Abstract Introduction: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with multisystem manifestations including seizures, mental retardation, autism, and tumors in the brain, heart, skin, and kidney. TSC is caused by germline inactivating mutations in the TSC1 or TSC2 genes, which encode hamartin (TSC1) and tuberin (TSC2). The TSC1 and TSC2 proteins function as a heterodimer to inhibit Rheb, which is the target of tuberin's GTPase activating domain. Rheb activates the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Therefore, tumors from TSC patients have hyperactivation of mTORC1. Rapamycin, an allosteric mTORC1 inhibitor, shrinks kidney angiomyolipomas in TSC patients. However, the impact of rapamycin is cytostatic and discontinuation of treatment leads to tumor regrowth. Our goal was to identify compounds that synergize with rapamycin to induce the death of tuberin-null, mTORC1 hyperactive cells. Methods: We screened the ICCB-Longwood Known Bioactives collection (6,671 compounds which include FDA-approved compounds and clinical trial level compounds) in 621-101 cells. 621-101 cells are patient-derived from a renal angiomyolipoma and have bi-allelic TSC2 inactivation. 1,250 cells per well were plated in 384-well plates and grown overnight followed by pretreatment with rapamycin (20 nM) or DMSO for two hours. Compounds (10-20 uM) were added and allowed to incubate for 48-72 h. ATP levels (CellTiter Glo, Promega) were used as an indicator of cell viability. Staurosporine (1-2 uM) was used as a positive control for cell death. Results: We identified and confirmed 5 compounds that synergize with rapamycin compared to drug treatment alone. Compound 1, a selective B-Raf inhibitor, induces a 2-fold decrease in ATP levels (n=4, p<0.01). Compound 2 is a calcium channel blocker, which also induces a 2-fold decrease in ATP levels (n=4, p<0.05). Compound 3, a G-protein coupled receptor inhibitor, induces a 2.5-fold decrease in ATP levels (n=4, p<0.05). Compound 4 is a JAK2 inhibitor that induces a 4-fold decrease in ATP levels in combination with rapamycin (n=4, p<0.05). Compound 5 is a pan-Aurora kinase inhibitor that induces a 4.5-fold decrease in ATP levels (n=4, p=0.06). Noteworthy, Compound 2 is FDA-approved and Compound 5 is in clinical trials. Additionally, we identified 14 compounds that decrease ATP levels more effectively alone than in combination with rapamycin, which we have classified as potential single agent therapies. Of these, Compound 6 a protein kinase-C inhibitor, has been confirmed to decrease cell viability in 621-101 cells and Tsc2-null mouse embryonic fibroblasts (MEFs) compared to 621-101 cells treated with rapamycin and Tsc2 wild-type MEFs, respectively. Conclusions: We identified novel therapeutic agents for mTORC1 hyperactive cells, including 5 that synergize with rapamycin and several with potential single-agent efficacy. Their biologic mechanisms of action may reveal novel cellular pathways that participate in the pathogenesis of TSC and other mTORC1 hyperactive cancers. Note:This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Doug Medvetz. A chemical screen in Tuberin-null cells identifies potential therapies for mTORC1 hyperactive cancers [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A3.