Exploiting a metabolic vulnerability in mTORC1-driven tumors Alexander J. Valvezan (1), Molly C. McNamara (2), Spencer K. Miller (2), Margaret E. Torrence (2), John M. Asara (3), Elizabeth P. Henske (4), and Brendan D. Manning (2); (1) Center for Advanced Biotechnology and Medicine, Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey, USA; (2) Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA; (3) Division of Signal Transduction, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA; (4) Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA. Mammalian Target of Rapamycin Complex 1 (mTORC1) is a master regulator of anabolic cell growth that is activated in the majority of human cancers across nearly all lineages. We find that mTORC1 activation creates dependence on nucleotide synthesis pathways, unveiling a targetable metabolic vulnerability. Tumor cells can acquire nucleotides in 2 ways: de novo synthesis, and/or uptake of nucleosides and nucleobases from the tumor microenvironment, which are then converted to nucleotides through salvage pathways. Inhibitors of inosine 5’-monophosphate dehydrogenase (IMPDH) block the rate-limiting step in de novo guanylate nucleotide synthesis, as well as guanylate salvage from hypoxanthine, the most abundant circulating purine nucleoside. Clinically approved IMPDH inhibitors selectively kill cells with uncontrolled mTORC1 activation and demonstrate robust anti-tumor efficacy at therapeutically relevant doses in mouse models of the mTORC1-driven genetic tumor syndrome Tuberous Sclerosis Complex (TSC). These effects stem from DNA replication stress and DNA damage caused by mTORC1-driven ribosomal RNA synthesis, which greatly increases cellular demand for nucleotides and thus renders nucleotide pools limiting. When compared directly to the mTORC1 inhibitor rapamycin, IMPDH inhibitor treatment provides a more durable anti-tumor response associated with tumor cell death. Comparison of all clinically approved IMPDH inhibitors in multiple cell and tumor models of TSC reveals mizoribine, an IMPDH inhibitor used as an immunosuppressant in Asia, as the best pre-clinical candidate for potential repurposing for the treatment of mTORC1-driven tumors.