Abstract 2972: Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven lung cancer

Abstract Standard care of lung cancer treatment has shifted away from non-specific, cytotoxic chemotherapy in favor of targeted therapies based on genetic mutations within tumors. In 2014, somatic mutations in the small GTPase RIT1 (Ras-like in all tissues) were discovered as oncogenic drivers of lung adenocarcinoma. Thousands of patients per year are diagnosed with RIT1-driven cancer, but treatment options are limited. A targeted therapy for RIT1-driven tumors would address a major unmet clinical need. Little is known about how RIT1 drives cellular transformation. To genetically dissect RIT1 function, we performed a genome-wide CRISPR/Cas9 screen in isogenic PC9 lung adenocarcinoma cells. This screen took advantage of the observation that RIT1-mutant cells are resistant to EGFR inhibition. We leveraged this drug resistance phenotype to identify genetic dependencies (gene knockouts that are detrimental to cell growth) and cooperating factors (gene knockouts that are beneficial to cell growth) in RIT1-mutant cells. From this screen, we found that one of the top essential genes was the deubiquitinase USP9X. This is intriguing given that the protein abundance of RIT1 is known to be important for its function. Therefore, we sought out to test the hypothesis that USP9X regulates RIT1 abundance and that inhibition of USP9X could be an effective therapeutic strategy for abrogating RIT1-driven tumor growth. Our model suggests that USP9X promotes proteasome-mediated degradation of RIT1. To test this, we assessed RIT1 abundance in the context of USP9X knockout (KO). We found that RIT1 protein abundance was decreased in USP9X KO PC9 cells compared to parental cells. Furthermore, cycloheximide (CHX)-chase experiments revealed that RIT1 stability was decreased in USP9X KO cells, and RIT1 degraded faster than in parental cells. The average half-life of RIT1 in USP9X KO cells was 3.4 hrs while the average half-life in parental PC9 cells was 12.3 hrs (95% CI = -12.5 to -5.4 hrs). Treatment with the proteasome inhibitor bortezomib (BTZ) rescued RIT1 degradation by 99% in parental cells and 190% in USP9X KO cells (95% CI = 57 to 126%). In addition to assessing protein abundance and stability, we performed co-immunoprecipitation experiments in RIT1-expressing HEK293T cells and found that RIT1 and USP9X physically interact. Taken together, these data support the hypothesis that RIT1 is a substrate of USP9X. In addition to providing better insight on the protein regulation of RIT1, this work has crucial therapeutic implications. The protein abundance of RIT1 is important for its function, and our model suggests that USP9X inhibition could be an effective means of reducing RIT1 protein abundance and abrogating tumor growth. Overall, this work is poised to significantly impact the field of RIT1 biology and address a major unmet clinical need for the treatment of RIT1-driven diseases. Citation Format: Amanda K. Riley, Athea Vichas, Naomi T. Nkinsi, Phoebe C. Parrish, Shriya Kamlapurkar, Alice H. Berger. Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven lung 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 2972.