Highlights d PHGDH expression accelerates mouse models of melanoma and breast cancer d Physiological serine levels can restrain tumor growth in breast cancer d Increased dietary serine levels can accelerate tumor progression d PHGDH expression only benefits tumors in tissues with low serine availability

Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples the folate and methionine metabolic cycles, methionine synthase (MTR), is required for cancer cell proliferation and tumor growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, MTR incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation.

Targeting folate metabolism can be an effective way to treat cancer. The enzyme methionine synthase catalyzes a key reaction in both folate and methionine metabolism. Early work suggested that inhibiting methionine synthase might restrain tumor growth, though the mechanism remains unclear. We find that due to its specific role in processing folates, methionine synthase is required for cancer proliferation. However, widely used cell culture conditions obscure the proliferative and metabolic consequences of methionine synthase inhibition. Complete dependence on methionine synthase only arises when 5-methyl tetrahydrofolate, the major folate found in circulation, is the predominant folate source provided to cells. In these physiological folate conditions, methionine synthase activity is necessary to maintain intracellular levels of nucleotides, but not methionine. These data reveal that the extracellular environment can alter the essentiality of methionine synthase and suggest that this enzyme plays a crucial cell-autonomous role in supporting nucleotide synthesis and cell proliferation in physiological contexts. Fellowship. A.M.D. was supported by a Jane Coffin Childs Postdoctoral Fellowship. M.G.V.H. acknowledges support from R35CA242379, R01CA201276, the Ludwig Center at MIT, the MIT Center for Precision Cancer Medicine, SU2C, the Emerald Foundation, the Lustgarten Foundation, and a Faculty Scholars Grant from HHMI. AUTHOR CONTRIBUTIONS Conceptualization, M.R.S., M.G.V.H.; Methodology, M.R.S., M.F.R., C.A.L.; Formal Analysis, M.R.S., A.M.D.; Investigation, M.R.S., A.M.D., M.F.R.; Resources, C.A.L.; Visualization, M.R.S., A.M.D.; Writing -Original Draft, M.R.S.; Writing -Review & Editing, M.R.S., A.M.D., M.G.V.H.; Funding Acquisition, M.R.S., A.M.D., M.G.V.H.