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Supplementary Methods include Enzyme Kinetic Assays and Computational Methods.

Supplementary Data and Methods

Supplementary Figures include Figure S1-S9.

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CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer., RIT1 mutations are mutually exclusive with other lung cancer drivers and lack targeted therapies. Here the authors examine genetic dependencies of mutant RIT1 with genome-wide CRISPR screens, revealing synergy between RIT1 and YAP1, and increased sensitivity to Aurora kinase inhibitors.

In recent work, we performed CRISPR/Cas9 screening in RIT1 (Ras-like in all tissues)-mutant cancer cells. We found that RIT1-mutant cells are vulnerable to loss of mitotic regulators, and mutant RIT1 synergizes with YAP1 (yes-associated protein 1) in oncogenesis. These findings can be leveraged to identify targeted therapies for RIT1-mutant cancer.

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.

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.

Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1‒6, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. The extent to which therapy actively drives tumor evolution by promoting mutagenic processes7 or simply provides the selective pressure necessary for the outgrowth of drug-resistant clones8 remains an open question. Here, we report that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Induction of A3A facilitated the formation of double-strand DNA breaks (DSBs) in cycling drug-treated cells, and fully resistant clones that evolved from drug-tolerant intermediates exhibited an elevated burden of chromosomal aberrations such as copy number alterations and structural variations. Preventing therapy-induced A3A mutagenesis either by gene deletion or RNAi-mediated suppression delayed the emergence of drug resistance. Finally, we observed accumulation of A3A mutations in lung cancer patients who developed drug resistance after treatment with sequential targeted therapies. These data suggest that induction of A3A mutagenesis in response to targeted therapy treatment may facilitate the development of acquired resistance in non-small-cell lung cancer. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to prevent or delay acquired resistance to lung cancer targeted therapy.

We present a cohort of 41 patients with osimertinib resistance biopsies, including 2 with an acquired CCDC6–RET fusion. Although RET fusions have been identified in resistant EGFR-mutant non–small cell lung cancer (NSCLC), their role in acquired resistance to EGFR inhibitors is not well described. To assess the biological implications of RET fusions in an EGFR-mutant cancer, we expressed CCDC6–RET in PC9 (EGFR del19) and MGH134 (EGFR L858R/T790M) cells and found that CCDC6–RET was sufficient to confer resistance to EGFR tyrosine kinase inhibitors (TKI). The selective RET inhibitors BLU-667 and cabozantinib resensitized CCDC6–RET-expressing cells to EGFR inhibition. Finally, we treated 2 patients with EGFR-mutant NSCLC and RET-mediated resistance with osimertinib and BLU-667. The combination was well tolerated and led to rapid radiographic response in both patients. This study provides proof of concept that RET fusions can mediate acquired resistance to EGFR TKIs and that combined EGFR and RET inhibition with osimertinib/BLU-667 may be a well-tolerated and effective treatment strategy for such patients. Significance: The role of RET fusions in resistant EGFR-mutant cancers is unknown. We report that RET fusions mediate resistance to EGFR inhibitors and demonstrate that this bypass track can be effectively targeted with a selective RET inhibitor (BLU-667) in the clinic. This article is highlighted in the In This Issue feature, p. 1494

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Atlantic killifish ( Fundulus heteroclitus) inhabiting the Atlantic Wood Industries region of the Elizabeth River, Virginia, have passed polycyclic aromatic hydrocarbon (PAH) resistance to their offspring as evidenced by early life stage testing of developmental toxicity after exposure to specific PAHs. Our study focused on environmentally relevant PAH mixtures in the form of Elizabeth River sediment extract (ERSE). Juvenile (5 month) F1 progeny of pollution-adapted Atlantic Wood (AW) parents and of reference site (King’s Creek [KC]) parents were exposed as embryos to ERSE. Liver alterations, including nonneoplastic lesions and microvesicular vacuolation, were observed in both populations. ERSE-exposed KC fish developed significantly more alterations than unexposed KC fish. Interestingly, unexposed AW killifish developed significantly more alterations than unexposed KC individuals, suggesting that AW juveniles are not fully protected from liver disease; rapid growth of juvenile fish may also be an accelerating factor for tumorigenesis. Because recent reports show hepatic tumor formation in adult AW fish, the differing responses from the 2 populations provided a way to determine whether embryo toxicity protection extends to juveniles. Future investigations will analyze older life stages of killifish to determine differences in responses related to chronic disease.

We present a cohort of 41 patients with osimertinib resistance biopsies, including two with an acquired CCDC6-RET fusion. While RET fusions have been identified in resistant EGFR-mutant NSCLC, their role in acquired resistance to EGFR inhibitors is not well described. To assess the biological implications of RET fusions in an EGFR-mutant cancer, we expressed CCDC6-RET in PC9 (EGFR del19) and MGH134 (EGFR L858R/T790M) cells and found that CCDC6-RET was sufficient to confer resistance to EGFR-TKIs. The selective RET inhibitors BLU-667 or cabozantinib resensitized CCDC6-RET-expressing cells to EGFR inhibition. Finally, we treated two patients with EGFR-mutant NSCLC and RET-mediated resistance with osimertinib and BLU-667. The combination was well-tolerated and led to rapid radiographic response in both patients. This study provides proof-of-concept that RET fusions can mediate acquired resistance to EGFR TKIs and that combined EGFR and RET inhibition with osimertinib/BLU-667 may be a well-tolerated and effective treatment strategy for such patients.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4–ALK. Under comparable conditions, N-ethyl-N-nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4–ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies. Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714–29. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 663

Recent studies show that perception of speaker's age is influenced by a listener's familiarity with the speaker's language. However, these results were based on young adult listeners. The current study examines whether language familiarity influences the perception of speaker's age in elderly listeners. The vowel stimuli was prepared from the sustained vowel /i/ collected from 30 native speakers of American English and 30 native speaker of Japanese. The sentence stimuli was taken from the reading of the North Wind and the Sun by these 60 speakers (in their native language). Fifteen elderly native speakers of English (mean age 73.5 years, range from 67 to 84 years) listened to both stimuli types and estimated the age of speakers. Correlation between perceived age and chronological age was moderate for the sentence stimuli, but weak for the vowel stimuli. Correlation between perceived age and chronological age was stronger when the listeners judged sentence stimuli in their native language than when they judged sentence stimuli in the foreign language (r=0.74 versus r=0.63). The vowel stimuli did not show the effect of speaker language. The results suggest that linguistic information has a critical role in age perception, regardless of listener's age.