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

“We identified this MTHFD1L variant in the ExAC database as a rare allele in the normal population, suggesting that certain individuals harbor heterozygous mutations 8 Cell Metabolism 23, 1–14, June 14, 2016 in MTHFD1L, which may, due to loss of heterozygosity, result in tumors defective in mitochondrial folate metabol…”

“These include DHFR (dihydrofolate reductase), an enzyme strongly inhibited by current antifolates, and TYMS (thymidylate synthase), the target of the important chemotherapeutic 5-fluorouracil (Huennekens, 1994; Longley et al., 2003).”

“Generation of CRISPR Knockouts Clonal CRISPR knockout cell lines were generated using the double nicking variant of the published protocol of Ran and colleagues (Ran et al., 2013b).”

“To confirm that the labeling does not involve conversion of NADH to NADPH by nicotinamide nucleotide transhydrogenase (NNT) (Leung et al., 2015), we knocked out NNT and observed no impact on proline labeling (Figure 3F).”

“While other substrates, including glycine and choline, can donate 1C units exclusively to the mitochondrial 1C pool, these substrates have not been shown to contribute significantly to the activated cytosolic 1C pool (Fan et al., 2014; Jain et al., 2012; Labuschagne et al., 2014).”

“These findings are consistent with additional recent work demonstrating a role for mitochondrial folate-mediated NADPH production in melanoma metastasis (Piskounova et al., 2015).”

“We previously established that deuterium tracers could be used to probe both the sources and sinks of NADPH (Fan et al., 2014).”

“Expression of both serine biosynthesis and the mitochondrial 1C pathway can be driven by the transcription factor ATF4, which can be activated by mTORC1 and NRF2-KEAP1 signaling (Ben-Sahra et al., 2016; DeNicola et al., 2015).”

“Expression of both serine biosynthesis and the mitochondrial 1C pathway can be driven by the transcription factor ATF4, which can be activated by mTORC1 and NRF2-KEAP1 signaling (Ben-Sahra et al., 2016; DeNicola et al., 2015).”

“We find, however, that, consistent with most prior literature on this topic, cytosolic 1C pathway fluxes in most proliferating cells are typically running in the direction from 10-formyl-THF to methylene-THF and therefore are consuming, not producing, NADPH (Tibbetts and Appling, 2010).”

“Equally upregulated are two genes of mitochondrial 1C transformations: SHMT2 (mitochondrial serine hydroxymethyl transferase) and MTHFD2 (mitochondrial methylenetetrahydrofolate dehydrogenase) (Jain et al., 2012; Lee et al., 2014; Nilsson et al., 2014).”

“While other substrates, including glycine and choline, can donate 1C units exclusively to the mitochondrial 1C pool, these substrates have not been shown to contribute significantly to the activated cytosolic 1C pool (Fan et al., 2014; Jain et al., 2012; Labuschagne et al., 2014).”

“Folate metabolism can produce mitochondrial NADPH via the enzyme ALDH1L2 and potentially also MTHFD2 (Fan et al., 2014; Lewis et al., 2014) and knockdown of SHMT2 in certain cancer cell lines increases their vulnerability to oxidative stressors (Kim et al., 2015; Ye et al., 2014).”

“Samples were analyzed by reversed-phase ion-pairing chromatography coupled with negative-mode electrospray-ionization high-resolutionMS on a stand-alone orbitrap (ThermoFisher Exactive) (Lu et al., 2010).”

“, 2014) and knockdown of SHMT2 in certain cancer cell lines increases their vulnerability to oxidative stressors (Kim et al., 2015; Ye et al., 2014).”

“Total mitochondrial NADH turnover can be estimated based on the cellular O2 uptake rate, with 80%of O2 uptake consumed by oxidative phosphorylation and 75% of oxidative phosphorylation driven by NADH (Fan et al., 2013; Rolfe and Brown, 1997).”

“Cells were fed [2,3,3-2H]serine and monitored for subsequent labeling of thymidine triphosphate (dTTP) (Herbig et al., 2002).”

“These include DHFR (dihydrofolate reductase), an enzyme strongly inhibited by current antifolates, and TYMS (thymidylate synthase), the target of the important chemotherapeutic 5-fluorouracil (Huennekens, 1994; Longley et al., 2003).”

“Finally, SHMT2 is constitutively found in not only mitochondria but also the nucleus (Anderson and Stover, 2009).”

“…that SHMT2 plays a role in mitochondrial health independent of 1C cycling between the cytosol and mitochondria, likely related to its ability to generate localized glycine and/or methylene-THF, with the latter potentially needed to support mitochondrial thymidine production (Anderson et al., 2011).”

“Mitochondrial 10-formyl-THF is needed to make formylmethionine for mitochondrial protein synthesis (Tucker et al., 2011).”

“Equally upregulated are two genes of mitochondrial 1C transformations: SHMT2 (mitochondrial serine hydroxymethyl transferase) and MTHFD2 (mitochondrial methylenetetrahydrofolate dehydrogenase) (Jain et al., 2012; Lee et al., 2014; Nilsson et al., 2014).”

“Embryonic defects induced by deletion of MTHFD1L are partially rescued by formate (Momb et al., 2013).”

“Similar to serine, and consistent with prior reports in mouse embryonic fibroblasts and Chinese hamster ovary cells (Patel et al., 2003; Stover et al., 1997), glycine was required to support (A) Western blot of folate metabolism genes in HCT-116 cells and two MTHFD2-deletion clones.”

“Consistent with a prior report using this tracer in engineered mouse embryonic fibroblasts (Pike et al., 2010), this result validates the tracing strategy and confirms that the M+1 form arises primarily from mitochondrial 1C production rather than MTHFD1 reversibility.”

“Together, these enzymes, both of which lie in a pathway essential for embryonic development (Di Pietro et al., 2002; Momb et al., 2013), transform serine into glycine and a formyl unit attached to THF (Figure 1A).”

“Serine has been reported to be the predominant 1C donor in cultured cells (Fu et al., 2001; Labuschagne et al., 2014), and we confirmed that in HEK293T cells it accounted for essentially all (>98%) of 1C units incorporated into biomass building blocks (thymidine and purines; there was no detectable synthesis of methio…”

“Similar to serine, and consistent with prior reports in mouse embryonic fibroblasts and Chinese hamster ovary cells (Patel et al., 2003; Stover et al., 1997), glycine was required to support (A) Western blot of folate metabolism genes in HCT-116 cells and two MTHFD2-deletion clones.”

“We did not detect expression of the adultspecific MTHFD2L isozyme in HEK293T cells (Bolusani et al., 2011).”

“To prove the link between AICAR and formyl unit availability, we fed cells 1 mM formate, which is present in human serum at 20–30 mM (Hanzlik et al., 2005; Lamarre et al., 2014).”

“To confirm that the labeling does not involve conversion of NADH to NADPH by nicotinamide nucleotide transhydrogenase (NNT) (Leung et al., 2015), we knocked out NNT and observed no impact on proline labeling (Figure 3F).”

“To prove the link between AICAR and formyl unit availability, we fed cells 1 mM formate, which is present in human serum at 20–30 mM (Hanzlik et al., 2005; Lamarre et al., 2014).”

“The relatively high NADPH/ NADP+ ratio in the cytosol may favor reduction of 1C units from 10-formyl-THF into methylene-THF, whereas low NADH levels in the mitochondria favor the opposite (Pelletier and MacKenzie, 1995).”

“At an average tumor size of 50 mm3, jugular venous catheters were placed following standard procedures (Kmiotek et al., 2012).”

“Intriguingly, the rate of formate elimination predicts resistance across species to the dietary poison methanol (Sweeting et al., 2010), suggesting such a detoxification role could be evolutionarily significant.”

“These measurements, which cannot distinguish between THF and methylene-THF due to abiotic exchange (Jägerstad and Jastrebova, 2014), revealed selective 10-formyl-THF depletion in the mitochondrial folate pathway enzyme knockouts (Figure 1H).”