Abstract 2628: Protein-level regulation of wild-type and mutant RIT1 by the deubiquitinase USP9X

Abstract

Abstract Somatic mutations and focal amplifications in the small GTPase RIT1 (Ras-like in all tissues) account for over 13,500 lung cancer diagnoses annually, but treatment options are limited. Of the currently identified mutations, the M90I variant (RIT1M90I) is most prevalent. A targeted therapy for RIT1-mutant and RIT1-amplified tumors would address a major unmet clinical need, but little is known about how RIT1 drives cellular transformation. Recent work suggests that the protein abundance of RIT1 is important for its function. In line with these findings, our lab performed a genome-wide CRISPR screen in RIT1-mutant lung adenocarcinoma cells and identified the deubiquitinase USP9X as a potential regulator of RIT1 abundance. We hypothesize that USP9X positively regulates wild-type and mutant RIT1, and USP9X inhibition could be a tractable means of abrogating RIT1-driven tumor growth. We have explored this hypothesis in numerous different cell line models, including NCI-H2110 cells—a human lung adenocarcinoma cell line that harbors an endogenous RIT1M90I mutation. siRNA-mediated knockdown of USP9X in NCI-H2110 cells resulted in a 56.29% reduction of RIT1 abundance (95% CI = -76.63 to -10.8) via Western Blot analysis. Furthermore, cycloheximide-chase experiments revealed that RIT1 stability was decreased in siUSP9X-treated cells compared to siCtrl-treated cells. We have also explored USP9X-mediated RIT1 regulation of endogenous wild-type RIT1 in PC9 lung adenocarcinoma cells and found that CRISPR knockout of USP9X reduced wild-type RIT1 abundance by 46.83% (95% CI = -73.84 to -32.51). In PC9 cells engineered to express RIT1M90I, siRNA-mediated knockdown of USP9X decreased RIT1M90I abundance by 58.56% (95% CI = -59.3 to -23.59). Furthermore, pharmacological inhibition of USP9X with two different small molecule inhibitors (WP1130 and G9) decreased RIT1 abundance in PC9 cells. Cells treated with 1μΜ of WP1130 showed a 56.21% (95% CI = -131.1 to 43.55) reduction in RIT1 abundance compared to DMSO-treated cells. In addition to assessing protein abundance and stability, we performed co-immunoprecipitation experiments in RIT1-mutant and RIT1-overexpressing HEK293T cells and found that both RIT1WT and RIT1M90I physically interact with USP9X. We also performed ubiquitination experiments in RIT1-overexpressing HEK293T cells and found that co-expression of wild-type USP9X decreased RIT1 ubiquitination, but a catalytically dead USP9X variant did not affect levels of RIT1-Ub. Taken together, these data support the hypothesis that USP9X controls the deubiquitination and stabilization of RIT1WT and RIT1M90I. This work provides more insight on the basic biology of RIT1 regulation and has important therapeutic implications given that USP9X inhibition could reduce RIT1 protein abundance and abrogate RIT1-driven tumor growth, thereby addressing a major unmet clinical need. Citation Format: Amanda K. Riley, Athea Vichas, Aidan Snell, Michael Grant, Lixin Wan, Alice H. Berger. Protein-level regulation of wild-type and mutant RIT1 by the deubiquitinase USP9X [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 2628.