Methionine synthase supports tumour tetrahydrofolate pools

ABSTRACT Mammalian cells require activated folates to generate nucleotides for growth and division. The most abundant circulating folate species is 5-methyl tetrahydrofolate (5-methyl-THF),

which is used to synthesize methionine from homocysteine via the cobalamin-dependent enzyme methionine synthase (MTR). Cobalamin deficiency traps folates as 5-methyl-THF. Here, we show using

isotope tracing that MTR is only a minor source of methionine in cell culture, tissues or xenografted tumours. Instead, MTR is required for cells to avoid folate trapping and assimilate

5-methyl-THF into other folate species. Under conditions of physiological extracellular folates, genetic MTR knockout in tumour cells leads to folate trapping, purine synthesis stalling,

nucleotide depletion and impaired growth in cell culture and as xenografts. These defects are rescued by free folate but not one-carbon unit supplementation. Thus, MTR plays a crucial role

in liberating THF for use in one-carbon metabolism. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

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Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS METHIONINE SYNTHASE IS ESSENTIAL FOR CANCER CELL PROLIFERATION IN PHYSIOLOGICAL FOLATE ENVIRONMENTS Article 18

November 2021 FOLATE METABOLISM: A RE-EMERGING THERAPEUTIC TARGET IN HAEMATOLOGICAL CANCERS Article Open access 11 March 2021 FORMATE OVERFLOW DRIVES TOXIC FOLATE TRAPPING IN MTHFD1

INHIBITED CANCER CELLS Article Open access 03 April 2023 DATA AVAILABILITY Source data are provided with this paper. All other data supporting the findings of this study are available from

the corresponding author on request. CODE AVAILABILITY The ‘Accucor’ package for natural isotope correction is publicly available through GitHub (https://github.com/lparsons/accucor).

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  Google Scholar  Download references ACKNOWLEDGEMENTS We thank W. Lu and other members of the Rabinowitz laboratory for helpful comments and suggestions. LentiCRISPR v.2 was a gift from F.

Zhang (Addgene plasmid no. 52961). This work was supported by National Institutes of Health grant nos. 1DP1DK113643 and R01 CA163591 to J.D.R. AUTHOR INFORMATION Author notes * Jonathan M.

Ghergurovich Present address: The Children’s Hospital of Philadelphia, Philadelphia, PA, USA * These authors contributed equally: Jonathan M. Ghergurovich, Xincheng Xu, Joshua Z. Wang.

AUTHORS AND AFFILIATIONS * Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA Jonathan M. Ghergurovich, Xincheng Xu, Joshua Z. Wang, Lifeng Yang, 

Rolf-Peter Ryseck, Lin Wang & Joshua D. Rabinowitz * Department of Molecular Biology, Princeton University, Princeton, NJ, USA Jonathan M. Ghergurovich & Lifeng Yang * Department of

Chemistry, Princeton University, Princeton, NJ, USA Xincheng Xu, Joshua Z. Wang, Lin Wang & Joshua D. Rabinowitz Authors * Jonathan M. Ghergurovich View author publications You can also

search for this author inPubMed Google Scholar * Xincheng Xu View author publications You can also search for this author inPubMed Google Scholar * Joshua Z. Wang View author publications

You can also search for this author inPubMed Google Scholar * Lifeng Yang View author publications You can also search for this author inPubMed Google Scholar * Rolf-Peter Ryseck View author

publications You can also search for this author inPubMed Google Scholar * Lin Wang View author publications You can also search for this author inPubMed Google Scholar * Joshua D.

Rabinowitz View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.M.G. conceived the study. J.M.G., X.X., J.Z.W. and J.D.R. designed the

experiments. J.Z.W., X.X., J.M.G., L.Y., R.-P.R. and L.W. conducted the experiments. J.Z.W., X.X., J.M.G. and J.D.R. wrote the paper with input from the other authors. CORRESPONDING AUTHOR

Correspondence to Joshua D. Rabinowitz. ETHICS DECLARATIONS COMPETING INTERESTS J.D.R. is a paid adviser and stockholder in Kadmon Pharmaceuticals, L.E.A.F. Pharmaceuticals and Rafael

Pharmaceuticals; a paid consultant of Pfizer; a founder, director, and stockholder of Farber Partners and Serien Therapeutics. J.D.R. and J.M.G. are inventors of patents in the area of

folate metabolism held by Princeton University. The other authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Metabolism_ thanks Kivanc Birsoy,

Jason Locasale and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: George Caputa. PUBLISHER’S NOTE Springer Nature remains

neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 MTR IS A MINOR SOURCE OF METHIONINE IN VITRO AND IN VIVO.

(A) Schematic of methionine labeling from [U-13C]methionine. Red circles indicate 13C atoms. MTR = methionine synthase. (B) Schematic of methionine labeling from [U-13C] or [3-13C]serine.

Blue circles indicate 13C atoms. MTHFR = methylenetetrahydrofolate reductase, SHMT = serine hydroxymethyltransferase. (C) Methionine labeling in cell lines after culturing for 4 h in media

containing [U-13C]serine (for 293 T) or [3-13C]serine (for HCT116 and HepG2) (mean ± SD, n = 2). (D) Methionine M + 1 fraction from 4 h [3-13C]serine tracing in HCT116 cultured in media

containing indicated methionine and folate concentrations (mean ± SD, n = 3 for each condition). Labeling of (E) serine and (F) methionine in serum, PDAC tumors, and normal tissues of male

C57BL/6 mice after [U-13C]serine infusion for 2.5 h. (mean ± SD, n = 3 mice; two technical replicates were included for each tumour). (G) Schematic of methionine labeling from

[13C5,15N]betaine. Orange circles indicate 13C atoms, green circles indicate 15N atoms. BHMT = betaine-homocysteine S-methyltransferase, DMG = dimethylglycine. Source data EXTENDED DATA FIG.

2 MTR IS IMPORTANT FOR CELL GROWTH IN PHYSIOLOGICAL FOLATES. (A) Expression of MTR in the HCT116, 8988 T, and HepG2 cell lines as reported in the Cancer Cell Line Encyclopedia.63 (B) Cell

growth curves in the media containing indicated folate sources (mean ± SD, n = 2). (C) Cell growth curves in media containing indicated folate and methionine concentrations (mean ± SD, n = 

3). (D) Individual tumor volumes for HCT116 xenografts in female CD-1 nude mice (n = 10 mice). (E) Terminal tumor mass of HCT116 xenografts in female CD-1 nude mice (mean ± SEM, n = 10

mice). _P_ values were determined by a two-sided paired Student’s _t_-test comparing ΔMTR-1 to wild-type, and ΔMTR-2 to CRISPR control (control-1). (F) Growth of subcutaneous HCT116

xenografts in male CD-1 nude mice on a standard folate (4ppm) or low folate diet (mean ± SEM, n = 10 mice). (G) Western blot analysis of MTR and eGFP in HCT116 wild-type (WT), CRISPR

control-1 or ΔMTR-1 which was also engineered to express a vector containing either eGFP or MTR cDNA. Loading control (COXIV) was analyzed on a separate gel from parallel experiments.

Results are representative of 2 independent biological replicates with similar results. SI = small intestine, SM = skeletal muscle. Source data EXTENDED DATA FIG. 3 LOSS OF MTR DISRUPTS

NUCLEOTIDE SYNTHESIS. (A) Water-soluble metabolite levels from HCT116 control and MTR knockout cells cultured in indicated media conditions. Each box reflects one independent biological

measurement, normalized to the average of control cells cultured in folic acid. (B) Relative nucleotide mono- and diphosphate abundances in HCT116 control and MTR knockout cells in indicated

media. Intensities are normalized to the average of control-1 cells in folic acid (mean ± SD, n = 3). (C) Relative thymidylate species abundances in HCT116 control and knockout cells in

indicated media. Intensities are normalized to the average of control-1 cells in folic acid (mean ± SD, n = 3). (D) Cell growth dose response curves for HCT116 WT and MTR knockout cells

treated with SHMT1/2 inhibitor SHIN2 under different folate conditions (mean ± SD, n = 5). For (B) and (C), _P_ values were determined by a one-way ANOVA comparing control to MTR knockout in

the same medium followed by Dunnett’s post hoc analysis. Source data EXTENDED DATA FIG. 4 METABOLOMICS OF MTR KNOCKOUT TUMORS. (A) Water-soluble metabolites levels from individual HCT116

control and MTR knockout subcutaneous tumors (normalized to wild-type tumor average). (B) Relative abundance of an S-ribosylhomocysteine isomer in HCT116 control and MTR knockout

subcutaneous tumors (mean ± SD, n = 10 tumors for control-1, and n = 9 tumors for each other group). Mice were fed standard chow. (C) MS/MS spectrum of m/z 268.0848 peak in positive-ion

mode. Fragmentation pattern suggests an S-ribosylhomocysteine (SRH) isomer. WT = wild-type. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Table 1. REPORTING

SUMMARY SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 2 Unprocessed western blots. SOURCE DATA FIG. 3 Statistical

source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA

EXTENDED DATA FIG. 2 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. RIGHTS AND PERMISSIONS

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