One-carbon (1C) metabolism, mediated by the folate cofactor, supports multiple physiological processes. These include biosynthesis (purines and thymidine), amino acid homeostasis (glycine, serine, and methionine), epigenetic maintenance, and redox defense. Both within eukaryotic cells and across organs, 1C metabolic reactions are compartmentalized. Here we review the fundamentals of mammalian 1C metabolism, including the pathways active in different compartments, cell types, and biological states. Emphasis is given to recent discoveries enabled by modern genetics, analytical chemistry, and isotope tracing. An emerging theme is the biological importance of mitochondrial 1C reactions, both for producing 1C units that are exported to the cytosol and for making additional products, including glycine and NADPH. Increased clarity regarding differential folate pathway usage in cancer, stem cells, development, and adult physiology is reviewed and highlights new opportunities for selective therapeutic intervention.

Summary One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. CRISPR-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-THF, and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis with the mitochondrial pathway required in nutrient poor conditions.

The enzyme serine hydroxymethyltransferse (SHMT) converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Folate one-carbon units support purine and thymidine synthesis, and thus cell growth. Mammals have both cytosolic SHMT1 and mitochondrial SHMT2, with the mitochondrial isozyme strongly up-regulated in cancer. Here we show genetically that dual SHMT1/2 knockout blocks HCT-116 colon cancer tumor xenograft formation. Building from a pyrazolopyran scaffold that inhibits plant SHMT, we identify small-molecule dual inhibitors of human SHMT1/2 (biochemical IC ∼ 10 nM). Metabolomics and isotope tracer studies demonstrate effective cellular target engagement. A cancer cell-line screen revealed that B-cell lines are particularly sensitive to SHMT inhibition. The one-carbon donor formate generally rescues cells from SHMT inhibition, but paradoxically increases the inhibitor's cytotoxicity in diffuse large B-cell lymphoma (DLBCL). We show that this effect is rooted in defective glycine uptake in DLBCL cell lines, rendering them uniquely dependent upon SHMT enzymatic activity to meet glycine demand. Thus, defective glycine import is a targetable metabolic deficiency of DLBCL.

SummaryOne-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. CRISPR-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-THF, and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis with the mitochondrial pathway required in nutrient poor conditions. eTOC blurbUsing genetic and metabolomic approaches, Ducker et al. dissect the roles of cytosolic and mitochondrial folate metabolism in cell proliferation, revealing that most cells default to mitochondria for making 1C units, simultaneously generating glycine, NADH and NADPH. Upon loss of the mitochondrial pathway, however, cytosolic metabolism supports tumor growth.

Myc is one of the most commonly deregulated oncogenes in human cancer, yet therapies directly targeting Myc hyperactivation are not presently available in the clinic. The evolutionarily conserved function of Myc in modulating protein synthesis control is critical to the Myc oncogenic program. Indeed, enhancing the protein synthesis capacity of cancer cells directly contributes to their survival, proliferation, and genome instability. Therefore, inhibiting enhanced protein synthesis may represent a highly relevant strategy for the treatment of Myc-dependent human cancers. However, components of the translation machinery that can be exploited as therapeutic targets for Myc-driven cancers remain poorly defined. Here, we uncover a surprising and important functional link between Myc and mammalian target of rapamycin (mTOR)-dependent phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 (4EBP1), a master regulator of protein synthesis control. Using a pharmacogenetic approach, we find that mTOR-dependent phosphorylation of 4EBP1 is required for cancer cell survival in Myc-dependent tumor initiation and maintenance. We further show that a clinical mTOR active site inhibitor, which is capable of blocking mTOR-dependent 4EBP1 phosphorylation, has remarkable therapeutic efficacy in Myc-driven hematological cancers. Additionally, we demonstrate the clinical implications of these results by delineating a significant link between Myc and mTOR-dependent phosphorylation of 4EBP1 and therapeutic response in human lymphomas. Together, these findings reveal that an important mTOR substrate is found hyperactivated downstream of Myc oncogenic activity to promote tumor survival and confers synthetic lethality, thereby revealing a unique therapeutic approach to render Myc druggable in the clinic.