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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.

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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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