Project 3: Abstract In this project, we will examine the transcription factors and expression pathways that drive tumor development in tuberous sclerosis complex (TSC). We have published already that transcription is a key dependence for TSC tumors, and that MITF is a driver transcription factor for angiomyolipoma (AMLK) development. In addition, we have recently generated a new model of TSC renal AML by inducing renal differentiation in TSC2-/- human induced pleuripotent stem cells (hIPSCs) to generate renal organoids. A major cell subset of TSC2-/- renal organoids have an AML expression phenotype in contrast to control renal-differentiated hIPSCs. In separate studies, we have used single cell RNA sequencing (scRNA-Seq) to characterize the composition of AML at high resolution, and have confirmed the importance of MITF-driven transcription in AML cells. In the current proposal, we will examine the JUN-AXL pathway in TSC tumor development, which leads to a novel kinase inhibitor sensitivity. Using a Drosophila model, we will dissect in further detail how mTORC1 regulates Mitf activity, and then translate key findings to mammalian cells. Last, we will use the TSC2-/- hiPSC renal organoid model to identify the transcriptional circuitry required for AML development, using scRNA-Seq, scATAC-Seq, and scChIP-Seq for H3K27ac and MITF. We will also determine the entire panel of transcription factors required for AML cell development using Perturb-Seq applied to the TSC2-/- hiPSC renal organoid model. Expected health-related outcomes include identification of multiple potential therapeutic approaches for both TSC patients and cancers with TSC2/TSC1 mutations.