Project Summary/Abstract The manifestation of a cancer resistant state during tumor evolution in response to therapy is not clearly explained by any one phenomenon but is partially attributed to intrinsic and adaptive phenotypic rewiring. Given the dynamic phenotypic switching of resistant cells in response to therapy, detailed examination of the sources of resistance is needed to identify vulnerabilities in the components of these resistance mechanisms. We concentrate on estrogen receptor-positive (ER+) breast cancer, which has one of the highest death rates worldwide. Roughly 30% of patients diagnosed with early-stage ER+ breast cancer develop resistance to initial endocrine and cell cycle inhibition therapy. We identified increased levels of the serine/threonine kinases AKT1 and AKT3 in resistant tumors relative to sensitive in patients with early-stage ER+ breast cancer treated with endocrine and CDK4/6 inhibitor therapy. We further observed increased AKT signaling pathways from BIOCARTA and REACTOME gene sets in resistant tumors during treatment. Thus, we hypothesize that upregulation of AKT is a cell survival component of resistance to endocrine and CDK4/6 therapy in early-stage ER+ breast cancer. At physiological conditions, the phosphatidylinositol-3-kinase (PI3K)/AKT pathway is essential for cell growth and plays a key role in regulating survival during cellular stress, which indicates its importance in cancer cell survival and transition to a resistant state during therapy. We will measure components of the AKT pathway that may modulate AKT expression, identify ligand-receptor relationships originating from cell crosstalk that leads to AKT activation, and the role AKT plays during the phenotypic transition to a resistance state. Our analyses will elucidate the benefits of adding AKT inhibitor treatment to endocrine and cell cycle inhibition on extending tumor response to therapy. Using our biorepository of serial and patient tumor samples and spheroid model system, we will test this hypothesis with the following aims. SA1: Using scRNAseq data from patient cancer cells, we will identify the signaling components driving AKT activity, and its downstream consequences, in resistant cancer tumors. We will identify intracellular or extracellular factors leading to AKT upregulation and its importance in the emergence of a resistant state during endocrine and cell cycle inhibition. SA2: Using spheroid assays with isogenic cell lines sensitive or resistant to ribociclib, or patient tumor cells either sensitive or resistant to cell cycle therapy, we will measure the ability of add-on AKT or PI3K inhibitor therapy to prolong response to cell cycle and endocrine therapy. We will test the degree to which AKT inhibition will prolong cancer cell response to endocrine and cell cycle inhibitor treatments and prevent resistant state transition. These studies will elucidate components important for emerging resistant states, reactivation of the cell cycle,...