Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway

Abstract

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.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism

“…Serine is the major source of formate in cells, contributing to around 50% of total formate production in mice13,17. The origin of the remaining 50% is unclear, however a significant fraction could come from endogenous formaldehyde.…”

“…1a. Although the 1C cycle is essential for cell growth and viability, supplementation with glycine and the nucleotide precursors hypoxanthine and thymidine (HT) can bypass this dependence13–16. Knockout cell lines were verified by sequencing, and protein expression when possible (Extended Data Fig.…”

Serine one-carbon catabolism with formate overflow

“…The residual activity of the remaining MTHFD1L protein or the cytosolic enzyme (MTHFD1) appears to be sufficient to satisfy the one-carbon unit demand for purine synthesis and thus to sustain growth rates similar to nonsilenced cells. On the basis of recent data from another group, the cytosolic pathway can compensate for the requirement of one-carbon units in cells, where a mitochondrial pathway enzyme (including SHMT2, MTHFD2, and MTHFD1L) has been knocked out ( 18 ). Together, these results indicate that the mitochondrial serine catabolism to glycine and formate is dispensable for purine synthesis and growth.…”

An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate

“…In HEK293T cells, cytosolic methylene-THF is produced from 10-formyl-THF (36). In cells fed with [ 13 C, 2 H]-formate, methylene-THF and its downstream products methyl-THF and dTTP were expected to be M+2 labeled.…”