靶向exportin 1单独及与5-氟尿嘧啶联合使用可减少皮肤鳞状细胞癌的生长

Background: Cutaneous squamous cell carcinoma (cSCC) affects approximately 1.8 million people annually in the US with an increasing number of cases. Nuclear-cytoplasmic transport of proteins and RNA is vital for cellular homeostasis and becomes dysregulated in skin cancer. Exportins like Exportin 1 (XPO1) transport cargo from the nucleus to the cytoplasm and are upregulated in multiple cancer types. We found increased expression of XPO1 in premalignant actinic keratosis and cSCC. The Exportin 1 inhibitor Selinexor is FDA approved for the treatment of relapsed multiple myeloma and refractory large diffuse B cell lymphoma. This project aimed to determine whether XPO1 targeting could be a useful strategy for the treatment of cSCC, as well as to identify additional targets for combination therapy with XPO1 inhibition. Thymidylate synthase (TYMS) inhibitor 5-Fluorouracil (5-FU), used clinically for the treatment of actinic keratosis and superficial basal cell carcinoma was identified as a potential agent in combination with Selinexor. Methods: XPO1-targeted or control shRNA was introduced into human cSCC cell line SCC-13 and validated. cSCC cells or subcutaneous tumors were treated with Selinexor or the vehicle. IC 50 s were established for Selinexor in HaCaT, KerCt, SCC-13, SCC-12B.2, COLO-16, and SRB1 cells, and for other inhibitors in SCC-13 cells. Flow cytometry and Caspase-Glo assays were used for cell cycle and apoptosis analyses. Genome-wide CRISPRi screen was performed in SCC-13 dCas9-KRAB cells and analyzed by MAGeCK followed by GSEA. In vitro synergy between 5-FU and Selinexor was evaluated using SynergyFinder 3.0. In addition, we assessed the effects of XPO1 inhibition and of 5-FU-Selinexor combination treatment on TYMS and key DNA damage repair proteins (MLH1, MSH2, CHEK1) using immunoblotting. Results: XPO1 knockdown or inhibition using Selinexor caused cell cycle arrest and apoptosis of SCC-13 cells. Selinexor was effective at killing cSCC cells with submicromolar IC 50 s and significantly reduced tumor growth in SCC-13 xenografts. The genome-wide CRISPRi screen identified 293 genes that increased cSCC cell sensitivity and 70 genes that increased resistance to Selinexor. The sensitizers were enriched in DNA damage repair pathways. XPO1 inhibition decreased TYMS and key DNA damage repair proteins (MLH1, MSH2, and CHEK1). 5-FU and Selinexor together decreased the expression of these DNA damage repair proteins compared to Selinexor alone and caused synergistic cell death of SCC-13 cells. Conclusions: Inhibition of XPO1 was effective at killing cSCC cells in culture and in xenografts. Our genome-wide CRISPRi screen, identified 293 potential sensitizers of Selinexor, one of which was TYMS, the target of 5-FU. 5-FU-Selinexor combination were more effective than either agent alone, suggesting this combination may have potential for the treatment of nonmelanoma skin cancer.