shRNA library screen to identify therapeutic vulnerabilities in ccRCC

Despite advances in immunotherapy for clear cell renal cell carcinoma, patients with metastatic disease continue to experience a bad outcome. This research investigates therapeutic vulnerabilities stemming from mutations in the SWI/SNF complex component PBRM1 – the second most frequently mutated gene in clear cell renal cell carcinoma. It employs a loss-of-representation shRNA screen using an shRNA library targeting 667 genes associated with epigenetic processes. This approach identified several synthetic lethal interactions that in the future may be exploited in clinical use. Two promising synthetic lethal candidate genes are CDK7 and CCNH, which together form the CDK-activating kinase (CAK) complex. Its main function is the regulation of the cell cycle and transcription through TFIIH. The identified interactions are validated through individual shRNA experiments in two cultured cell lines. Notably, CDK7 inhibitors are under clinical investigation for various cancers. The findings of this study demonstrate that CDK7 inhibitor treatment induces a significant proliferation disadvantage in PBRM1-deficient cells indicating a potential therapeutic benefit of CDK7 inhibitors in patients with clear cell renal cell carcinoma with a PBRM1 mutation after failure of conventional treatment failure. Subsequently, this study delves into the molecular connection between PBRM1 and CDK7/CCNH. Analysis reveals that CDK7 inhibition does not significantly alter phosphorylation of RNA polymerase 2, implicated in translation, between PBRM1-deficient and wildtype cells. Moreover, CDK4 and CDK6 expression remains comparable after CDK7 Inhibition between PBRM1-deficient and wildtype cells. Similarly, analysis of cell cycle phases shows no significant differences after CDK7 inhibitor treatment. DNA damage response pathway analysis leads to the hypothesis that the increased levels of 53BP1 in PBRM1-deficient cells result in higher sensibility for CDK7 inhibition. Additionally, RNA sequencing and ATAC sequencing unveil distinct gene regulation patterns between PBRM1 knockout and wildtype cells. In summary, these results suggest a broader interplay between CAK and PBRM1, potentially affecting transcription and epigenetics as well as impacting the DNA damage pathway of 53BP1. Such interactions may underlie a state of synthetic lethality, warranting their therapeutic exploration for clear cell renal cell carcinoma.