ABSTRACT Glioblastoma multiforme (GBM) is the most frequent and malignant primary tumor of the central nervous system in adults. Despite surgical resection followed by radiotherapy and chemotherapy, the median survival after diagnosis is 12 months and fewer than 5% of patients survive more than 5 years. Protein phosphatase 6 (PP6) and the mammalian target of rapamycin complex 2 (mTORC2) are enzyme complexes that are often overexpressed in GBM tumors, and their overexpression is negatively correlated with outcome. PP6 is a conserved protein phosphatase that forms active trimeric complexes consisting of catalytic (PP6c), SAPS (SAPS1-3), and ankyrin-repeat (ANR28, ANR44, ANR52) subunit. mTORC2 is a protein kinase complex consisting of the catalytic subunit mTOR as well as Rictor, Sin1, and LST8. While the roles of PP6 and mTORC2 in GBM oncogenesis are well demonstrated, no specific PP6 or mTORC2 inhibitors have been identified, and little is known about their functional interaction or other pathway components that could be surrogate targets. Here based on exciting preliminary data connecting these pathways, we propose a dual targeting strategy. In Aim 1, we will determine the molecular mechanism by which PP6 regulates mTORC2 stability and activity and use proteomic approaches to determine targetable interaction surfaces within the specific PP6 complex implicated in mTORC2 control. In Aim 2, we will use quantitative phosphoproteomics to identify mTORC2 substrates and validate them as surrogates of mTORC2 activity and potential targets for therapeutic intervention in GBM. Successful completion of the proposed research will illuminate the molecular mechanism underlying the regulation of mTORC2 by PP6, identify strategies for PP6 inhibition, and elucidate these complex signaling pathway interactions. Our studies will uncover and validate new targets in the PP6- mTORC2 pathway, linking two critical, but so far elusive, contributors to GBM tumorigenesis. They will also provide a new series of targeting opportunities for the development of highly specific therapeutic strategies for GBM. Being part of iTarget COBRE will be very beneficial for realizing these aims, by providing strong mentoring support, excellent research core infrastructure, and outstanding scientific and intellectual interactions with other iTarget projects. Furthermore, by obtaining additional data, allowing us to refine our hypotheses, and further strengthening our track record of publications, iTarget support will accelerate my competitiveness for independent extramural funding. It will enhance my scientific contribution to the critical mass of investigators at Dartmouth exploring biomolecular targeting at the interface between discovery and translational application.