Serine restriction alters sphingolipid diversity to constrain tumour growth

AbstractSerine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1,2,3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7—or in conditions of low serine availability8,9—to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.

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Download referencesAcknowledgementsWe thank M. Gantner, M. Friedlander and all members of the laboratory of C.M.M. for support and helpful discussions; and N. Mainolfi, V. Suri, A. Friedman and M. Manfredi of Raze Therapeutics for providing PH-755. This work was supported by the NIH (R01CA188652 and R01CA234245 to C.M.M.; U54CA132379), a Camille and Henry Dreyfus Teacher-Scholar Award (to C.M.M.), the National Science Foundation (NSF) Faculty Early Career Development (CAREER) Program (1454425 to C.M.M.), the Helmsley Center for Genomic Medicine (to A.F.M.P) and funding from Ferring Foundation (to A.S.). This work was also supported by NIH grants to the Salk Institute Mass Spectrometry Core (P30CA014195, S10OD021815).Author informationAuthor notesThese authors contributed equally: Thekla Cordes, Michal K. Handzlik, Le YouAffiliationsDepartment of Bioengineering, University of California San Diego, La Jolla, CA, USAThangaselvam Muthusamy, Thekla Cordes, Michal K. Handzlik, Le You, Esther W. Lim, Jivani Gengatharan, Mehmet G. Badur, Martina Wallace & Christian M. MetalloMass Spectrometry Core, Salk Institute for Biological Studies, La Jolla, CA, USAAntonio F. M. PintoClayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, CA, USAMatthew J. Kolar & Alan SaghatelianMoores Cancer Center, University of California, San Diego, La Jolla, CA, USAChristian M. MetalloContributionsC.M.M. and T.M. designed the study. T.M., T.C., L.Y., E.W.L. and J.G. performed in vitro cell studies and independently repeated spheroid growth assays. T.M. and M.K.H. performed xenograft experiments. T.M., T.C., M.K.H., L.Y., M.G.B. and A.F.M.P. generated and analysed targeted metabolomics data. A.F.M.P., M.J.K. and M.G.B. generated and analysed untargeted lipidomics data. A.S. and M.W. guided experimental design and analysis. C.M.M. and T.M. wrote the manuscript with input from all authors.Corresponding authorCorrespondence to
Christian M. Metallo.Ethics declarations

Competing interests
The authors declare no competing interests.

Additional informationPeer review information Nature thanks Sarah-Maria Fendt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended data figures and tablesExtended Data Fig. 1 Mitochondrial pyruvate transport and amino acid metabolism influence spheroid growth.a, HCT116 spheroid growth from 3-, 5- and 8-day cultures. Scale bars, 100 μm. b, Metabolite levels in HCT116 adherent and spheroid cultures (n = 3 culture wells each). c, Alanine levels in adherent and spheroid cultures (n = 3 culture wells each condition and cell line). d, Isotopic labelling (M2 citrate/M3 pyruvate) in HCT116 and MCF7 cells cultured with [U-13C6]glucose for 24 h (n = 3 culture wells each). e, Protein expression of phosphorylated PDH (pPDH), total PDH (tPDH) and β-actin in HCT116 cells. Each lane derived from a single culture well, processed in parallel, and used for quantification. For gel source data, see Supplementary Information. f, Citrate labelling in HCT116 cells cultured with [U-13C5]glutamine in HCT116 (n = 3 culture wells each condition). g, Metabolite levels upon UK5099 treatment in HCT116 spheroid cultures (n = 6 culture wells each). h, Abundances of alanine and serine in A549 spheroid cultures upon treatment with UK5099 (n = 3 culture wells each). i, Spheroid growth in cells upon UK5099 treatment (n = 3 culture wells each). j, k, Adherent growth of A549 (j) and HCT116 (k) cells upon treatment with UK5099 (n = 3 culture wells each). l, m, Adherent growth of A549 (l) and HCT116 (m) cells upon MPC1 or MPC2 knockdown compared to control (shNT) (n = 3 culture wells each). n, o, Isotopologue distributions of serine (n) and citrate (o) in HCT116 spheroid cultures traced with [U-13C6]glucose for 24 h (n = 3 culture wells each). p, Alanine abundances in HCT116 spheroids in the presence of 1 mM alanine and UK5099 (n = 3 culture wells each). q, r, Spheroid growth of HCT116 (q) and MCF7 (r) cells grown in the presence of UK5099 and alanine (n = 3 culture wells each condition). s, Cell number of adherent HCT116 cells in the presence of UK5099 and alanine (n = 3 culture wells each). t–v, Spheroid biomass in HCT116 (t), MCF7 (u) and A549 (v) cells grown in the presence or absence of 0.4 mM serine, 0.4 mM glycine, 1 mM alanine and 1 mM formate (n = 3 culture wells for each cell line and condition). Two-sided Student’s t-test (b–i, n, o), one-way ANOVA (t–v) or two-way ANOVA (j–m, p–s) was performed for each comparison, with no adjustment for multiple comparison. Similar results obtained in two (d, e, h, p, r), three (c, f, g, i, n, o, q), or four (b) independent experiments. Data are mean ± s.e.m. *P 
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