ABSTRACT The major growth factor signaling pathways in normal cells (e.g., PI3K and RAS) are also the ones that are most frequently genetically activated in cancer cells, leading to cell autonomous growth and proliferation. mTOR complex 1 (mTORC1) is a shared downstream effector of these pathways and a central driver of cell growth and is aberrantly activated in the majority of human cancers. This activation occurs through a network of upstream oncogenes and tumor suppressors that converge on a small G protein switch directly upstream of mTORC1. This switch involves the tuberous sclerosis complex (TSC) tumor suppressors, which form a protein complex (the TSC complex) that regulates a member of the Ras family of GTPases, called Rheb, an essential direct activator of mTORC1. Our previous studies have found that the TSC complex and Rheb serve as the key molecular link between the PI3K pathway and mTORC1 signaling and that this regulation promotes changes in key metabolic pathways underlying cell growth in both normal and cancer cells. Supported by the last 6 years of funding from this R35, we have greatly advanced and expanded this area of research, opening up several previously unforeseen new avenues of investigation through both published and ongoing work. Taking advantage of the long-term, stable funding afforded by this mechanism, we have also developed innovative new genetic mouse models and methodologies that set us up for new breakthrough discoveries over the next cycle of this grant, especially related to PI3K-mTOR signaling within the poorly understood nutrient and metabolic niche of the tumor microenvironment, which is a major focus of this renewal. Four major areas of research will include defining A) the biochemical and pathophysiological mechanisms underlying the regulation and function of the TSC complex, B) the capacity of mTORC1 to properly integrate oncogenic and nutrient signals within the tumor microenvironment, C) the metabolic consequences of PI3K- mTOR activation and inhibition in tumors of different origins, stages, and niches, and D) targetable metabolic vulnerabilities accompanying its aberrant regulation in tumors. While key mechanistic questions regarding this ubiquitous signaling network will continue to be answered through rigorous biochemical and cell biological studies, much of our efforts will combine novel genetic models with state-of-the-art analytical tools to define the salient in vivo features of the PI3K-mTOR network as they apply to tumor metabolism, growth, and progression. The overarching goals of our research are to define the precise roles of this signaling network in cancer and how best to therapeutically target the high percentage of tumors with uncontrolled mTORC1 signaling, beyond the single-agent use of mTOR inhibitors. I am confident that, if given the resources, we will continue to gain a deeper understanding of cancer cell biology, the tumor microenvironment, and therapeutic vulnerabilities, while a...