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The unique catalytic properties of PSAT1 mediate metabolic adaptation to glutamine blockade

Nature Metabolism volume 6, pages 1529–1548 (2024)Cite this article

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Abstract

Cultured cancer cells frequently rely on the consumption of glutamine and its subsequent hydrolysis by glutaminase (GLS). However, this metabolic addiction can be lost in the tumour microenvironment, rendering GLS inhibitors ineffective in the clinic. Here we show that glutamine-addicted breast cancer cells adapt to chronic glutamine starvation, or GLS inhibition, via AMPK-mediated upregulation of the serine synthesis pathway (SSP). In this context, the key product of the SSP is not serine, but α-ketoglutarate (α-KG). Mechanistically, we find that phosphoserine aminotransferase 1 (PSAT1) has a unique capacity for sustained α-KG production when glutamate is depleted. Breast cancer cells with resistance to glutamine starvation or GLS inhibition are highly dependent on SSP-supplied α-KG. Accordingly, inhibition of the SSP prevents adaptation to glutamine blockade, resulting in a potent drug synergism that suppresses breast tumour growth. These findings highlight how metabolic redundancy can be context dependent, with the catalytic properties of different metabolic enzymes that act on the same substrate determining which pathways can support tumour growth in a particular nutrient environment. This, in turn, has practical consequences for therapies targeting cancer metabolism.

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Fig. 1: Breast cancer cells adapt to glutamine blockade and develop altered NEAA dependencies.
Fig. 2: The SSP is upregulated in glutamine-independent breast cancer cells.
Fig. 3: The SSP is required for adaptation to glutamine blockade.
Fig. 4: PSAT1 permits sustained α-KG production during glutamine blockade.
Fig. 5: SSP inhibition collapses α-KG levels in CB-839-resistant breast cancer cells.
Fig. 6: Combination therapy targeting GLS and the SSP synergistically suppresses breast cancer growth.
Fig. 7: AMPK signalling drives PHGDH upregulation upon glutamine blockade.

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Data availability

The RNA-seq data are deposited in the Gene Expression Omnibus (GEO) database under accession number GSE263696. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Lukey Laboratory, our colleagues in the Demerec building and C. Thompson for helpful discussions and insights. We are grateful to C. Amor Vegas for providing advice and reagents for senescence staining. We also thank the Cold Spring Harbor Laboratory (CSHL) Cancer Center Animal, Mass Spectrometry, Next-Generation Sequencing and Organoid Shared Resources, which are funded in part by a National Institutes of Health Cancer Center Support Grant (5P30CA045508). This work was supported by grants from the Department of Defense Breast Cancer Research Program (BC200599), National Institutes of Health (R01GM149957 and 5P30CA045508), METAvivor, Simons Foundation and The Elsa U. Pardee Foundation to M.J.L.; the Leslie C. Quick, Jr. Fellowship from the CSHL School of Biological Sciences to J.d.R.S.; National Cancer Institute (5P01CA013106-Project 3) to D.L.S.; and National Institutes of Health NIAID R25 training grant (AI140472) to J.R.C. Schematic images were created with BioRender.com.

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Author notes

  1. Ana S. H. Costa

    Present address: Matterworks Inc., Somerville, MA, USA

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Yijian Qiu, Olivia T. Stamatatos, Qingting Hu, Jed Ruiter Swain, Suzanne Russo, Ava Sann, Ana S. H. Costa, David L. Spector & Michael J. Lukey

  2. Graduate Program in Genetics, Stony Brook University, Stony Brook, NY, USA

    Qingting Hu

  3. CSHL School of Biological Sciences, Cold Spring Harbor, NY, USA

    Jed Ruiter Swain

  4. Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Sara Violante & Justin R. Cross

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Contributions

Y.Q., O.T.S., Q.H. and J.d.R.S. generated and analysed the majority of data. A.S., A.S.H.C., S.V. and J.R.C. performed MS and data analysis for metabolomics. D.L.S. and S.R. provided patient-derived breast cancer organoids. M.J.L. directed the work, interpreted the data and drafted the manuscript, with input from all authors.

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Correspondence to Michael J. Lukey.

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Nature Metabolism thanks Richard Possemato, Mercedes Tome and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Short tandem repeat (STR) profiling.

a, STR profiling report for parental MDA-MB-231 cells. b, STR profiling report for CB839RS MDA-MB-231 cells. c, STR profiling report for GlnIND MDA-MB-231 cells. CB839RS, CB-839-resistant; GlnIND, glutamine-independent.

Extended Data Fig. 2 NEAA dropout analysis.

a, Sensitivity of BT-549 cells to 6 days deprivation of individual NEAAs. Cells were cultured in complete media (parental) or glutamine-free media (GlnIND) lacking individual NEAAs. Viable cell counts under control conditions were set as 100%. Red triangle, dependence unique to GlnIND cells. Green triangle, dependence unique to parental cells. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. b, NEAA sensitivity analysis as in panel ‘a’, but with MDA-MB-231 cells cultured in complete media (parental) or complete media supplemented with 500 nM CB-839 (CB839RS). Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Parental MDA-MB-231 data are shared between Fig. 1i and Extended Data Fig. 2b for ease of comparison. CB839RS, CB-839 resistant; NA, not applicable; NEAA, nonessential amino acids.

Source data

Extended Data Fig. 3 Sequence of GLS protein based on cDNA sequencing data.

The amino acid sequences are translated from sequencing data of full-length GLS cDNA, prepared from parental, GlnIND, and CB839RS MDA-MB-231 cells. The gray box marks a flexible loop located at the dimer-dimer interface of the tetrameric forms of GLS, the binding site of CB-839. CB839RS, CB-839-resistant; GlnIND, glutamine-independent; GLS, glutaminase.

Extended Data Fig. 4 Western blot analysis of adapted and modified breast cancer cells.

a, Western blot analysis of indicated proteins in parental, CB839RS, and GlnIND MDA-MB-231 whole cell lysates (upper panels). [U-13C6]-glucose stable-isotope tracing data for de novo synthesized serine (m + 3) after 16 h (lower panels). Data are presented as mean values ± SD, n = 3 biological replicates. b, Western blot analysis of PHGDH in parental and adapted GlnIND breast cancer cell lines. c, Pearson correlation coefficient analysis of CB-839 sensitivity and serine dependence across cell lines. A value of 1000 nM CB-839 is shown when IC50CB-839 > 1000 nM. Each data point represents the mean from 3 independent experiments. This is a repeat of Fig. 2f, but with the cell lines labeled. d, Sensitivity of BT-549 cells to NCT-503 (upper panel) and BI-4916 (lower panel). Parental cells cultured in complete medium and GlnIND cells in glutamine-free medium. Data are presented as mean values ± SD, n = 3 biological replicates. e, Western blot validation of knockdown of PHGDH or PSAT1 by independent shRNAs. f, Growth of BT-549 cells harboring a control vector, a PHGDH shRNA vector, or a PSAT1 shRNA vector. Parental and GlnIND cells cultured in complete medium or glutamine-free medium, respectively for 6 days. Viable control cell count in each condition was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. *** P ≤ 0.001; NS, not significant. g, Western blot validation of knockdown of PHGDH or PSAT1 by shRNAs. Tubulin control blots in Fig. 2b, c are shared with Extended Data Fig. 4a because the data were obtained from the same samples. Parental BT-549 NCT-503 dose curve is shared between Fig. 3e and Extended Data Fig. 4d, for different comparisons. CB839RS, CB-839-resistant; GlnIND, glutamine-independent; GLS, glutaminase; GLS2, glutaminase 2; GLUL, glutamine synthetase; PHGDH, D-3-phosphoglycerate dehydrogenase; PSAT1, phosphoserine aminotransferase 1; PSPH, phosphoserine phosphatase; xCT, cystine/glutamate antiporter.

Source data

Extended Data Fig. 5 α-KG is the key SSP product for glutamine-independent growth.

a, Western blot validation of CRISPR/Cas9-mediated knockout of PHGDH or PSAT1 using independent sgRNAs. b, Sensitivity of breast cancer cell lines to 6 days BI-4916 treatment. Data are presented as mean values ± SD, n = 3 biological replicates. c, Western blot validation of knockdown of PHGDH or PSAT1 by independent shRNA constructs. d, Western blot validation of knockdown of PSPH by shRNA. e, Sensitivity of adapted Hs 578T cells to 6 days CB-839 treatment. Data are presented as mean values ± SD, n = 3 biological replicates. f, Growth of GlnIND BT-549 cells treated with 12 µM NCT-503 in glutamine-free medium supplemented with dimethyl α-KG (500 µM) or serine at the indicated concentrations over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. ** P ≤ 0.01; NS, not significant. g, Serine supplementation fails to rescue the growth of CB839RS Hs 578T cells treated with 12 µM NCT-503 or 2 µM BI-4916, in medium also containing 500 nM CB-839, over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. *** P ≤ 0.001; NS, not significant. h, Dimethyl α-KG supplementation rescues the growth of CB839RS Hs 578T cells treated with 12 µM NCT-503 or 2 µM BI-4916, in medium also containing 500 nM CB-839, over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. i, Growth of adapted BT-549 cells in glutamine-free medium over 6 days. Dimethyl α-KG was used at 500 µM. Viable control cell count in the absence of dimethyl α-KG was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. ** P ≤ 0.01; *** P ≤ 0.001; NS, not significant. j, Glutamate supplementation fails to rescue the growth of CB839RS Hs 578T cells treated with 12 µM NCT-503 or 2 µM BI-4916, in medium also containing 500 nM CB-839, over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. *** P ≤ 0.001; NS, not significant. k, Supplementation with 500 µM dimethyl α-KG, 4 mM serine, or 4 mM glutamate leads to increased intracellular abundance of these metabolites in GlnIND MDA-MB-231 cells. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. *** P ≤ 0.001. Parental MDA-MB-231 BI-4916 dose curve is shared between Fig. 3a, b and Extended Data Fig. 5b, and parental BT-549 BI-4916 dose curve is shared between Extended Data Fig. 4d and Extended Data Fig. 5b, for different comparisons. Control and NCT-503 only (no supplementation) data are shared between Extended Data Fig. 5g, h because the data were obtained from the same experiment. Control and BI-4916 only data are shared between Extended Data Fig. 5g, h, j because the data were obtained from the same experiment. CB839RS, CB-839-resistant; GlnIND, glutamine-independent; SSP, serine synthesis pathway; DMα-KG, dimethyl α-ketoglutarate; PHGDH, D-3-phosphoglycerate dehydrogenase; PSPH, phosphoserine phosphatase.

Source data

Extended Data Fig. 6 Kinetic analysis of human PSAT1, GOT2, and GPT2.

a, Representative Lineweaver–Burk plot of the inverse initial velocity (1/V0) as a function of the inverse of the glutamate concentration (1/[S]) for PSAT1. b, Representative Lineweaver–Burk plot of the inverse initial velocity (1/V0) as a function of the inverse of the glutamate concentration (1/[S]) for GOT2. c, Representative Lineweaver–Burk plot of the inverse initial velocity (1/V0) as a function of the inverse of the glutamate concentration (1/[S]) for GPT2. d, Representative Lineweaver–Burk plot of the inverse initial velocity (1/V0) as a function of the inverse of the 3-PHP concentration (1/[S]) for PSAT1. PSAT1, phosphoserine aminotransferase 1, GOT2, glutamic-oxaloacetic transaminase 2; GPT2, glutamic–pyruvic transaminase 2; 3-PHP, 3-phosphohydroxypyruvate; Km, Michaelis constant; V0, initial velocity, [S], substrate concentration.

Extended Data Fig. 7 SSP inhibition suppresses de novo serine biosynthesis.

a, [U-13C6]-glucose stable-isotope tracing data showing the effect of 16 h treatment with 12 μM NCT-503 on de novo synthesized serine (m + 3) in parental and CB839RS MDA-MB-231 cells. Data are presented as mean values ± SD, n = 4 biological replicates. Schematic shows incorporation of labeled carbons from glucose into serine and glycine via the SSP. b, [U-13C6]-glucose stable-isotope tracing data showing the effect of 16 h treatment with 10 μM BI-4916 on de novo synthesized serine (m + 3) in parental and GlnIND cells. Data are presented as mean values ± SD, n = 3 biological replicates. CB839RS, CB-839 resistant derivative; GlnIND, glutamine-independent; 3-PG, 3-phosphoglycerate; PHGDH, D-3-phosphoglycerate dehydrogenase.

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Extended Data Fig. 8 CB-839 synergizes with PHGDH inhibitors against breast cancer.

a, Drug combination analysis using the Chou-Talalay method to score drug interactions in breast cancer cell lines. b, Drug combination analysis using the Chou-Talalay method to score drug interactions in patient-derived TNBC organoids HCM-CSHL-0366-C50, NH85T, NH93T. Data are presented as mean values ± SD, n = 3 biological replicates.

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Extended Data Fig. 9 Combination therapy targeting GLS and the SSP suppresses breast cancer growth.

a, Body weight measurement of tumor-bearing mice in the indicated treatment groups. n = 6 (MDA-MB-231) or n = 7 (BT-549) biological replicates, error bars represent SEM. b, Growth of control and PSAT1-knockout MDA-MB-231 orthotopic breast tumor xenografts in NU/J mice administered the indicated treatments (left panel), and body weight measurements of tumor-bearing mice under the indicated treatment (right panel). Data are presented as mean values ± SEM, n = 3 biological replicates. Two-tailed unpaired t-test. ** P ≤ 0.01. c, Western blot analysis of PSAT1 in cultured cells and orthotopic breast tumor xenografts harvested from untreated or CB-839 treated mice. PSAT1, phosphoserine aminotransferase 1.

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Extended Data Fig. 10 AMPK signaling mediates PHGDH upregulation upon glutamine blockade.

a, Western blot analysis of ATF4 and NRF2 in nuclear fractions of parental and GlnIND MDA-MB-231 cells (left panel). Western blot analysis of HIF-1α in whole-cell lysates (right panel). BAY-3827 was used at 5 µM and samples collected at 48 h. b, Western blot analysis of lysates of parental and GlnIND BT-549 cells cultured under the indicated conditions. BAY-3827 was used at 5 µM and samples collected at 48 h. c, Western blot analysis of cultured BT-549 cells and BT-549 orthotopic breast tumor xenografts, harvested from untreated or CB-839-treated mice. d, Western blot analysis of lysates of the indicated breast cancer cell lines and patient-derived TNBC organoids. The Tubulin control blot is shared between Fig. 6c and Extended Data Fig. 10c because the data were obtained from the same experiment.

Supplementary information

Reporting Summary

Supplementary Table 1

Breast cancer cell lines used in this study, with their receptor status, molecular subtype and IC50 value for CB-839 indicated.

Supplementary Table2

Sequences of primers used in this study.

Supplementary Table 3

Gene expression data from RNA-seq analysis showing differentially expressed genes between parental and GlnIND MDA-MB-231 cells.

Supplementary Table 4

Antibodies used in this study, with dilutions indicated.

Supplementary Table 5

Plasmids generated and/or used in this study.

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Qiu, Y., Stamatatos, O.T., Hu, Q. et al. The unique catalytic properties of PSAT1 mediate metabolic adaptation to glutamine blockade. Nat Metab 6, 1529–1548 (2024). https://doi.org/10.1038/s42255-024-01104-w

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