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Polyamine pathway activity promotes cysteine essentiality in cancer cells

Nature Metabolism volume 2, pages 1062–1076 (2020)Cite this article

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Abstract

Cancer cells have high demands for non-essential amino acids (NEAAs), which are precursors for anabolic and antioxidant pathways that support cell survival and proliferation. It is well-established that cancer cells consume the NEAA cysteine, and that cysteine deprivation can induce cell death; however, the specific factors governing acute sensitivity to cysteine starvation are poorly characterized. Here, we show that that neither expression of enzymes for cysteine synthesis nor availability of the primary precursor methionine correlated with acute sensitivity to cysteine starvation. We observed a strong correlation between efflux of the methionine-derived metabolite methylthioadenosine (MTA) and sensitivity to cysteine starvation. MTA efflux results from genetic deletion of methylthioadenosine phosphorylase (MTAP), which is frequently deleted in cancers. We show that MTAP loss upregulates polyamine metabolism which, concurrently with cysteine withdrawal, promotes elevated reactive oxygen species and prevents cell survival. Our results reveal an unexplored metabolic weakness at the intersection of polyamine and cysteine metabolism.

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Fig. 1: Cancer cell lines are generally sensitive to cysteine starvation, and some are highly sensitive.
Fig. 2: Methylthioadenosine efflux correlates with sensitivity to cysteine starvation.
Fig. 3: Impact of polyamine metabolism on cysteine-starvation sensitivity.
Fig. 4: Inhibition of polyamine metabolism protects highly sensitive cells from cysteine starvation.
Fig. 5: Impact of methionine levels and acute MTAP deletion on sensitivity to cysteine starvation.
Fig. 6: MTAP status is linked to AMD1 expression, polyamine-pathway activity and sensitivity to cysteine starvation.

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Source data for figures have been deposited at: http://researchdata.gla.ac.uk. Source data are provided with this paper.

References

  1. DeBerardinis, R. J. & Chandel, N. S. Fundamentals of cancer metabolism. Sci. Adv. 2, e1600200 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    CAS  PubMed  Google Scholar 

  3. Maddocks, O. D. et al. Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells. Nature 493, 542–546 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Maddocks, O. D. K. et al. Modulating the therapeutic response of tumours to dietary serine and glycine starvation. Nature 544, 372–376 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Eagle, H. The specific amino acid requirements of a mammalian cell (strain L) in tissue culture. J. Biol. Chem. 214, 839–852 (1955).

    Article  CAS  PubMed  Google Scholar 

  6. Eagle, H. The specific amino acid requirements of a human carcinoma cell (Stain HeLa) in tissue culture. J. Exp. Med. 102, 37–48 (1955).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Eagle, H. Nutrition needs of mammalian cells in tissue culture. Science 122, 501–514 (1955).

    Article  CAS  PubMed  Google Scholar 

  8. Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yang, W. S. et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc. Natl Acad. Sci. USA 113, E4966–E4975 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. De Sanctis, G., Spinelli, M., Vanoni, M. & Sacco, E. K-Ras activation induces differential sensitivity to sulfur amino acid limitation and deprivation and to oxidative and anti-oxidative stress in mouse fibroblasts. PLoS ONE 11, e0163790 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Poursaitidis, I. et al. Oncogene-selective sensitivity to synchronous cell death following modulation of the amino acid nutrient cystine. Cell Rep. 18, 2547–2556 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Cramer, S. L. et al. Systemic depletion of l-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth. Nat. Med. 23, 120–127 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. McCormick, P. N. et al. Assessment of tumor redox status through (S)-4-(3-[(18)F]fluoropropyl)-l-glutamic acid PET imaging of system xc (–) activity. Cancer Res. 79, 853–863 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Dixon, S. J. et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. eLife 3, e02523 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Harris, I. S. et al. Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression. Cancer Cell. 27, 211–222 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. DeNicola, G. M. et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 475, 106–109 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sayin, V. I. et al. Activation of the NRF2 antioxidant program generates an imbalance in central carbon metabolism in cancer. eLife 6, e28083 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Kang, Y. P. et al. Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer. eLife 8, e45572 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Muir, A. et al. Environmental cystine drives glutamine anaplerosis and sensitizes cancer cells to glutaminase inhibition. eLife 6, e27713 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Romero, R. et al. Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis. Nat. Med. 23, 1362–1368 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Shin, C. S. et al. The glutamate/cystine xCT antiporter antagonizes glutamine metabolism and reduces nutrient flexibility. Nat. Commun. 8, 15074 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Alvarez, S. W. et al. NFS1 undergoes positive selection in lung tumours and protects cells from ferroptosis. Nature 551, 639–643 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kim, J. et al. Expression of cystathionine beta-synthase is downregulated in hepatocellular carcinoma and associated with poor prognosis. Oncol. Rep. 21, 1449–1454 (2009).

    CAS  PubMed  Google Scholar 

  24. Zhao, H. et al. Frequent epigenetic silencing of the folate-metabolising gene cystathionine-beta-synthase in gastrointestinal cancer. PLoS ONE 7, e49683 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhu, J. J. et al. Transsulfuration activity can support cell growth upon extracellular cysteine limitation. Cell Metab. 30, 865 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Brosnan, J. T. & Brosnan, M. E. The sulfur-containing amino acids: an overview. J. Nutr. 136, 1636S–1640S (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Kryukov, G. V. et al. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells. Science 351, 1214–1218 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Marjon, K. et al. MTAP deletions in cancer create vulnerability to targeting of the MAT2A/PRMT5/RIOK1 axis. Cell Rep. 15, 574–587 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Mavrakis, K. J. et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5. Science 351, 1208–1213 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Zilka, O. et al. On the mechanism of cytoprotection by ferrostatin-1 and liproxstatin-1 and the role of lipid peroxidation in ferroptotic cell death. ACS Cent. Sci. 3, 232–243 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shlomi, T., Fan, J., Tang, B., Kruger, W. D. & Rabinowitz, J. D. Quantitation of cellular metabolic fluxes of methionine. Anal. Chem. 86, 1583–1591 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zabala-Letona, A. et al. mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer. Nature 547, 109–113 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  34. Zhang, T., Labuschagne, C. F., Vousden, K. H. & Maddocks, O. D. K. Direct estimation of metabolic flux by heavy isotope labeling simultaneous with pathway inhibition: metabolic flux inhibition assay. Methods Mol. Biol. 1862, 109–119 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Asimakopoulou, A. et al. Selectivity of commonly used pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). Br. J. Pharmacol. 169, 922–932 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Andreu-Perez, P. et al. Methylthioadenosine (MTA) inhibits melanoma cell proliferation and in vivo tumor growth. BMC Cancer 10, 265 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Subhi, A. L. et al. Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites. J. Biol. Chem. 278, 49868–49873 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Subhi, A. L. et al. Loss of methylthioadenosine phosphorylase and elevated ornithine decarboxylase is common in pancreatic cancer. Clin. Cancer Res. 10, 7290–7296 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Wishart, D. S. et al. HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Res. 46, D608–D617 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Krall, A. S., Xu, S., Graeber, T. G., Braas, D. & Christofk, H. R. Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor. Nat. Commun. 7, 11457 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Harris, P. N., Krahl, M. E. & Clowes, G. H. p-Dimethylaminoazobenzene carcinogenesis with purified diets varying in content of cysteine, cystine, liver extract, protein, riboflavin, and other factors. Cancer Res. 7, 162–175 (1947).

    CAS  PubMed  Google Scholar 

  42. Lockart, R. Z. Jr. & Eagle, H. Requirements for growth of single human cells. Science 129, 252–254 (1959).

    Article  PubMed  Google Scholar 

  43. Ye, C., Sutter, B. M., Wang, Y., Kuang, Z. & Tu, B. P. A metabolic function for phospholipid and histone methylation. Mol. Cell. 66, 180–193 e188 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Casero, R. A. Jr. & Marton, L. J. Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat. Rev. Drug Discov. 6, 373–390 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Ramani, D., De Bandt, J. P. & Cynober, L. Aliphatic polyamines in physiology and diseases. Clin. Nutr. 33, 14–22 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. Bistulfi, G. et al. The essential role of methylthioadenosine phosphorylase in prostate cancer. Oncotarget 7, 14380–14393 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Gerner, E. W. & Meyskens, F. L. Jr. Polyamines and cancer: old molecules, new understanding. Nat. Rev. Cancer 4, 781–792 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Bello-Fernandez, C., Packham, G. & Cleveland, J. L. The ornithine decarboxylase gene is a transcriptional target of c-Myc. Proc. Natl Acad. Sci. USA 90, 7804–7808 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bertino, J. R., Waud, W. R., Parker, W. B. & Lubin, M. Targeting tumors that lack methylthioadenosine phosphorylase (MTAP) activity: current strategies. Cancer Biol. Ther. 11, 627–632 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Lien, E. C., Ghisolfi, L., Geck, R. C., Asara, J. M. & Toker, A. Oncogenic PI3K promotes methionine dependency in breast cancer cells through the cystine-glutamate antiporter xCT. Sci. Signal. 10, eaao6604 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Hayano, M., Yang, W. S., Corn, C. K., Pagano, N. C. & Stockwell, B. R. Loss of cysteinyl-tRNA synthetase (CARS) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation. Cell Death Differ. 23, 270–278 (2016).

    Article  CAS  PubMed  Google Scholar 

  52. Sanderson, S. M., Mikhael, P. G., Ramesh, V., Dai, Z. & Locasale, J. W. Nutrient availability shapes methionine metabolism in p16/MTAP-deleted cells. Sci. Adv. 5, eaav7769 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. 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 

  54. Zhang, T. et al. Changeover from signalling to energy-provisioning lipids during transition from colostrum to mature milk in the giant panda (Ailuropoda melanoleuca). Sci. Rep. 6, 36141 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hirakawa, D. A., Olson, L. M. & Baker, D. H. Comparative utilization of a crystalline amino-acid diet and a methionine-fortified casein diet by young-rats and mice. Nutr. Res. 4, 891–895 (1984).

    Article  CAS  Google Scholar 

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Acknowledgements

We wish to thank the staff of the Biological Services facility at the Cancer Research UK (CRUK) Beatson Institute, funded by CRUK (A18076 & A17196). We thank D. Tennant, S. Tardito, K. Ryan and A. Chalmers for assistance with biological resources. K.B. and D.A. are core funded by CRUK (A17196 & A29799). O.D.K.M., T.Z., C.B. and A.C.N. were funded by a Cancer Research UK Career Development Fellowship awarded to O.D.K.M. (C53309/A19702).

Author information

Author notes

  1. Tong Zhang

    Present address: Novartis Institutes for BioMedical Research, Shanghai, China

  2. Christin Bauer

    Present address: AstraZeneca R&D, Cambridge, UK

Authors and Affiliations

  1. Institute of Cancer Sciences, Wolfson Wohl Cancer Research Centre, University of Glasgow, Glasgow, UK

    Tong Zhang, Christin Bauer, Alice C. Newman, Alejandro Huerta Uribe, Karen Blyth & Oliver D. K. Maddocks

  2. Cancer Research UK Beatson Institute, Glasgow, UK

    Dimitris Athineos & Karen Blyth

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Contributions

T.Z. performed cell-culture experiments, mass spectrometry and data analysis. C.B. performed cell-culture experiments and analysis and live-cell imaging experiments, plus data analysis. A.C.N. performed cell-culture experiments, designed CRISPR constructs and generated CRISPR clones. A.H.U. performed cell culture, mass spectrometry and associated data analysis. D.A. performed and analysed data for in vivo experiments. K.B. contributed to designing, supervising and analysing in vivo work. O.D.K.M. performed cell-culture experiments, contributed to experimental design, data analysis and interpretation and wrote the manuscript. All authors contributed to finalizing the manuscript.

Corresponding author

Correspondence to Oliver D. K. Maddocks.

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Competing interests

O.D.K.M. contributed to CRUK Cancer Research Technology filing of UK Patent Application no. 1609441.9, relating to dietary modulation of amino acids, and is a co-founder and shareholder in Faeth Therapeutics. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Cellular response to cysteine starvation.

a, Cell lines were grown in complete medium, or matched medium lacking the stated nutrients for 24 h. MDA-MB-231 data is replicated from Fig. 1b for comparison with other cell lines. b, MDA-MB-231 cells were either grown in complete medium containing all amino acids (Ctr), or matched medium lacking cysteine, with (-Cys +GSH) or without (-Cys) glutathione 5 mM for the stated times. Metabolites were extracted and analysed by LCMS. c, Cell lines were grown with (Ctr) or without cysteine (-Cys) and Ferrostatin (+F) 1 μM for 24 h and 72 h. d, Colorectal (HCT116 & SW480) and breast (MDA-MB-231 & MDA-MB-468) cancer cells were either grown in complete medium containing all amino acids (Control), or matched medium lacking cysteine, with (-Cys +HC) or without (-Cys) homocysteine 0.8 mM for 24 h. Cell lysates were probed for transsulfuration pathway enzyme expression by western blot. e, Cells were either grown in complete medium containing all amino acids (+), or matched medium lacking cysteine (-) for 24 h. Cell lysates were probed for transsulfuration pathway enzyme expression by western blot. Western blots are representative of two independent experiments. Bands were quantified on a LiCor scanner, actin corrected band intensity (arbitrary units) are shown. Statistical comparisons in b were done by an ordinary one-way ANOVA with Sidak’s multiple comparison test. Other than western blots, all data are averages of n = 3 biological replicates, error bars are SD.

Source data

Extended Data Fig. 2 Rescue with cysteine precursors homocysteine and cystathionine.

a, MDA-MB-231 cells were grown in medium lacking cysteine (-Cys), supplemented either with vehicle (Veh), homocysteine 0.2 mM (HC) or cystathionine 0.2 mM (CTH) for the stated times. Metabolites were extracted and analysed by LCMS. b, MDA-MB-231 cells were grown in medium lacking cysteine, supplemented with vehicle (Veh), homocysteine (HC) 0.2 mM or 0.8 mM for 8 h. Metabolites were extracted and analysed by LCMS. Statistical comparisons were done by an ordinary one-way ANOVA with Sidak’s multiple comparison test. All data are averages of n = 3 biological replicates, error bars are SD.

Source data

Extended Data Fig. 3 Correlation between polyamine pathway activity and cysteine starvation sensitivity.

a, Cells were grown with (+) or without (-) cysteine for 24 h. Cell lysates were probed for MTAP expression by western blot and quantified with a LiCor scanner. b, Cells were grown with (+) or without (-) cysteine for 24 h. Cell lysates were probed for AMD1 and ODC1 enzyme expression by western blot and quantified with a LiCor scanner. Western blots are representative of two independent experiments. c, Pearson correlation coefficients (R2) for a range of biological parameters versus sensitivity of 11 cell lines to cysteine starvation are shown. P-value = two-sided Student’s t-distribution Metabolite levels are all intracellular, except for ‘Extracellular MTA’. Multiple parameters were assessed under fed (Ctr) and cysteine starved (-Cys) conditions. All metabolite levels are normalized to cell number. Protein expression was quantified by western blot using a LiCor scanner, and are all normalized to actin. Iron uptake was assessed by ICP-OES. Steady state ROS levels were detected by live cell imaging of CellROX stain, steady state lipid peroxidation levels were evaluated by immunocytochemistry staining for MDA (malonyldialdehyde), both quantified using an automated microscope. d, Cells were grown in complete medium (with 13C315N1-serine 0.4 mM substituted for serine) for 48 h. Metabolites were extracted and analysed by LCMS. e, Using peak area data shown in panel (d); GSH levels are shown as % of total GSH pool. In the top panel all isotopologues are shown, in the lower panel only cysteine derived isotopologues (m+4 and m+7) are shown. Supplementary data file contains analysis underlying correlation coefficients in (c). Other than western blots, all data are averages of n = 3 biological replicates, error bars are SD.

Source data

Extended Data Fig. 4 Impact of polyamines on ROS levels.

a, Cells were grown in complete (Ctr) or medium lacking cysteine (-Cys), with or without (Veh) CSE inhibitor beta-cyano-L-Alanine (0.5 mM and 1 mM) for 48 h. b, Cells were either grown in complete medium containing all amino acids (Control), or matched medium lacking cysteine (-Cysteine), with increasing amounts of MTA for 20 h and 72 h. c, Cells were grown in complete medium with vehicle or MTA 500 μM for 24 h. Metabolites were extracted and analysed by LCMS. d, Top panel: Cells were grown in complete medium containing either putrescine (+Putrescine), spermidine (+Spermidine) or spermine (+Spermine), all 20 μM or without (+Vehicle). Bottom panel: Cells were grown in medium without cysteine containing either putrescine (+Putrescine), spermidine (+Spermidine) or spermine (+Spermine), all 20 μM or without (+Vehicle). Reactive oxygen species (ROS) were detected in real time by an Operetta automated microscope in live cells treated with CellROX deep red. e, Cells were grown in complete medium with or without 0.1 mM MTOB for 16 h, then either grown in complete medium (Ctr), or matched medium lacking cysteine (-Cys) with or without 0.1 mM MTOB for 32 h. f, Cells were grown in complete medium with or without 0.1 mM MTOB for 5 h (with 13C515N1-methionine 0.2 mM substituted for methionine). All data are averages of n = 3 biological replicates, error bars are SD.

Source data

Extended Data Fig. 5 Labelling transsulfuration intermediates with 34S-methionine.

a, MDA-MB-231 cells were grown in complete medium (with 34S1-methionine 0.2 mM substituted for methionine) for 24 h and then treated with AMD1 inhibitor sardomozide 20 μM for 2 h, 4 h and 8 h. b, MDA-MB-231 cells and c, MIAPaCa-2 (right) were grown in complete medium with or without AMD1 inhibitor sardomozide 20 μM for 16 h (with 34S1-methionine 0.2 mM substituted for methionine). All data are averages of n = 3 biological replicates, error bars are SD.

Source data

Extended Data Fig. 6 Impact of polyamine pathway modulation on ROS levels.

a, Cells were grown in complete medium with or without PAOX inhibitor 10 μM, 40 μM or 80 μM for 16 h, then grown in complete medium (Ctr), or matched medium lacking cysteine with or without PAOX inhibitor 10 μM, 40 μM or 80 μM for 24 h. b, Cell lines were grown in complete medium with (+SMOXi) or without (+Vehicle) SMOX inhibitor MDL72527 50 μM, then grown in complete medium (Ctr) or matched medium lacking cysteine (-Cys) with or without 50 μM SMOX inhibitor. Reactive oxygen species (ROS) were detected in real time by an Operetta automated microscope in live cells treated with CellROX deep red. ROS staining intensity is shown for 16 h timepoint. c, MDA-MB-231 cells were grown in either complete medium (black bars) or medium lacking cysteine (orange bars) under either normoxia (Nor) or hypoxia (Hyp); 1% oxygen, for stated times. d, Cell lines were grown in complete medium (Ctr) or matched medium lacking cysteine (-Cys) with or without 20 μM AMD1 inhibitor. Reactive oxygen species (ROS) were detected in real time by an Operetta automated microscope in live cells treated with CellROX deep red. ROS staining intensity is shown for 16 h timepoint. e, Cell lines highly sensitive to cysteine starvation (MDA-MB-231, PANC-1, MIAPaCa-2) were grown in medium without cysteine containing increasing concentrations of Methionine (0 to 1 mM) for 20 h. f, g, Cell lines sensitive to cysteine starvation (MDA-MB-231, PANC-1, MIAPaCa-2) were grown in medium without cysteine containing increasing concentrations of Methionine (0 to 10 μM) for 17 h (f) and 41 h (g). h, Cell lines were grown in complete medium (Ctr) or matched medium lacking cysteine (-Cys) with or without methionine (Met) at the stated concentrations. Reactive oxygen species (ROS) were detected in real time by an Operetta automated microscope in live cells treated with CellROX deep red. ROS staining intensity is shown for 18 h timepoint. Statistical comparisons in b, c, d & h were done by an ordinary one-way ANOVA with Sidak’s multiple comparison test. All data are averages of n = 3 biological replicates, error bars are SD.

Source data

Extended Data Fig. 7 Impact of methionine restriction on polyamine and transsulfuration pathways.

a, PANC-1 cells were grown in medium lacking methionine supplemented with varying levels of 13C515N1-methionine for 5 h. Metabolites were extracted and analysed by LCMS. b, Levels of amino acids were assessed using LCMS data from the experiments shown in Fig. 5a and Extended Data Fig. 7a. Relative quantity (peak area, relative to 50uM condition) is shown for each amino acid. c, MIAPaCa-2 cells were cultured in either DMEM or RPMI for two weeks. Experiments were performed by growing cells either in complete medium (Ctr) or matched medium lacking cysteine (-Cys) for 32 h and 48 h. All data are averages of n = 3 biological replicates with error bars as SD.

Source data

Extended Data Fig. 8 Consequences of acute MTAP deletion in HCT116 cells.

a, MTAP positive (Parental/Par, NTC) and negative (M1, M2) HCT116 cells were grown in complete medium (with 13C515N1-methionine 0.2 mM substituted for methionine) for 30 h. Metabolites were extracted and analysed by LCMS. Data are averages of n = 3 biological replicates with error bars as SD. b, CD-1 Nude mice were injected with MTAP positive (NTC) and MTAP deleted (M2) HCT116 cells. Once measurable xenograft tumours had formed, mice were transferred to a diet & drinking water regime either containing all amino acids (Complete), or lacking cysteine and cystine, but containing all other amino acids (-Cys), data are averages, bars are SEM (NTC Control diet n = 8, NTC –Cys diet n = 7, MTAP-KO2 Control diet n = 9, MTAP-KO2 –Cys n = 10). c, Metabolites were extracted from xenograft tumours and serum and subjected to LCMS analysis for MTA levels, data are averages, bars are SD, unpaired Ttest, 2-tails, NTC n = 16, MTAP KO n = 19.

Source data

Extended Data Fig. 9 Consequences of acute MTAP restoration in HCT116 cells.

a, HCT116 cells in which MTAP had been deleted by CRISPR/Cas9 (M2), were either stably transfected with an empty vector (clones M2.EV1 & M2.EV2) or a plasmid for MTAP expression (clones M2.MX1 & M2.MX2). MTAP expression was validated by western blot. Western blot is representative of two independent experiments. Cells were grown in complete medium containing all amino acids (Ctr), or matched medium lacking cysteine (-Cys). After 20 h, cells were fixed, stained and counted. b, The stated HCT116 clones were grown in complete medium with or without the xCT inhibitor and ferroptosis inducer erastin 10 μM for 18 h. c, MTAP expressing (NTC & M2.MX2) and MTAP deleted (M2 & M2.EV1) HCT116 cells were grown in complete medium (with 13C515N1-methionine 0.2 mM substituted for methionine) for 30 h. Metabolites were extracted and analysed by LCMS. d, MTAP expressing (NTC & M2.MX2) and MTAP deleted (M2) HCT116 cells were seeded at a range of cell densities in complete medium in 24-well plates. After 24 h, cells were washed with PBS and medium was changed for medium lacking cysteine or complete medium containing all amino acids. After 20 h, cells were fixed, stained and counted. e, Glioblastoma (GBM) cell lines were grown in complete medium for 24 h. Cell lysates were probed for MTAP, AMD1 and Actin expression by western blot and quantified with a LiCor scanner. Western blot is representative of two independent experiments. f, GBM cell lines were grown in either complete medium (Ctr) or medium lacking cysteine (-Cys) for 48 h. g, The breast cancer cell lines MDA-MB-231 (highly sensitive) and MDA-MB-468 (resistant) were grown in either control (Ctr) or medium lacking cysteine (-Cys) for 48 or 72 h. Cells for this timepoint were fixed and counted, while other cells treated the same way were re-fed with complete medium for an additional 48 h (Ctr + 48 h, -Cys 48 h), before cells were counted. All data except western blots are averages of n = 3 biological replicates, error bars are SD.

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Zhang, T., Bauer, C., Newman, A.C. et al. Polyamine pathway activity promotes cysteine essentiality in cancer cells. Nat Metab 2, 1062–1076 (2020). https://doi.org/10.1038/s42255-020-0253-2

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