Abstract 5715: CDK7 modulation of Hippo-YAP activity by RNAPII phosphorylation regulates CDK4/6 inhibitor resistance in hormone positive breast cancer

Abstract

Abstract Breast cancer is the leading cause of cancer in women and poses a massive social economic burden worldwide. CDK4/6 inhibitors have roles as standard of care in advanced and adjuvant setting for hormone positive (HR (+)), HER2 negative breast cancer. CDK6 amplification has been proposed to confer resistance to CDK4/6 inhibitors. By a small molecule screen, we discovered a multi-kinase inhibitor PIK-75 that suppressed the Hippo-YAP pathway and significantly downregulated CDK6, resulting in reversal of CDK6 amplification mediated CDK4/6 inhibitor resistance. To further probe the mechanism, we discovered that PIK-75 suppressed phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII) which then resulted in downregulation of YAP expression and the Hippo pathway. Chromatin immunoprecipitation (ChIP) confirmed suppression of RNAPII Ser5 occupancy at YAP promoter after PIK-75 or shCDK7 treatment. Integrating results from RNA sequencing and proteomic analysis in breast cancer cells treated with PIK-75 revealed widespread transcription suppression and substantial spliceosome defect. Alternative splicing analysis by PIK-75 treated cells revealed a widespread splicing defect affecting multiple genes, including YAP, providing an alternative pathway for PIK-75-CDK7 mediated transcriptional regulation. We then investigated the phenotypic impact mediated by PIK-75 and CDK7 inhibition. We discovered that CDK7 silenced cells showed downregulation of YAP intranuclear translocation, impairment of cell migration, and cell cycle progression. ChiP-sequencing of RNAPII was also performed to investigate changes in global RNAPII occupancy and impact on promoter proximal pausing. In vivo studies confirmed that PIK-75 treatment demonstrated tumoricidal efficacy in CDK6 amplified HR (+) breast cancer cells, while the CDK4/6 inhibitor abemaciclib showed minimal cytotoxicity. In conclusion, we discovered a novel CDK7-RNAPII phosphorylation-Hippo/YAP-CDK6 regulation axis that when perturbed, could regulate CDK4/6 inhibitor resistance in HR (+) breast cancer. Molecular studies and multi-omic profiling elucidated the role of CDK7 in hippo pathway and characterizes multiple roles of PIK-75. Our study provides intriguing novel insights into the link between "transcriptional CDKs" and the hippo pathway that could potentially be further probed for therapeutics in HR (+) breast cancer. Citation Format: Chung-Jen Yu, Yong-Ji Zhuang, Chih-Yi Lin, Yi-Ru Tseng, Ting-Yi Lin, Hsiu-Man Shih, Chun-Yu Liu, Ta-Chung Chao, Ling-Ming Tseng, Chi-Cheng Huang, Yi-Fang Tsai, Feng chiao Tsai, Jiun-I Lai. CDK7 modulation of Hippo-YAP activity by RNAPII phosphorylation regulates CDK4/6 inhibitor resistance in hormone positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5715.