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Methionine synthase is essential for cancer cell proliferation in physiological folate environments

Nature Metabolism volume 3, pages 1500–1511 (2021)Cite this article

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Abstract

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 folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the 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.

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Fig. 1: Cells can be cultured in medium with 5-methyl THF as the folate source.
Fig. 2: MTR is only essential for proliferation when 5-methyl THF is the folate source.
Fig. 3: MTR is required for nucleotide synthesis in 5-methyl THF medium.
Fig. 4: MTR knockout reduces SAM but not methionine levels.
Fig. 5: Physiological folates prevent growth of MTR knockout tumours and cause methotrexate resistance.

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Data availability

Unprocessed western blot images, integrated peak areas for metabolite level quantification and processed data from comparative analysis of barcode counts of 489 human cancer cell lines in each condition in the PRISM multiplexed growth assay are provided as source data for Figs. 1–5 and Extended Data Figs. 1–5. PCR amplicon sequencing results including raw barcode counts for each cancer cell line in the PRISM 489 cell line pool in each condition/replicate are provided in Supplementary Table 1. Source data are provided with this paper.

Code availability

Any code used to analyse or plot data in this manuscript is available from the corresponding author upon request.

References

  1. Chiang, P. K. et al. S-adenosylmethionine and methylation. FASEB J. 10, 471–480 (1996).

    Article  CAS  PubMed  Google Scholar 

  2. Lane, A. N. & Fan, T. W. Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res. 43, 2466–2485 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ducker, G. S. & Rabinowitz, J. D. One-carbon metabolism in health and disease. Cell Metab. 25, 27–42 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Pfeiffer, C. M. et al. Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2. Br. J. Nutr. 113, 1965–1977 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Goldman, I. D. The characteristics of the membrane transport of amethopterin and the naturally occurring folates. Ann. NY Acad. Sci. 186, 400–422 (1971).

    Article  CAS  PubMed  Google Scholar 

  6. Ritchie, C., Cordova, A. F., Hess, G. T., Bassik, M. C. & Li, L. SLC19A1 Is an Importer of the Immunotransmitter cGAMP. Mol. Cell 75, 372–381 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Muir, A., Danai, L. V. & Vander Heiden, M. G. Microenvironmental regulation of cancer cell metabolism: implications for experimental design and translational studies. Dis. Model. Mech. https://doi.org/10.1242/dmm.035758 (2018).

  8. Rancati, G., Moffat, J., Typas, A. & Pavelka, N. Emerging and evolving concepts in gene essentiality. Nat. Rev. Genet. 19, 34–49 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Banerjee, R. V. & Matthews, R. G. Cobalamin-dependent methionine synthase. FASEB J. 4, 1450–1459 (1990).

    Article  CAS  PubMed  Google Scholar 

  10. Stover, P. J. Vitamin B12 and older adults. Curr. Opin. Clin. Nutr. Metab. Care 13, 24–27 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chanarin, I., Deacon, R., Lumb, M., Muir, M. & Perry, J. Cobalamin-folate interrelations: a critical review. Blood 66, 479–489 (1985).

    Article  CAS  PubMed  Google Scholar 

  12. Fujii, K., Nagasaki, T. & Huennekens, F. M. Accumulation of 5-methyltetrahydrofolate in cobalamin-deficient L1210 mouse leukemia cells. J. Biol. Chem. 257, 2144–2146 (1982).

    Article  CAS  PubMed  Google Scholar 

  13. Walker, P. R. et al. Induction of apoptosis in neoplastic cells by depletion of vitamin B12. Cell Death Differ. 4, 233–241 (1997).

    Article  CAS  PubMed  Google Scholar 

  14. McLean, G. R. et al. Cobalamin analogues modulate the growth of leukemia cells in vitro. Cancer Res. 57, 4015–4022 (1997).

    CAS  PubMed  Google Scholar 

  15. Corcino, J. J., Zalusky, R., Greenberg, M. & Herbert, V. Coexistence of pernicious anaemia and chronic myeloid leukaemia: an experiment of nature involving vitamin BI2 metabolism. Br. J. Haematol. 20, 511 (1971).

    Article  CAS  PubMed  Google Scholar 

  16. Eastwood, D. W., Green, C. D., Lambdin, M. A. & Gardner, R. Effect of nitrous oxide on the white-cell count in leukemia. N. Engl. J. Med. 268, 297–299 (1963).

    Article  CAS  Google Scholar 

  17. Ikeda, K. et al. Antileukemic effect of nitrous oxide in a patient with chronic myelogenous leukemia. Am. J. Hematol. 30, 114 (1989).

    Article  CAS  PubMed  Google Scholar 

  18. Matthews, J. H. Cyanocobalamin [c-lactam] inhibits vitamin B12 and causes cytotoxicity in HL60 cells: methionine protects cells completely. Blood 89, 4600–4607 (1997).

    Article  CAS  PubMed  Google Scholar 

  19. Liteplo, R. G., Hipwell, S. E., Rosenblatt, D. S., Sillaots, S. & Lue-Shing, H. Changes in cobalamin metabolism are associated with the altered methionine auxotrophy of highly growth autonomous human melanoma cells. J. Cell. Physiol. 149, 332–338 (1991).

    Article  CAS  PubMed  Google Scholar 

  20. Boss, G. R. Cobalamin inactivation decreases purine and methionine synthesis in cultured lymphoblasts. J. Clin. Invest. 76, 213–218 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lumb, M. et al. Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-adenosylmethionine metabolism in the rat. Biochim. Biophys. Acta 756, 354–359 (1983).

    Article  CAS  PubMed  Google Scholar 

  22. van der Westhuyzen, J., Fernandes-Costa, F. & Metz, J. Cobalamin inactivation by nitrous oxide produces severe neurological impairment in fruit bats: protection by methionine and aggravation by folates. Life Sci. 31, 2001–2010 (1982).

    Article  PubMed  Google Scholar 

  23. Scott, J. M., Dinn, J. J., Wilson, P. & Weir, D. G. Pathogenesis of subacute combined degeneration: a result of methyl group deficiency. Lancet 2, 334–337 (1981).

    Article  CAS  PubMed  Google Scholar 

  24. Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hansen, M. F., Jensen, S. O., Fuchtbauer, E. M. & Martensen, P. M. High folic acid diet enhances tumour growth in PyMT-induced breast cancer. Br. J. Cancer 116, 752–761 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Farber, S. et al. The action of pteroylglutamic conjugates on man. Science 106, 619–621 (1947).

    Article  CAS  PubMed  Google Scholar 

  27. Farber, S., Diamond, L. K., Mercer, R. D., Sylvester, R. F. & Wolff, J. A. Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-aminopteryl-glutamic acid (aminopterin). N. Engl. J. Med. 238, 787–793 (1948).

    Article  CAS  PubMed  Google Scholar 

  28. Yu, C. et al. High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nat. Biotechnol. 34, 419–423 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Palmer, A. M., Kamynina, E., Field, M. S. & Stover, P. J. Folate rescues vitamin B12 depletion-induced inhibition of nuclear thymidylate biosynthesis and genome instability. Proc. Natl Acad. Sci. USA 114, E4095–E4102 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen, L., Ducker, G. S., Lu, W., Teng, X. & Rabinowitz, J. D. An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate. Anal. Bioanal. Chem. https://doi.org/10.1007/s00216-017-0514-4 (2017).

  31. Stover, P. & Schirch, V. Evidence for the accumulation of a stable intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate. Biochemistry 31, 2148–2155 (1992).

    Article  CAS  PubMed  Google Scholar 

  32. Labuschagne, C. F., van den Broek, N. J., Mackay, G. M., Vousden, K. H. & Maddocks, O. D. K. Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells. Cell Rep. 7, 1248–1258 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Misselbeck, K. et al. A hybrid stochastic model of folate-mediated one-carbon metabolism: Effect of the common C677T MTHFR variant on de novo thymidylate biosynthesis. Sci. Rep. 7, 797 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Bjursell, G. & Reichard, P. Effects of thymidine on deoxyribonucleoside triphosphate pools and deoxyribonucleic acid synthesis in chinese hamster ovary cells. J. Biol. Chem. 248, 3904–3909 (1973).

    Article  CAS  PubMed  Google Scholar 

  35. Gao, X. et al. Dietary methionine influences therapy in mouse cancer models and alters human metabolism. Nature 572, 397–401 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sullivan, M. R. et al. Increased serine synthesis provides an advantage for tumors arising in tissues where serine levels are limiting. Cell Metab. 29, 1410–1421 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cynober, L. A. Plasma amino acid levels with a note on membrane transport: characteristics, regulation, and metabolic significance. Nutrition 18, 761–766 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Ghergurovich, J. M. et al. Methionine synthase supports tumor tetrahydrofolate pools. Nat. Metab. https://doi.org/10.1038/s42255-021-00465-w (2021).

  39. Mato, J. M., Alvarez, L., Ortiz, P. & Pajares, M. A. S-adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol. Ther. 73, 265–280 (1997).

    Article  CAS  PubMed  Google Scholar 

  40. Maddocks, O. D., Labuschagne, C. F., Adams, P. D. & Vousden, K. H. Serine metabolism supports the methionine cycle and DNA/RNA methylation through de novo ATP synthesis in cancer cells. Mol. Cell 61, 210–221 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Su, X., Wellen, K. E. & Rabinowitz, J. D. Metabolic control of methylation and acetylation. Curr. Opin. Chem. Biol. 30, 52–60 (2016).

    Article  CAS  PubMed  Google Scholar 

  42. Cho, H. S. et al. Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B. Nat. Commun. 3, 1072 (2012).

    Article  PubMed  Google Scholar 

  43. Gu, X. et al. SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway. Science 358, 813–818 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Reeves, P. G. Components of the AIN-93 Diets as Improvements in the AIN-76A Diet. J. Nutr. 127, 838–841 (1997).

    Article  Google Scholar 

  45. Bailey, R, L. et al. Total folate and folic acid intake from foods and dietary supplements in the United States: 2003–2006. Am. J. Clin. Nutr. 91, 231–237 (2010).

    Article  Google Scholar 

  46. Gaukroger, J. et al. Paradoxical response of malignant melanoma to methotrexate in vivo and in vitro. Br. J. Cancer 47, 671–679 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chu, E., Drake, J. C., Boarman, D., Baram, J. & Allegra, C. J. Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells. J. Biol. Chem. 265, 8470–8478 (1990).

    Article  CAS  PubMed  Google Scholar 

  48. Sanderson, S. M., Gao, X., Dai, Z. & Locasale, J. W. Methionine metabolism in health and cancer: a nexus of diet and precision medicine. Nat. Rev. Cancer 19, 625–637 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Chaturvedi, S., Hoffman, R. M. & Bertino, J. R. Exploiting methionine restriction for cancer treatment. Biochem. Pharmacol. 154, 170–173 (2018).

    Article  CAS  PubMed  Google Scholar 

  50. Sakura, T. et al. High-dose methotrexate therapy significantly improved survival of adult acute lymphoblastic leukemia: a phase III study by JALSG. Leukemia 32, 626–632 (2017).

    Article  PubMed  Google Scholar 

  51. Baldwin, C. M. & Perry, C. M. Pemetrexed: a review in its use in the management of advanced non-squamous non-small cell lung cancer. Drugs 69, 2279–2302 (2009).

    Article  CAS  PubMed  Google Scholar 

  52. Gonen, N. & Assaraf, Y. G. Antifolates in cancer therapy: structure, activity and mechanisms of drug resistance. Drug Resist. Updat. 15, 183–210 (2012).

    Article  CAS  PubMed  Google Scholar 

  53. Visentin, M., Zhao, R. & Goldman, I. D. The antifolates. Hematol. Oncol. Clin. North Am. 26, 629–648 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Li, X., Wei, S. & Chen, J. Critical appraisal of pemetrexed in the treatment of NSCLC and metastatic pulmonary nodules. Onco. Targets Ther. 7, 937–945 (2014).

    PubMed  PubMed Central  Google Scholar 

  55. Otake, Y. et al. Expression of thymidylate synthase in human non-small cell lung cancer. Jpn. J. Cancer Res. 90, 1248–1253 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ceppi, P. et al. Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNA and protein levels for thymidylate synthase. Cancer 107, 1589–1596 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Takezawa, K. et al. Thymidylate synthase as a determinant of pemetrexed sensitivity in non-small cell lung cancer. Br. J. Cancer 104, 1594–1601 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Li, Y. et al. Transcriptomic and functional network features of lung squamous cell carcinoma through integrative analysis of GEO and TCGA data. Sci. Rep. 8, 15834 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Gherasim, C., Lofgren, M. & Banerjee, R. Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin. J. Biol. Chem. 288, 13186–13193 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Oltean, S. & Banerjee, R. Nutritional modulation of gene expression and homocysteine utilization by vitamin B12. J. Biol. Chem. 278, 20778–20784 (2003).

    Article  CAS  PubMed  Google Scholar 

  61. Jarrett, J. T., Goulding, C. W., Fluhr, K., Huang, S. & Matthews, R. G. Purification and assay of cobalamin-dependent methionine synthase from Escherichia coli. Methods Enzymol. 281, 196–213 (1997).

    Article  CAS  PubMed  Google Scholar 

  62. Yamada, K., Gravel, R. A., Toraya, T. & Matthews, R. G. Human methionine synthase reductase is a molecular chaperone for human methionine synthase. Proc. Natl Acad. Sci. USA 103, 9476–9481 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Olteanu, H. & Banerjee, R. Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. J. Biol. Chem. 276, 35558–35563 (2001).

    Article  CAS  PubMed  Google Scholar 

  64. Muir, A. et al. Environmental cystine drives glutamine anaplerosis and sensitizes cancer cells to glutaminase inhibition. eLife https://doi.org/10.7554/eLife.27713 (2017).

  65. Sullivan, L. B. et al. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells. Cell 162, 552–563 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Zhao, H., French, J. B., Fang, Y. & Benkovic, S. J. The purinosome, a multi-protein complex involved in the de novo biosynthesis of purines in humans. Chem. Commun. 49, 4444–4452 (2013).

    Article  CAS  Google Scholar 

  67. Okesli, A., Khosla, C. & Bassik, M. C. Human pyrimidine nucleotide biosynthesis as a target for antiviral chemotherapy. Curr. Opin. Biotechnol. 48, 127–134 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).

    Article  CAS  PubMed  Google Scholar 

  70. Kanarek, N. et al. Histidine catabolism is a major determinant of methotrexate sensitivity. Nature 559, 632–636 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Jin, X. et al. A metastasis map of human cancer cell lines. Nature 588, 331–336 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank N. Kanarek for her comments and technical advice on folate measurements. We thank all members of the Vander Heiden laboratory for input and advice on the manuscript. M.R.S. was supported by grant no. T32-GM007287 and by an MIT Koch Institute Graduate Fellowship. A.M.D. was supported by a Jane Coffin Childs Postdoctoral Fellowship. M.G.V.H. acknowledges support from grant nos. R35CA242379, R01CA201276 and P30CA014051, the Ludwig Center at MIT, the MIT Center for Precision Cancer Medicine, Stand Up To Cancer, the Emerald Foundation, the Lustgarten Foundation and a Faculty Scholars Grant from Howard Hughes Medical Institute.

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Author notes

  1. These authors contributed equally: Mark R. Sullivan, Alicia M. Darnell.

Authors and Affiliations

  1. Koch Institute for Integrative Cancer Research, Cambridge, MA, USA

    Mark R. Sullivan, Alicia M. Darnell, Montana F. Reilly, Ahmed Ali & Matthew G. Vander Heiden

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Mark R. Sullivan, Alicia M. Darnell & Matthew G. Vander Heiden

  3. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Tenzin Kunchok & Caroline A. Lewis

  4. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA

    Lena Joesch-Cohen, Daniel Rosenberg, Ahmed Ali, Matthew G. Rees, Jennifer A. Roth & Matthew G. Vander Heiden

  5. Dana-Farber Cancer Institute, Boston, MA, USA

    Matthew G. Vander Heiden

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Contributions

M.R.S. and M.G.V.H. were responsible for study conceptualization. M.R.S., A.M.D., M.F.R., T.K. and C.A.L. were responsible for the study methodology. M.R.S., A.M.D. and L.J.-C. conducted the formal analysis. M.R.S., A.M.D. and M.F.R. were responsible for the investigation. T.K., C.A.L., D.R., M.R., A.A., L.J.-C. and J.A.R. were responsible for the resources. M.R.S. and A.M.D. were responsible for the data visualization.M.R.S. and A.M.D wrote the original draft. M.R.S., A.M.D. and M.G.V.H. wrote the second draft, and reviewed and edited it. M.R.S., A.M.D., J.A.R. and M.G.V.H. were responsible for the acquisition of funding.M.G.V.H. supervised the study.

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Correspondence to Matthew G. Vander Heiden.

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M.G.V.H. is on the scientific advisory board of Agios Pharmaceuticals, Aeglea Biotherapeutics, iTeos Therapeutics, Faeth Therapeutics, Sage Therapeutics, DRIOA Ventures and Auron Therapeutics. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Characterization of cells cultured in 5-methyl THF.

(A-B) Histogram (A) or box plot (B) of log2 MTR mRNA levels for cells in the Cancer Cell Line Encyclopedia (CCLE)24 (n = 1019/1457 cell lines with detectable MTR). Median (box center line), interquartile range (IQR) (box), 1.5*IQR (whiskers), and outliers (points) are plotted; dashed line represents the overall median. (C) Proliferation rate of T.T cells in media containing no folate source (−), folic acid (+FA), or 5-methyl THF (+5-MeTHF) before (0 days, n = 3 independent samples) or after (3 days, n = 6 independent samples) of culture with no folic acid (“prestarved”). (D) LC/MS measurement of folic acid and 5-methyl THF concentrations in dialyzed fetal bovine serum. (E-H) LC/MS measurement of intracellular methionine, serine, lysine (E), SAM (F), AMP, ADP, ATP, GMP, GDP, GTP, GAR, AICAR, IMP (G), dUMP and dTMP (H) in A549 and T.T cells cultured for up to 3 days in medium lacking folates (n = 3 independent samples). Data are normalized to protein concentration and an internal standard. (I) Proliferation rate of A549 or T.T cells cultured in medium lacking folates for up to 3 days (n = 4 independent samples). (J-L) Correlation between the proliferation rate in 5-methyl THF and folic acid medium (J; dashed line: y=x), log2 MTR mRNA level and log2 relative viability in 5-methyl THF versus folic acid medium (K), or log2 MTR mRNA level and proliferation rate in 5-methyl THF / folic acid (L) for 489 barcoded cell lines (Pearson’s product moment correlation coefficient / p-value for J: R = 0.255, p = 2e-07; K: R = -0.041 p = 0.4; L: 5-methyl THF R = 0.08, p = 0.11 and folic acid R = 0.1, p = 0.05). Purple line is a linear regression fit, shading represents a 95% confidence interval around the mean. (M) Western blot analysis of MTR expression in A549 or T.T cells cultured in medium with the indicated folate source (representative of 1 experiment). (A-M) Mean +/- SD error bars are displayed.

Source data

Extended Data Fig. 2 MTR knockout affects levels of intracellular folate species and proliferation in different folate sources.

(A) Proliferation rates of A549 MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured in folic acid (+FA) or 96% 5-methyl THF, 4% folic acid (phys ratio)4 for 4 days after a folate pre-starvation period (n = 3 independent samples). (B) LC/MS measurement of intracellular folate, 5-methyl THF, combined 5,10-methenyl THF/10-formyl THF, and 5-formyl THF levels in A549 and T.T MTR knockout cells +EV or +MTR cultured up to 3 days in medium lacking folates (n = 1 independent sample for each time point). Data are normalized to protein concentration and an internal standard. (C) Proliferation rate of A549 or T.T MTR knockout cells +EV or +MTR cultured in medium lacking folates (prestarved) or with folic acid for 3 days (n = 3 independent samples; A549: p = 0.011; T.T: p = 0.046). p values indicated are derived from a two-tailed, unpaired Welch’s t test (* = p <0.05). (A,C) Mean +/- SD error bars are displayed.

Source data

Extended Data Fig. 3 MTR knockout results in folate insufficiency and impaired nucleotide synthesis in 5-methyl THF.

(A-D) LC/MS measurement of intracellular AMP, ADP, ATP (A), GMP, GDP, GTP (B), GAR, AICAR, IMP (C), and dTMP (D) levels in T.T MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in folic acid (+FA) or 5-methyl THF (+5-MeTHF) after a prestarvation period (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: AMP: 0.014, ADP: 0.002, ATP: 0.014, GMP: 0.26, GDP: 0.008, GTP: 0.017, GAR: 0.004, AICAR: 0.005, IMP: 0.014, dTMP: 0.01; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: AMP: 0.012, ADP: 0.0097, ATP: 0.007, GMP: 0.0006, GDP: 0.008, GTP: 0.012, GAR: 0.002, AICAR: 0.003, IMP: 0.022, dTMP: 2.51*10-5; +EV +FA vs +EV +5-methyl THF p-values: AMP: 0.007, ADP: 0.006, ATP: 5.26*10-5, GMP: 0.0004, GDP: 0.006, GTP: 2.94*10-5, GAR: 0.002, AICAR: 0.003, IMP: 0.008, dTMP: 0.005). Data are normalized to protein concentration and to an internal standard. Mean +/- SEM error bars are displayed. (E) Proliferation rates of T.T MTR knockout cells +EV or +MTR cultured as in A-D in the indicated folate with or without the addition of 100 µM each of the indicated nucleotide precursors hypoxanthine (H), uridine (U), and thymidine (T) (n = 3 independent samples; except +MTR +H,U,T n = 6). (F) Proliferation rates of A549 MTR knockout cells +EV or +MTR passaged in the indicated folate with the addition of nucleotide precursors for up to 16 days (n = 3 independent samples). (G) Proliferation rates of T.T MTR knockout cells +EV or +MTR cultured in the indicated folate as in A-D, with or without 1 mM serine or formate (n = 3 independent samples). (E-G) Mean +/- SD error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test (* = p <0.05, ** = p <0.01, *** = p <0.001).

Source data

Extended Data Fig. 4 Neither SAM nor methionine levels are limiting for proliferation in MTR knockout cells in 5-methyl THF.

(A-B) Proliferation rates of A549 and T.T MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured in folic acid (+FA), 5-methyl THF (5-MeTHF), or no folate (−) for 4 days after a prestarvation period, with or without 900 uM added methionine (A) or T.T MTR knockout cells +EV or +MTR cultured in the indicated folate or 100 uM each nucleotide precursor (hypoxanthine, uridine, and thymidine) at varied extracellular methionine concentrations (B). (C-D) LC/MS measurement of intracellular methionine, serine, lysine (C), SAM, and SAH levels (D) in T.T MTR knockout cells +EV or +MTR cultured in the indicated folate source as in A (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: methionine: 0.002, serine: 0.57, lysine: 9.94*10-6, SAM: 0.007, SAH: 0.024; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: methionine: 0.01, serine: 2.71*10-5, lysine: 0.0001, SAM: 0.003, SAH: 0.47; +EV +FA vs +EV +5-methyl THF p-values: methionine: 0.011, serine: 5.57*10-5, lysine: 0.0001, SAM: 0.24, SAH: 0.27). Data are normalized to protein concentration and an internal standard. (E) Western blots to assess phosphorylation of mTORC1 targets and levels of mono- or dimethyl lysine-containing proteins in T.T MTR knockout cells +EV or +MTR cultured in the indicated folate as in A. The ratio of phospho-protein to total protein signal from 3 independent replicates is shown. (F) Proliferation rates of T.T MTR knockout cells +EV or +MTR cultured in the indicated folate for 16 hours, with or without the addition of 1 mM SAM (n = 3 independent samples for +EV, n = 6 for +MTR; +EV −folate vs +EV +SAM p = 1.6*10-6, +EV +SAM vs +MTR +SAM p = 0.11). (A,B,F) Mean +/- SD error bars are displayed. (C-E) Mean +/- SEM error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test (* = p <0.05, ** = p <0.01, *** = p <0.001).

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Extended Data Fig. 5 5-methyl THF medium blunts methotrexate efficacy but not import.

(A-B) Proliferation rates of A549 and T.T cells (A) or T.T MTR knockout cells without (+EV) or with MTR expression (+MTR) (B) cultured in folic acid (+FA) or 5-methyl THF (+5-MeTHF) for 4 days across a range of methotrexate doses (n = 3 independent samples). (C) Log2 fold change in relative viability of 489 cell lines cultured in 5-methyl THF versus folic acid as in A-B (log2 fc 5-MeTHF/FA) at the indicated methotrexate doses, grouped by tissue of origin (note that the 0 dose is the same data plotted in Fig. 1G). Median (box center line), interquartile range (IQR) (box), and 1.5*IQR (whiskers) are plotted. (D) Mean barcode read counts for the total pool of cancer cell lines, before (day 0) or after (day 4) culture in the indicate folate/methotrexate doses as in A-B. Tukey HSD p-value was calculated by correcting a two-way ANOVA test for multiple comparisons between the dose response curves in the two folates (n = 489 cell lines; folic acid vs. 5-methyl THF dose response p = 0.049). (E-G) LC/MS measurement of intracellular AMP, ADP, ATP, GMP, GDP, GTP, GAR, AICAR, IMP, dUMP, dTMP, dTTP (E), methionine, serine, lysine (F), and intracellular methotrexate (Mtx) (G) levels in A549 MTR knockout cells +EV or +MTR cultured in the indicated folate across a range of doses of methotrexate as in A-B (n = 3 independent samples). Data are normalized to cell number and an internal standard. (A-G) Mean +/- SD error bars are displayed, except in D where mean +/- SEM is displayed.

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Supplementary information

Reporting Summary

Supplementary Table 1

Raw PCR amplicon sequencing barcode counts for each cancer cell line in our PRISM barcoded pool or control spike-in, in folic acid or 5-methyl THF medium across a range of methotrexate doses. Individual biological and technical replicates, population-level cell counts and mean-normalized gDNA yields for correcting cell counts (as described in the Methods) are included. No sequenced barcodes have been removed; therefore, not all are exact matches to the barcodes associated with each cell line/condition/replicate group due to, for example, sequencing errors.

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Sullivan, M.R., Darnell, A.M., Reilly, M.F. et al. Methionine synthase is essential for cancer cell proliferation in physiological folate environments. Nat Metab 3, 1500–1511 (2021). https://doi.org/10.1038/s42255-021-00486-5

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