Abstract
Exportin-1 (XPO1) is known to be overexpressed or mutated in several cancer types. XPO1 is the target of selinexor, which has received approval in hematologic cancers but not in solid tumors. We recently performed a large-scale genomic analysis of 42,793 patients with cancer spanning 322 cancer types (Taylor et. al. Cancer Discovery 2019). We identified a highly statistically significant hotspot mutation in XPO1 R749Q occurring mainly in colorectal cancer patients. We have generated 2 different isogenic colorectal cancer cell lines bearing the XPO1 R749Q mutation (HCT116 and LS174T) using CRISPR-CAS9. We observed that in vitro and in vivo, these cell lines did not reveal a role for XPO1 R749Q on cell growth and proliferation. A chemical compound library screen of >200 FDA-approved cancer therapies revealed a strong therapeutic resistance of XPO1 R749Q cells relative to wildtype (XPO1 WT) cells, specifically to chemotherapies used in the treatment of colon cancer. However, XPO1 R749Q remained sensitive to inhibition with the selective XPO1 inhibitor selinexor.
Mass spectrometry analysis of nuclear and cytoplasmic fractionated proteins revealed XPO1 R749Q mutant cells had increased export of proteins from the nucleus compared to XPO1 WT cells. Upon ionizing radiation or chemotherapy, XPO1 R749Q cells had less DNA damage as measured by comet assay and γ-H2Ax immunofluorescence. XPO1 R749Q cells are resistant to chemotherapy but respond to selinexor in vitro. Selinexor effectively blocked nuclear export of canonical cargoes of XPO1 in XPO1 R749Q and XPO1 WT cells. The combination of selinexor and irinotecan showed high levels of synergy in vitro. In vivo observations in mice xenografted with XPO1 R749Q mutant HCT116 cells showed moderate tumor response to selinexor or irinotecan monotherapies but prolonged tumor responses to combination therapy. Single-cell intracellular signaling protein analysis using the Isoplexis platform discovered increased mTOR signaling in the XPO1 R749Q vs. WT cells. Western blot validation confirmed increased mTOR signaling in XPO1 R749Q mutants.
In conclusion, our current study sheds novel insights into therapeutic targeting of cancers where XPO1 mutations occur, such as colorectal cancer. Specifically, these mutations confer resistance to DNA-damaging therapies such as chemotherapy and radiation; however, they may predict for sensitivity to targeted agents such as XPO1 inhibition. We have generated unique genetic models for study of XPO1 that are currently lacking. Our study established the biological and mechanistic consequences of XPO1 R749Q mutations in cancer, thereby having a significant positive impact on the treatments of patients with cancer. Further work is ongoing for the identification of cargo proteins transported by mutated XPO1 that connect the effects of XPO1 R749Q mutation on nuclear export to response to DNA damage, mTOR signaling and XPO1 inhibition in tumors in vitro and in vivo.
Citation Format: Tulasigeri M. Totiger, Monika Chojnacka, Jumana Afaghani, Sana Chaudhry, Skye Montoya, Maurizio Affer, Justin Taylor. The biological role and mechanisms of XPO1 R749Q mutation in colon cancer [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 853.