Abstract B018: Novel therapeutics for targeting the aberrant nuclear export machinery in colorectal cancer

Abstract

Abstract XPO1 is a nuclear export receptor responsible for exporting many key proteins critical for cell survival from the nucleus to the cytoplasm. Recently, we identified a highly statistically significant hotspot mutation in XPO1 R749Q occurring in patients with colorectal cancer. Out of 96 patients identified to have XPO1 R749Q mutations, 27 (28%) were in colorectal cancer (CRC). 95% of XPO1 R749Q mutant CRC were tumor mutation burden-high (TMB-H; >10 mut/Mb) and 0 were mis-match repair deficient (dMMR)/micro satellite instability-high (MSI-H). Co-mutation analysis showed that 95% of XPO1 R749Q CRC were POLE mutated, which was significantly enriched over XPO1 WT tumors. To investigate whether XPO1 R749Q might play a role in CRC biology, we have generated two different isogenic colorectal cancer cell lines bearing the XPO1 R749Q mutation (HCT116 and LS174T) using CRISPR-CAS9 to better understand the role of this aberrant XPO1 mutation on nuclear export and tumorigenesis. Immunofluorescence (IF) and Stochastic Optical Reconstruction Microscopy (STORM) super-resolution experiments showed that R749Q mutant cells had significantly increased localization of XPO1 in the cytoplasm of the XPO1 R749Q mutant cells when compared to wildtype (XPO1 WT) cells, particularly at the edge of the nuclear pores of the XPO1 R749Q mutant cells suggesting increased nuclear export. Mass spectrometry analysis of nuclear and cytoplasmic fractionated proteins confirmed that XPO1 R749Q mutant cells had increased export of proteins from the nucleus compared to XPO1 WT cells. Structural modeling using published structures of XPO1 bound to Ran-GTP predicted that XPO1 R749Q mutation increased affinity of the regulatory H9-loop of XPO1 to Ran, thus favoring increased shuttling and retention in the cytoplasmic compartment. Therefore, we hypothesize that XPO1 R749Q may represent a novel mechanism of nuclear export alteration involved with kinetic tuning of global transport. Despite the increased nuclear export function, XPO1 R749Q cells did not show a proliferative advantage. We thus set out to test whether XPO1 R749Q might be enriched in cancer as an adaptive response to chemotherapy treatment. Using a chemical compound library of >200 FDA-approved cancer therapies we observed a strong therapeutic resistance of XPO1 R749Q cells relative to XPO1 WT cells, specifically to chemotherapies used in the treatment of colon cancer, such as irinotecan. However, these cells remained sensitive to treatment with the XPO1 inhibitor selinexor. Mice xenografted with XPO1 R749Q mutant HCT116 cells showed moderate tumor response to selinexor or irinotecan monotherapies but prolonged tumor responses to combination therapy. Recent clinical data has shown POLE mutations to be a biomarker for response to PD-1 immune checkpoint blockade, especially in CRC. Since our genetic data identify POLE mutations commonly co-occurring with XPO1 R749Q in colon cancer, we are planning to test the combination of PD-1 inhibitors and selinexor to target both these mutations, as well as CRC tumors without these mutations. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Monika Chojnacka, Gabriel Gaidosh, Jumana Afaghani, Maurizio Affer, Jenna Zabroski, Jacob Jahn, Vindhya Nawaratne, Ryan Notti, Ramiro Verdun, Daniel Bilbao, Josean Rodriguez, Justin Taylor. Novel therapeutics for targeting the aberrant nuclear export machinery in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B018.