Abstract 5343: Proximity labeling reveals role of 14-3-3 protein in CDK4/6 inhibitors resistance for hormone positive breast cancer

Abstract CDK4/6 inhibitors are currently standard of care for advanced hormone positive (HR+) breast cancer, and CDK6 overexpression induces resistance against CDK4/6 inhibitors. We incidentally discovered that overexpression of a CDK6 D224Y mutant mitigated this resistance. Mechanistic studies revealed that CDK6 overexpression resulted in suppressed apoptosis and increased G1/S progression after treatment with CDK4/6 inhibitors; these features were abolished when CDK6 D224Y was overexpressed. APEX2- based proximity labeling proteomics revealed CDK6 overexpression, but not CDK6 D224Y, induced proximity of the 14-3-3 protein YWHAB. Co-immunoprecipitation experiments confirmed YWHAB bound overexpressed wild type CDK6 but not CDK6 D224Y, and YWHAB knockdown decreased CDK6 protein in a proteosomal dependent manner, suggesting that YWHAB served as a chaperone role for CDK6 stablization. Knockdown of YWHAB reversed the resistance conferred by CDK6 amplification. Molecular modeling studies predicted that the aspartate-to-tyrosine mutation of CDK6 D224Y would result in destabilizing binding between CDK6 and YWHAB. Xenograft studies confirmed that CDK6 D224Y overexpression reversed the resistance conferred by CDK6 overexpression in vivo. Analysis of independent clinical cohorts revealed an association of 14-3-3 proteins with survival in HR (+) breast cancer. Both 14-3-3 beta (YWHAB) and zeta (YWHAZ) were associated with activation of G1/S pathway genes, suggesting a role for 14-3-3 proteins in CDK4/6 inhibitors treatment outcome. In a clinical cohort, high YWHAZ expression was associated with worse outcome to CDK4/6 inhibitors treatment. Transcriptomic studies revealed that high YWHAB/YWHAZ expression was associated with upregulated G1/S pathway signature, confirming a role of 14-3-3 proteins in CDK4/6-cyclinD-Rb-E2F axis. In summary, by studying CDK6 D224Y, we uncovered a role for the 14-3-3 protein YWHAB in CDK4/6 inhibitor resistance. Citation Format: Wei-Ting Chung, Yi-Ru Tseng, Tsan-Jan Chen, Wenching Wang, Muh-Hwa Yang, Chun-Yu Liu, Yi-Fang Tsai, Chi-Cheng Huang, Ling-Ming Tseng, Ta-Chung Chao, Jiun-I Lai. Proximity labeling reveals role of 14-3-3 protein in CDK4/6 inhibitors resistance for hormone positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5343.