Project Summary Cyclin-dependent kinase CDK4/6 inhibitor (CDK4/6i) in combination with aromatase inhibitors is the first-line treatment for ER+ advanced or metastatic breast cancer. Despite CDK4/6 inhibitors significantly improve overall survival of such patients, not all patients respond to these drugs and most patients whose tumors initially respond to CDK4/6i eventually develop acquired resistance. Although many efforts have been invested into the studying mechanism of resistance, CDK4/6i resistance remains a big challenge for HR+ breast cancer. Thus, it is urgent to develop new approaches to overcome resistance in CDK4/6i resistant breast cancer (CRBC). O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is one type of glycosylation that occurs when a monosaccharide, O-GlcNAc, is added onto serine or threonine residues of proteins by O-GlcNAc transferase (OGT). O-GlcNAcylation is involved in a range of cellular activities and aberrant O-GlcNAcylation has been implicated in a host of diseases including cancer. However, the role of O-GlcNAcylation in cancer drug resistance remains largely unknown. Through an innovative quantitative high throughput combination screen (qHTCS) and follow-up extensive preliminary studies using cellular and molecular approaches, we identified a novel OGT-mediated mechanism regulating the resistance to CDK4/6i in ER+ breast cancer. The major objective of this proposal is to determine the role of OGT-mediated pathway in the regulation of CDK4/6i resistance in CRBC cells. Specifically, we will (1) investigate the molecular mechanism of how OGT-mediated pathway regulates CDK4/6i resistance in CRBC cells, (2) evaluate the effects of newly identified drug combinational treatments on palbociclib resistance using resistant PDX and syngeneic models, (3) conduct clinical study to further evaluate the correlation between OGT-mediated pathway and CDK4/6i resistance in tumors from patients. The completion of proposed studies will not only elucidate a novel mechanism regulating CDK4/6i resistance in ER+ breast cancer, but also provide an innovative therapeutic strategy to treat CRBC patients.