Proximity labeling and thermal profiling proteomic analysis unveil mechanistic role of YWHAZ and YWHAB in CDK4/6 inhibitor resistance

CDK4/6 inhibitors are currently standard of care in both early and metastatic hormone positive breast cancer. With the expanded use of CDK4/6 inhibitors in early breast cancer, identifying and addressing resistance mechanisms become increasingly important for improving patient outcome. In our prior work, using proximity labeling approaches, we identified the YWHAB/YWHAZ complex (both members of the 14-3-3 family) as important mediators for CDK4/6 inhibitors resistance. Genetic silencing of YWHAB sensitized resistant hormone positive breast cancer cells towards CDK4/6 inhibitors. We further demonstrated that YWHAB-YWHAZ existed as a dimer and suppressed proteosomal function for CDK6 degradation, leading to CDK4/6 inhibitor resistance. To elucidate the interactome of YWHAZ, and to probe for proteosomal roles in CDK4/6 inhibitor resistance, we performed Thermal Proximity Co-aggregation (TPCA) proteomics on cells with/without YWHAZ silencing. TPCA analysis identified proteins involved in proteosomal processing and confirmed a role of YWHAZ in mediating proteosomal degradation. Further functional studies validate novel mechanisms for CDK4/6 inhibitors. Our findings were correlated with TCGA and other public cohorts to demonstrate a prognostic and predictive role of YWHAZ in CDK4/6 inhibitors. In summary, our findings indicate a role for proteosomal selectivity that mediates CDK4/6 inhibitor resistance and could serve as future therapeutic targets for hormone positive breast cancer.