Abstract 526: Identification of CDK6 binding proteins by proximity labeling reveals novel mechanisms for CDK4/6 inhibitor resistance in breast cancer

Abstract Breast cancer is one of the most prevalent cancer type in human, and the majority (~70%) is the hormone positive (HR+) subtype. The current frontline treatment of choice in metastatic HR(+) breast cancer is CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy. Current guidelines recommend CDK4/6i treatment as 1st or 2nd line, as CDK4/6i provides survival benefit and good quality of life. However, resistance eventually emerges, leading to discontinuation of CDK4/6i and switching to subsequent treatment. Investigation of mechanisms to CDK4/6i resistance is an active area of intense research. CDK6 overexpression has been reported as an important mechanism for CDK4/6i resistance by several independent groups. We incidentally discovered a point mutation in the CDK6 protein that abolishes the CDK4/6i resistance conferred by CDK6 overexpression. When treated with CDK4/6i, overexpression of wild type CDK6 did not suppress G1/S, while both non-transfected and overexpression of mutant CDK6 both had similar suppression of G1/S. We hypothesized that a binding partner to CDK6 demonstrated differential binding activity to the mutant which abolished the resistance by CDK6 overexpression. To assess this hypothesis, we performed proximity labeling (PL) by APEX2. Both wild type and mutant CDK6 were cloned into APEX-Flag/V5 vectors with either cytoplasm (NES) or nuclear (NLS) signals. Mass spectrometry following treatment by H2O2 induced biotinylation produced candidate peptides that bound with overexpressed CDK6. We identified leading candidates of differentially binding partners that bound to mutant CDK6. Further experimental validation was performed to confirm physical proximity between CDK6 and the candidate protein. Our research uncovered novel insights to how CDK6 conferred resistance towards CDK4/6i and expands knowledge into further therapeutic development. Citation Format: Chung Wei Ting, Yong-Ji Zhuang, Chih-Yi Lin, Chung-Jen Yu, Ta-Chung Chao, Ling-Ming Tseng, Chun-Yu Liu, Yi-Fang Tsai, Chi-Cheng Huang, Jiun-I Lai. Identification of CDK6 binding proteins by proximity labeling reveals novel mechanisms for CDK4/6 inhibitor resistance in 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 526.