the tras-treated group however, in the combination group, PARP was dramatically reduced. We have previously shown that selinxor modulates DNA Damage Response (DDR) proteins suggesting that one mechanism by which the combination enhances cancer cell death is by accumulation of DNA damage that cannot be resolved.
Conclusion: Sel-tras combination is a novel candidate therapy to HER2+ BC modulating DDR and apoptosis. It synergizes to induce survival benefit and TG inhibition. These data provide rational support for study of sel-tras combination in clinical trials.
Citation Format: Sivan Elloul, Hua Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4646.