Abstract Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by germline loss-of-function mutations in TSC1 or TSC2. Renal disease, which includes cysts, angiomyolipomas, and renal cell carcinoma, is a major source of morbidity and mortality for both children and adults with TSC. mTORC1 inhibitors have partial, cytostatic effects on TSC-associated tumors, so continuous and potentially lifelong therapy is required, often beginning in early childhood. The death of two children from serious infection in a recent trial of mTORC1 inhibition for TSC highlights the unmet need for novel therapies that eliminate TSC-associated tumor cells. The TSC1/TSC2 protein complex inhibits mTORC1. Multiple components of the TSC signaling network can localize to the lysosomal membrane, including mTOR, Rheb, TSC1, and TSC2. Lysosomes are highly dynamic organelles with both degradative and signaling functions. We have found that nuclear levels of the transcription factor TFEB, a master regulator of lysosomal biogenesis, are elevated in TSC1-deficient and TSC2-deficient cells, in a mouse model of TSC renal disease, and in human TSC tumors. This is unexpected, because mTORC1 phosphorylates TFEB and TFE3, leading to their cytoplasmic sequestration. We have also found that TFEB is required for the proliferation of TSC2-deficient cells, in vitro and in vivo. These and other preliminary data lead to our central hypothesis that hyperactivation of TFEB and TFE3 leads to a lysosome-dependent increase in cell proliferation in TSC. This hypothesis will be tested in three Aims: Aim 1. To identify the mechanisms of nuclear localization of TFEB and TFE3 in TSC2-deficient cells. Aim 2. To identify compounds that induce TFEB and TFE3 to move to the cytoplasm in TSC2-deficient cells. Aim 3. To determine the in vivo impact of TFEB and TFE3 on cell proliferation and renal cystogenesis in TSC. We expect this project to have scientific and preclinical impact by elucidating the roles of TFEB and lysosomal biogenesis in TSC-associated renal disease, with the potential for clinical translation to prevent and/or eliminate TSC-associated renal tumors.