Project Summary/Abstract ROS1 fusions have recently been found to be oncogenic drivers of 1-2% of all non-small cell lung cancers (NSCLC). While targeted ROS1 kinase inhibitors like crizotinib have shown some initial success in the clinic, most patients have incomplete responses, and some do not respond at all (i.e., are de novo resistant to therapy). Thus far, no examples of de novo resistance have been published, no mechanisms for this upfront resistance have been discovered, and the only published mechanism for acquired resistance is an on-target mutation in the ROS1 kinase domain that prevents drug binding. Elucidating what downstream pathways drive ROS1 fusion-mediated tumors and contribute to resistance to targeted therapies is critical for development of rational polytherapies to improve patient response and survival. Preliminary studies from our lab, based on data from two ROS1 fusion-positive patients exhibiting de novo resistance to crizotinib and a crizotinib- resistant patient-derived ROS1 fusion-positive NSCLC cell line, suggest that genetic activation of the PI3K/mTOR pathway may not only cooperate with ROS1 fusions in driving oncogenesis, but also contribute to upfront resistance to targeted kinase inhibition. In these ROS1 fusion-positive cases, we have discovered two novel mutations in the PI3K/mTOR pathway that we suspect cause hyperactivation of mTOR signaling. In this study, I propose to functionally and biochemically characterize these PI3K/mTOR mutations and determine how activation of this pathway contributes to ROS1 fusion-mediated oncogenesis (Aim 1). Additionally, I will determine whether mTOR pathway activation is necessary and sufficient to drive resistance to ROS1-targeted therapy in ROS1 fusion-driven NSCLC, and whether combinatorial inhibition of ROS1 and mTOR enhances tumor cell shrinkage and death in vivo (Aim 2).