PROJECT SUMMARY Targeted therapies have considerably improved survival from many cancers, including breast cancer. A major limitation of such therapies is resistance that occurs as a consequence of the extreme molecular evolution and adaptability of tumor cells. For targeted therapies to achieve their full potential of inducing sustained cures, they must be paired with additional agents that preclude activation of alternative growth and survival pathways. Identifying the most appropriate combinations requires a concerted effort to uncover mechanisms underlying activation of compensatory pathways. The PI3K/AKT/mTOR pathway is commonly activated in breast cancer. This proposal focuses on discovering new intermediaries of this signaling loop that can be targeted to improve the efficacy of mTOR inhibitors. Of the two mTOR complexes, rapalogs inhibit mTORC1 and protein translation. As single agents, rapalogs have limited efficacy in breast cancer, primarily due to feedback activation of AKT and induction of survival signals. Inhibitors of both mTORC1 and mTORC2 also cause AKT activation. Lastly, while dual inhibitors of PI3K and mTOR block this feedback pathway, they suffer from dose- limiting toxicities. These outcomes indicate that strictly targeting PI3K/AKT/mTOR is insufficient. We postulate that discerning the key elements within the negative feedback pathway that extends from mTOR to AKT will reveal novel targetable proteins for impeding rebound activation of AKT in response to mTOR inhibition. This should improve therapeutic outcomes without increasing toxicity. We used a computational method to reveal dasatinib, an inhibitor of Src Family Kinases (SFK) and Abl kinase, as a drug that may be highly synergistic with rapalogs. In breast cancer cells, dasatinib completely blocked the rebound activation of AKT that occurred with the rapalog, rapamycin. These two drugs also synergistically inhibited growth of triple negative breast cancer cells in vitro, and elicited tumor regression in multiple mouse models of breast cancer. These and other data revealed that at least one SFK resides within the pathway leading from mTOR to AKT and suggests that dual mTOR/SFK inhibition may be a novel approach to curtail resistance to mTOR inhibitors. Using phospho- proteomics and transcriptomics, we also found that focal adhesion signaling may be a major gateway for mTOR negative feedback signaling to AKT. We propose three aims to address these possibilities. In Aim 1, we will assess whether simultaneous blockade of SFKs and mTOR inhibits growth of patient derived xenografts of breast cancers as well as tumors that are resistant to current targeted therapies for HER2 and estrogen receptor. In Aim 2, we will interrogate the role of focal adhesion signaling in mediating mTOR feedback with the goal of leveraging this pathway for therapeutic benefit. Lastly, Aim 3 will uncover molecular signatures of resistance to mTOR/SFK inhibitors with the goal of revealing additional signaling inputs that control mTOR negative feedback. Together, these aims represent a comprehensive approach to discerning the molecular interplay between mTOR, elements of focal adhesions, and AKT in dictating tumor cell survival and growth.