Summary Tuberous Sclerosis Complex (TSC) is a hamartomatous disease that manifests in multiple organ systems. In TSC, mutations in the TSC1/2 genes cause the hyperactivation of mTORC1, a central hub regulating cell signaling and metabolism. mTORC1 hyperactivation results in upregulation of DNA/RNA, protein and lipid synthesis. Macropinocytosis is a highly conserved, actin-dependent endocytic process for the uptake of extracellular material, including proteins and lipids. In proliferating cells macropinocytosis can deliver extracellular nutrients to the lysosome, where they are processed into critical macromolecule building blocks. We have found that macropinocytosis is increased six-fold in TSC2-deficient cells, and that tryptophan (an essential amino acid) stimulates this nutrient uptake mechanism. In preliminary studies, we identified a novel role of tryptophan metabolism via the kynurenine pathway in macropinocytosis. Interestingly, inhibition of the kynurenine pathway selectively inhibits the proliferation of TSC2- deficient cells. Nutrient uptake via macropinocytosis in TSC2-deficient cells is poorly understood, representing an important knowledge gap. Our central hypothesis is that mTORC1-hyperactive cells depend on macropinocytosis via the kynurenine pathway to maintain enhanced anabolic metabolism and cell proliferation, and that inhibition of tryptophan catabolism via kynurenine would lead to decreased cell proliferation and tumor formation in TSC and LAM. The mechanistic link between mTORC1, kynurenine metabolism, and the therapeutic potential of targeting this novel metabolic vulnerability will be addressed in two aims: Aim 1: Identify the mTOR-driven mechanisms that regulate macropinocytosis in TSC and LAM. Aim 2: Determine the in vivo impact of targeting the Kyn pathway in TSC. We will utilize cutting-edge metabolic and proteomic profiling to investigate the novel hypothesis that targeting the kynurenine pathway inhibits macropinocytosis and triggers selective cell death in TSC2-deficient cells. We will then perform sensitive nutrient tracing assays to investigate how the enhanced kynurenine catabolism via AHR/TDO/IDO impacts the metabolism and survival of TSC2-deficient cells. Novel approaches of image-based high throughput drug screening using FDA-approved compound libraries and cutting-edge spatial transcriptomics will be employed to study the role of kynurenine in TSC2-dependent macropinocytosis. Importantly, we will test the therapeutic potential of inhibiting tryptophan metabolism via the kynurenine pathway in established preclinical models of TSC.