Abstract 5848: Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein
Authors: Tripathi Brajendra K. , Hirsh Nicole , Qian Xiaolan , Wang Dunrui , Durkin Marian E. , de Miguel Fernando J. , Politi Katerina , Doroshow James H. , Lowy Douglas R. 
Published: June 2022

Abstract
Mutant KRAS is found in &gt;30% of lung adenocarcinomas, and tumors with mutant KRAS usually have poor outcomes with current therapies. The rationale for the new treatment approach described here for tumors with mutant KRAS arises from the critical antitumor role that we have uncovered for reactivating the DLC1 tumor suppressor protein, which is frequently downregulated in a wide range of human cancers. Although several mechanisms account for DLC1 downregulation, here we have focused on a subset of mutant KRAS lung tumor lines that express wild type DLC1 mRNA but lack detectable DLC1 protein. We have recently reported that cytoplasmic EZH2 protein directly methylates Lysine-678 of DLC1 protein leading to its ubiquitination by CUL-4A and consequently, proteasomal degradation. In this study, we have found a specific inhibitor of CRM1 protein (Chromosomal Region Maintenance 1, also known as Exportin 1 [XPO1]) substantially reduces cytoplasmic EZH2 level in several mutant KRAS lung cancer cell lines demonstrating that cytoplasmic EZH2 depends on its nuclear export by CRM1. CRM1, which is the major mammalian nuclear export regulator, is frequently overexpressed and/or mutated in human cancer, including lung adenocarcinomas, and these phenotypes are associated with poor prognosis. Endogenous CRM1 and EZH2 protein colocalized and formed a complex in several lung cancer lines and patient-derived tumor xenografts implying the cancer relevance of this interaction. In addition to blocking the cytoplasmic export of EZH2, pharmacologic inhibition of CRM1 increased the steady-state level of DLC1 protein in several mutant KRAS lung cancer lines and patient-derived xenografts. The tumor suppressor activity of the post-translationally stabilized DLC1 protein was further increased by kinase inhibitors of AKT and SRC, which reverse the direct phosphorylation and attenuation of DLC1 tumor suppressor functions by these kinases. The three-drug combination of inhibitors of CRM1 (Selinexor), AKT (MK-2206), and SRC (Saracatinib), which is well-tolerated in mice, induced potent inhibition of tumor xenograft growth and was associated with induction of markers of apoptosis and senescence. Although each of these inhibitors has multiple targets, CRISPR/Cas9 knockout of DLC1 abolished most of the tumor growth inhibitory activity of the three-drug combination, strongly suggesting that DLC1 is the predominant target for the antitumor activity of the drug combination. Interestingly, KRAS siRNA knockdown phenocopied CRM1 inhibition, as it reduced cytoplasmic EZH2, DLC1 methylation, and DLC1 degradation. Our findings suggest CRM1 inhibitors, when given in conjunction with AKT and/or SRC kinase inhibitors, may have clinical efficacy against lung tumors with mutant KRAS.
Citation Format: Brajendra K. Tripathi, Nicole Hirsh, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Fernando J. de Miguel, Katerina Politi, James H. Doroshow, Douglas R. Lowy. Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5848.