A Chemical-Genetic Platform for Interrogating Specific mTOR Functions

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Project Summary / Abstract Mechanistic Target of Rapamycin (mTOR) is a protein kinase that acts through two distinct protein complexes to control critical cellular processes including protein synthesis, autophagy, and cell motility. Hyperactivation of mTOR drives oncogenesis through increases in cell growth and survival. In recent years, new pharmacological tools have helped make sense of the intricate mTOR signaling network, revealing the necessity of certain downstream mTOR effectors in cancerous tissue and the importance of mTOR complex 2 (mTORC2) as a therapeutic target. The goal of this proposal is to develop chemical-genetic tools for studying specific aspects of mTOR signaling. In particular, we propose to develop a method by which we can specifically inhibit mTORC2 to study its role in cancer. Currently there are no pharmacological inhibitors specific to mTORC2. Genetic methods for inhibiting mTORC2 require knockout or knockdown of genes encoding proteins in mTORC2. This approach disrupts the balance between the two mTOR complexes and suffers from slow onset, leading to the activation of complex feedback networks. In Aim 1, we propose to develop a small molecule that inhibits mTOR if and only if an auxiliary protein is present. This system would allow us to express the auxiliary protein in a specific cell type or even subcellular compartment, thereby sensitizing mTOR to small molecule inhibition in only those cells or compartments. This approach has several advantages; namely, it functions through a gain-of-function mechanism where an inert auxiliary protein is introduced, leaving the endogenous system intact and unchanged until the small molecule inhibitor is introduced. Preliminary data show that we have successfully identified a small molecule that selectively inhibits mTOR in yeast or mammalian cells exogenously expressing an auxiliary protein. In Aim 2, we propose to apply this tool to specifically inhibit mTORC2 and study how it contributes to cell growth and survival in cancer. We hypothesize that phosphorylation of particular mTORC2 substrates is critical for tumor growth and survival, and these substrates can be revealed using specific mTORC2 inhibitors. Completion of this proposal will provide a new chemical-genetic tool to address a previously unmet need in the area of mTOR biology: the ability to selectively inhibit subpopulations of mTOR. This will shed light on the critical roles of mTOR dysregulation in cancer and provide novel therapeutic strategies.