SUMMARY The mechanistic target of rapamycin complex 1 (mTORC1) supports proliferation through parallel induction of key anabolic processes, including protein, lipid, and nucleotide synthesis. We hypothesized that these processes are coupled to maintain anabolic balance in cells with mTORC1 activation, a common event in human cancers. Loss of the tuberous sclerosis complex (TSC) tumor suppressors results in activation of mTORC1 and development of the tumor syndrome TSC. We find that pharmacological inhibitors of guanylate nucleotide synthesis have selective deleterious effects on TSC-deficient cells, including in mouse tumor models. This effect stems from replication stress and DNA damage caused by mTORC1-driven ribosomal RNA synthesis, which renders nucleotide pools limiting. These findings reveal a metabolic vulnerability downstream of mTORC1 triggered by anabolic imbalance.

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Financial support: This research was supported by grants from the National Institutes of Health (P01CA120964 to J.M.A. E.P.H. and B.D.M., P30CA006516 to J.M.A., and R35CA197459 to B.D.M.) and a Rothberg Courage Award from the TS Alliance to B.D.M. Conflict of interest statement: B.D.M. is a shareholder and scientific advisory board member for Navitor Pharmaceuticals and LAM Therapeutics. Word count: 5,171 Number of figures: 6 main figures, 4 supplemental figures Structured AbstractPurpose: mTORC1 is a master regulator of anabolic cell growth and proliferation that is activated in the majority of human tumors. We recently demonstrated that elevated mTORC1 activity in cells and tumors can confer dependence on IMPDH, the rate-limiting enzyme in de novo guanylate nucleotide synthesis, to support increased ribosome biogenesis and cell viability. Pharmacological agents that inhibit IMPDH, such as mizoribine and mycophenolic acid (CellCept), are in wide clinical use as immunosuppressants. However, whether these agents can be repurposed for anti-tumor therapy requires further investigation in preclinical models, including direct comparisons to identify the best candidate(s) for advancement.Experimental Design: Distinct IMPDH inhibitors were tested on cell and mouse tumor models of tuberous sclerosis complex (TSC), a genetic tumor syndrome featuring widespread lesions with uncontrolled mTORC1 activity. Growth and viability were assessed in cells and tumors lacking the TSC2 tumor suppressor, together with drug pharmacokinetics and pharmacodynamics, target inhibition, and effects on tumor, tissue, and plasma metabolic biomarkers.Results: Mizoribine, used throughout Asia, exhibited greater selectivity in specifically targeting TSC2-deficient cells with active mTORC1 compared to the FDA-approved IMPDH inhibitors mycophenolic acid or ribavirin, or approved inhibitors of other nucleotide synthesis enzymes. In distinct tumor models, mizoribine demonstrated robust anti-tumor efficacy that is superior to mycophenolic acid, despite similar immunosuppressive effects.Conclusions: These results provide pre-clinical rationale for repurposing mizoribine as an antitumor agent in tumors with active mTORC1, such as in TSC. Our findings also suggest that IMPDH inhibitors should be revisited in cancer models where MMF has shown modest efficacy.