Abstract B028: Pharmacological inhibition of USP9X as a novel targeted therapy for RIT1-driven lung adenocarcinoma and other cancers
Authors: Riley Amanda , Vichas Athea , Berger Alice 
Published: May 2023

Since its discovery as an oncogene in 2014, mutations and amplifications in the small GTPase RIT1 (Ras-like in all tissues) have been associated with lung adenocarcinoma, myeloid malignancies, and uterine carcinosarcoma. Genome-sequencing studies in lung adenocarcinoma tumors have found that RIT1 is mutated in 2% of cases and amplified in another 14%. Several oncogenic mutations have been characterized, with the M90I variant (RIT1M90I) being most prevalent. Although targeted therapies have revolutionized the treatment of many cancers, there are no targeted therapies available for RIT1-driven diseases, and cytotoxic chemotherapy remains the only treatment option. Given its structure as a small GTPase, RIT1 itself may be difficult to drug, but identifying therapeutic vulnerabilities of RIT1-driven cancers could reveal druggable targets. Indeed, through CRISPR screening our lab recently identified the deubiquitinase USP9X as a potential regulator of RIT1 abundance and function. We hypothesize that USP9X positively regulates RIT1 protein abundance, meaning that USP9X inhibition would decrease RIT1 protein levels. It has previously been found that the protein abundance of RIT1 is important for its function, thus USP9X inhibitors could represent a novel targeted therapy for RIT1-driven diseases. There are currently two small molecule inhibitors—WP1130 and G9—that have been shown to target USP9X. We've tested these inhibitors in PC9 lung adenocarcinoma cells and found that WP1130 significantly reduced the abundance of endogenous wild-type RIT1 to 56.21% (p=0.0125, 95% CI = -71.96 to -15.61) compared to DMSO-treated cells. In a USP9X knockout (KO) PC9 cell line, treatment with WP1130 did not significantly affect RIT1 protein abundance (p=0.6001). These effects appear to be due to USP9X-targeted regulation of RIT1 given that the protein abundance of CDC20—a known target of USP9X—was reduced to 71.7% in response to WP1130 treatment (p=0.0380, 95% CI = -52.74 to -3.861), but CDC20 levels were not significantly affected in USP9X KO PC9 cells (p=0.4510). We have also tested G9 in PC9 cells and found a similar trend, but further work is ongoing to identify appropriate dosing of this inhibitor. In addition to testing the effects of WP1130 in PC9 cells, we also assessed how USP9X inhibition affects RIT1-mutant cells. For this we turned to the NCI-H2110 cell line, which is the only commercially available cell line harboring an endogenous RIT1M90I mutation. A dose response curve revealed that PC9 cells may be more sensitive to WP1130 than NCI-H2110 cells (IC50 in PC9 cells = 0.5059μM; IC50 in NCI-H2110 cells = 1.344 μM). This suggests that USP9X inhibition may be more effective at targeting wild-type RIT1, but this hypothesis must be further explored in vitro and in vivo. In summary, this work is poised to address a major unmet clinical need for the treatment of diseases characterized by RIT1 mutations and amplifications. USP9X inhibition could be a tractable means of reducing RIT1 protein abundance and abrogating the growth of RIT1-driven tumors.

Citation Format: Amanda Riley, Athea Vichas, Alice Berger. Pharmacological inhibition of USP9X as a novel targeted therapy for RIT1-driven lung adenocarcinoma and other cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B028.