Summary A majority of patients with Tuberous Sclerosis Complex (TSC) develop benign kidney tumors known as renal angiomyolipoma (AML) that can cause renal insufficiency and spontaneous life-threatening hemorrhages. The main therapy for AML is everolimus, a rapamycin analog inhibitor of the kinase mTOR with cytostatic activity that only partially reduces tumor size. AMLs become stable over time, and tumor re-growth is often occurs after treatment is interrupted due to side effects. Therefore, there is an urgent need to elucidate mechanisms of tumor resistance for the development of more efficacious therapies. Efforts to recapitulate AML experimentally have failed for the past 20+ years, precluding the study of AML biology. To address this problem, we have used genetically engineered patient-derived induced pluripotent stem cells (iPSCs) to generate AML organoids. Organoids generated from iPSCs carrying biallelic inactivating mutations in the TSC2 locus (i.e. TSC2-/-) faithfully recapitulated key anatomical and molecular features of human kidney AML (reported in Hernandez JOR et al. Nat Commun. 2021 Nov 11;12(1):6496). Some of those features included the presence of myomelanocytic AML-like cells co-expressing smooth muscle and melanocyte markers, and the transcriptional activation of signaling pathways shared with kidney AML. Transplantation of TSC2-/- AML organoids into the kidneys of immunodeficient rodents resulted in fully vascularized human AML xenografts for mechanistic studies and for drug testing testing in vivo. Using these novel tools we identified potential mechanisms of tumor resistance driven by p21CIP1 and by BCL-2 apoptosis modulators, preventing AML cell death induced by rapalogs. Our in vivo experiments also indicated that drug delivery via nanocarriers may increase the efficacy of anti-tumor therapy while reducing undesired effects in other tissues. The objective of this proposal is to elucidate anti-apoptotic mechanisms driven by p21CIP1 and BCL-2 proteins for the development of novel anti-tumor therapies combining BCL-2 protein inhibitor drugs and rapalogs, that can be co-delivered using tissue-targeting nanoparticles. Our long-term goal is to design new therapies for AML with increased efficacy and specificity. The central hypothesis is that antiapoptotic mechanisms driven by p21CIP1 and BCL-2 apoptosis inhibitors sustain AML cell survival promoting tumor resistance to rapalog therapy. Our three aims are: Aim 1: To investigate anti-apoptotic mechanisms of tumor resistance driven by p21CIP1 in renal AML; Aim 2: To elucidate the role of IGFBP2 in stabilizing p21CIP1 promoting AML cell survival; Aim 3: To study the contribution of BCL-2 proteins to AML cell survival through pharmacologic blockage of BH3 domain interaction. Collectively, these studies will provide much needed insight into the mechanisms of AML and will assess the efficacy of BCL-2 inhibitor therapy alone or in combination with rapalogs in AML-targeting nanoparticl...