Project Summary/Abstract Lung cancer is the leading cause of cancer-related deaths in the US and disproportionally affects Veterans. Lung cancer subtypes that are currently refractory to targeted therapies include tumors carrying activated K-Ras or drug-resistant EGFR mutations, as well as tumors whose genetic alterations are “unknown”. Among the “unknown” category, Rictor (a unique component of mTORC2) amplification was recently identified as a driver genetic alteration in 11-13% of non-small cell lung cancer (NSCLC). In the case of drug-resistant mutant EGFR tumors, targeting mutant EGFR or MAP kinase pathway downstream of K-Ras often results in induction of multiple “bypass” receptor tyrosine kinase (RTK) signaling. As mTOR is a common signaling node downstream of these “bypass” kinases, targeting mTOR represents a promising approach in multiple settings of drug resistance. While pharmacological inhibition of mTORC1 is possible through rapamycin and the rapalogs, an mTORC2-specific inhibitor has yet to be developed. Selective inhibition of mTORC2 has the advantage of not perturbing the mTORC1-dependent negative feedback loops and mTORC1- mediated inhibition of macropinocytosis in mutant Ras tumors. However, effort to specifically target mTORC2 has been largely unsuccessful thus far because limited structural knowledge of the complex. Base on the new Cryo-EM studies and co-crystal structure of mTOR-mLST8, we discovered that while mLST8 is a component of both complexes, loss of mLST8 selectively inhibits mTORC2, but not mTORC1. We found that point mutations disrupting mLST8-mTOR binding specifically destabilize mTORC2, pointing to a viable strategy for inhibitor design. The overall goal of this proposal is (1) to gain a rigorous understanding of the contribution of mTORC2 to subtypes of lung cancer that are currently refractory to targeted therapies, and (2) to further investigate if targeting mLST8 can be used to selectively inhibit mTORC2. We will test targeting mLST8 in Rictor amplified tumor (Aim 1). We will also dissect the relative contribution of mTORC1 and mTORC2 in tumor resistance to EGFR tyrosine kinase inhibitors and assess therapeutic potential of targeting mLST8/mTORC2 in pre-existing TKI-resistant human lung cancer (Aim 2). Success of this project will have significant translational potential for the veteran populations. The proposed studies will provide data on target validation and pave the way for screening selective mTORC2 inhibitors for treatment of lung cancer subtypes that carry Rictor amplification, or drug- resistant EGFR mutations. In parallel to this study, we have completed an in silico screen of pharmacological compounds that may prevent binding of mTOR and mLST8, thereby specifically inhibiting mTORC2. Although out of the scope of this proposal, future studies will include testing of these compounds for mTORC2 specific inhibition that can be further translated for treatment of NSCLC.