Abstract
Abstract Background: Advanced non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related deaths in the United States and worldwide. This is largely due to intrinsic or acquired therapy resistance. The current study investigates the potential of a repurposed antimuscarinic overactive bladder drug to resensitize chemotherapy-resistant NSCLC to chemotherapy-induced apoptosis. Methods: The chemotherapy drug, docetaxel (DTX), resistant human NSCLC cell cultures (A549R) were established to recapitulate the clinical emergence of chemoresistance. These cultures were used to harvest total RNA for RNA-seq and qPCR. DTX-induced apoptosis was measured with crystal violet survival assay, colony formation assay, and TUNEL assay. In vivo experiments were performed by injecting A549R cells into the flanks of immunocompromised mice. Mice were treated with various agents, tumor sizes were monitored, and tumor mass was measured at the end of the experiment. Results: RNA-seq data of DTX-resistant NSCLC (A549R) cells show a 13.5-fold increase in expression of the muscarinic acetylcholine receptor 3 (CHRM3) compared to chemonaive A549 cells which was supported by qPCR analysis (16-fold increase). Oncomine clinical data from TCGA and Bild Lung suggest an increased mortality risk at 3 years is associated with increased CHRM3 expression. In vitro survival experimental results show resensitization of A549R cells to DTX-induced cell death when co-treated with the CHRM3 antagonist, darifenacin. Crystal violet cell survival assays show a 70% reduction in survival when cells are treated with darifenacin + DTX compared to DTX alone. This is supported by colony formation assay results with complete absence of colony growth in the darifenacin + DTX combination therapy group. Furthermore, cells treated with darifenacin + DTX show increased apoptosis measured by TUNEL assay compared to cells treated with DTX alone (% TUNEL positive: DTX alone = 0.9%, darifenacin + DTX = 88%). In vivo tumor measurements of injected A549R cells revealed a 91% decrease in tumor growth in the darifenacin + DTX treated mice compared to the DTX alone treated mice. Conclusion: We show that chemoresistant NSCLC has increased expression of CHRM3 which is associated with poorer clinical outcomes. Furthermore, the currently available antimuscarinic, darifenacin, which is used in the clinic to treat overactive bladder, effectively resensitizes DTX-resistant cells to DTX-induced cell death both in vitro and in vivo. This novel therapy has unique advantages over alternate approaches as it aims to save chemotherapy-eligible patients at multiple levels: 1) to resensitize chemotherapy-resistant tumors, 2) to synergize with chemotherapy for improved efficacy, and 3) to reduce the effective dose of chemotherapy, thus lowering toxic side effects. Funding provided by an INBRE Pilot Grant, and the UI start-up fund (to T.B.) Citation Format: Tyler Bland, Ethan Overfelt. Chemoresistant non-small cell lung cancer: A resensitizing strategy utilizing antimuscarinics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1744.