ABSTRACT: Estrogen receptor-positive (ER+) disease represents greater than 70% of new breast cancer cases and is treated with endocrine-therapy and CDK4/6 inhibitors in the metastatic setting. While these approaches have clinical impact, veritably all patients ultimately progress on treatment. Conventionally, CDK4/6 inhibitors function by inhibiting cellular proliferation by inhibiting progression through G1/S phase of the cell cycle. However, an ever-increasing collection of data indicate that the systemic delivery of CDK4/6 inhibitors has broad effects on both the tumor (e.g. adaptive signaling and metabolism) and the associated microenvironment (e.g. immunological milieu). This complexity in mechanism of action is likely relevant for the response to therapy and acquisition of resistance and disease progression. The corollary of which, is that by fully understanding the functional drivers of resistance new interventions could be developed to enhance the durability of response or treat progressed disease. One of the challenges in studying ER+ breast cancer is the lack of robust mouse models. As shown in the preliminary data, using intraductal injection a robust mouse model for ER+ luminal breast cancer has been developed. We will use this model to interrogate the impact of CDK4/6 inhibition on the multi-focal tumors that arise in this model with an emphasis on both the canonical effects on cell cycle as well as re- programming within the tumor microenvironment. For this work we will use multi-spectral imaging and single cell sequencing approaches as we have recently published for tumor characterization and immunological features of the disease. These models are amenable to somatic genetic manipulation that also enable drivers of therapeutic resistance in vivo (Aim 1). Since many patients are being treated with CDK4/6 inhibitors, there are progressively more patients whose tumors have progressed on therapy. In recognition that understanding the real world experience is important, we initiated a clinical study to longitudinally follow patients who are being treated or whose tumors have progressed on CDK4/6 inhibitor based therapy. In metastatic breast cancer it is common to biopsy progressive disease to evaluate routine clinical biomarkers; therefore, for many patients we are collecting multiple tissue specimens. Here we will use this collection of tissues to define biological features of the tumors with a focus on proteins and spatial features of the tumor that are lost with typical molecular analyses (Aim 2). The data from these two Aims will be integrated to rigorously delineate the mechanisms of resistance to CDK4/6 inhibition in ER+ breast cancer.