PROJECT SUMMARY Drug resistance continues to be the major limiting factor in achieving cures for cancer patients. In breast cancer, cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have been approved recently to treat patients with advanced estrogen receptor-positive tumors. However, most patients exhibit resistance due to a lack of predictive biomarker. Understanding the molecular basis underlying drug resistance is required to provide a critical breakthrough in identifying a predictive biomarker and developing effective therapeutic strategies. Based on our previous work and preliminary data, we propose that a cytoskeletal protein keratin 19 (K19), which currently serves as one of the most reliable diagnostic and prognostic markers, regulates signaling events to impact resistance against CDK4/6 inhibitors. We found that K19 binds to and inhibits a multifunctional kinase GSK to stabilize a CDK4/6 partner cyclin D3 in breast cancer cells. K19-GSK3 interaction was associated with decreased accumulation of GSK3 in the nucleus that is crucial to GSK3 function. Moreover, K19 promoted proliferation and maintained the sensitivity of cells to CDK4/6 inhibitors. We hypothesize that cyclin D3 stabilized by K19-dependent inhibition of GSK3 causes tumors to become dependent on the CDK4/6 pathway for growth and sensitizes them to CDK4/6 inhibitors. At the molecular level, we surmise that K19 filaments serve as cytoplasmic scaffolds for GSK3 to prevent its entry into the nucleus where it phosphorylates cyclin D3 for degradation. To address our hypotheses, we propose to 1) determine K19 effects on the GSK3 signaling network, 2) characterize the interaction between K19 and GSK3, and 3) determine the impact of K19 on drug resistance and tumor growth. To this end, in Aim 1, we will identify upstream regulators of K19-GSK3 interaction as well as GSK3 targets whose activities affect K19- dependent phenotypes. In Aim 2, GSK3-binding deficient K19 mutant will be characterized to determine how K19 interacts with GSK3 and affects the nuclear entry of GSK3. In Aim 3, we will assess how K19 affects sensitivities of various breast cancer subtypes to CDK4/6 inhibitors and test the role of K19 and GSK3 on the sensitivity of tumors to a CDK4/6 inhibitor in vivo. Upon successful completion, the knowledge gained from the study can help design new drugs targeting K19-dependent signaling pathways, establish K19 as a predictive biomarker for response to CDK4/6 inhibitors, and develop effective therapeutic strategies to combat drug resistance.