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Glucose limitation protects cancer cells from apoptosis induced by pyrimidine restriction and replication inhibition

Nature Metabolism volume 6, pages 2338–2353 (2024)Cite this article

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Abstract

Cancer cells often experience nutrient-limiting conditions because of their robust proliferation and inadequate tumour vasculature, which results in metabolic adaptation to sustain proliferation. Most cancer cells rapidly consume glucose, which is severely reduced in the nutrient-scarce tumour microenvironment. In CRISPR-based genetic screens to identify metabolic pathways influenced by glucose restriction, we find that tumour-relevant glucose concentrations (low glucose) protect cancer cells from inhibition of de novo pyrimidine biosynthesis, a pathway that is frequently targeted by chemotherapy. We identify two mechanisms to explain this result, which is observed broadly across cancer types. First, low glucose limits uridine-5-diphosphate-glucose synthesis, preserving pyrimidine nucleotide availability and thereby prolonging the time to replication fork stalling. Second, low glucose directly modulates apoptosis downstream of replication fork stalling by suppressing BAK activation and subsequent cytochrome c release, key events that activate caspase-9-dependent mitochondrial apoptosis. These results indicate that the low glucose levels frequently observed in tumours may limit the efficacy of specific chemotherapeutic agents, highlighting the importance of considering the effects of the tumour nutrient environment on cancer therapy.

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Fig. 1: Metabolism-focused CRISPR screen reveals resistance of cancer cells to loss of pyrimidine biosynthesis genes in low glucose.
Fig. 2: Low glucose is protective against the consequences of inhibition of de novo pyrimidine synthesis.
Fig. 3: The effect of low glucose on pyrimidine availability is independent of pyrimidine salvage activity.
Fig. 4: Glucose shunts UTP into the UDP-glucose synthesis pathway, increasing replication stress upon DHODH inhibition.
Fig. 5: Low glucose blocks apoptosis downstream of replication fork stalling.
Fig. 6: Low glucose-mediated protection from the consequences of pyrimidine nucleotide depletion in cancer cells.

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All data needed to interpret, verify and extend the research discussed in the article are available in the main text, supplementary materials or the source data. Source data are provided with this paper.

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Acknowledgements

We thank H. An and J. W. Harper for reagents and advice on the Keima system, D. Jones for assistance with the metabolomics measurements and D. Green, K. Birsoy, H. D. Ryoo and the Possemato lab members for helpful discussion and advice. plentiCRISPRv2 and lentiCas9-Blast were a gift from F. Zhang (research resource identifier (RRID): Addgene_52961, Addgene_52962). psPAX2 and pMD2.G were a gift from D. Trono (RRID: Addgene_12260, Addgene_12259). BPK1520 was a gift from K. Joung (RRID: Addgene_65777). pDONR223_CFLAR_WT_V5 was a gift from J. Boehm, M. Meyerson and D. Root (RRID: Addgene_82936). pCW57-MCS1-P2A-MCS2 (Blast) was a gift from A. Karpf (RRID: Addgene_80921). Funding was provided by the National Institutes of Health (grant nos. R01CA286141, R01CA214948 and R01GM132491 to R.P., and grant no. R35GM139610 to T.T.H.), and the Pew Charitable Trusts, Alexander and Margaret Stewart Trust and the American Cancer Society (all to R.P.). A Cancer Center Support Grant (no. P30CA016087) provides funding to the metabolomics shared resource.

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Authors and Affiliations

  1. Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA

    Minwoo Nam, Abdul Hannan Mir & Richard Possemato

  2. Laura & Isaac Perlmutter Cancer Center, New York, NY, USA

    Minwoo Nam, Wenxin Xia, Abdul Hannan Mir, Tony T. Huang & Richard Possemato

  3. Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY, USA

    Wenxin Xia & Tony T. Huang

  4. University of Massachusetts Chan Medical School, Worcester, MA, USA

    Alexandra Jerrett & Jessica B. Spinelli

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Contributions

R.P. and M.N. conceptualized the study. M.N. curated the data. R.P. acquired the funding. M.N., A.H.M., W.X., A.J., J.B.S. and T.T.H. carried out the investigation. R.P. and M.N. devised the methodology. R.P. supervised the study. R.P. and M.N. wrote the original manuscript draft. R.P. and M.N. reviewed and edited the manuscript.

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Correspondence to Richard Possemato.

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Nature Metabolism thanks Cristina Munoz-Pinedo, Juan Manuel Schvartzman 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 The effects of pyrimidine/purine synthesis inhibitors and aminopterin on proliferation of cancer cells.

a,b, Dose-response curves for proliferation of Jurkat (a) and Raji (b) cells treated with brequinar or PALA in high and low glucose. c, Uridine-mediated rescue of proliferative defects of Jurkat cells treated with brequinar or PALA. n = 3 biological replicates. d,e, Determination of the IC50 of mizoribine (n = 4 independent experiments in d, n = 3 in e), MPA (n = 5 in d, n = 3 in e) and aminopterin (n = 3) in Jurkat (d) and Raji (e) cells in high and low glucose. f,g, Dose-response curves for proliferation of Jurkat (f) and Raji (g) cells treated with mizoribine, MPA or aminopterin. Data are mean ± SEM except (c; mean ± SD). Statistical significance is determined by two-sided Student’s t-test (d-e), ns: not significant.

Source data

Extended Data Fig. 2 The effects of pyrimidine/purine synthesis inhibitors on metabolism and DNA synthesis of cancer cells.

a,b, LC-MS-based measurement of metabolites in Jurkat cells (a) or Raji cells (b) treated with brequinar (400 nM) for 24 hr in high and low glucose, n = 4 biological replicates. c, Illustrations of glutamine tracer-labeled purine bases (left) and LC-MS-based assessment of fractional labeling of purine nucleotides (right), n = 4 biological replicates. d, Cell cycle profile of Jurkat cells cultured under high and low glucose conditions for 24 hr, n = 3 biological replicates. e,f, Assessment of replication fork dynamics using DNA fiber assay in Raji cells treated with brequinar for 24 hr (n = 200 replication forks, 2 independent experiments). Data are mean ± SEM except (e; mean ± SD). Statistical significance is determined by 2-way ANOVA (d) or Mann-Whitney test (e). ns: not significant.

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Extended Data Fig. 3 Glucose restriction specifically represses replication stress and apoptosis caused by pyrimidine synthesis inhibition.

a, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with brequinar ± uridine in high and low glucose for 48 hr. b, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with brequinar (400 nM), RTX (20 nM) or HU in high and low glucose for 48 hr. c, Immunoblot analysis of replication stress and apoptosis in Jurkat cells co-treated with RTX (10 nM) and multiple doses of 3-DAU for 48 hr. d, Proliferation of Jurkat cells following RTX washout (left) and immunoblot analysis of replication stress and apoptosis in those cells (right), n = 3 biological replicates. e, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with brequinar (400 nM), RTX (10 nM), HU (0.5 mM), or aphidicolin (2 μg/ml) for 48 hr in HPLM containing high or low glucose. f, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with MPA ± 50 μM guanosine (left) or 20 μM MPA ± 50 μM Z-VAD (right) in high and low glucose for 48 hr. g, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with actinomycin D (ActD) ± Z-VAD (50 μM) in high and low glucose for 48 h. h, Colonies formed from RTX-treated HCT116 cells in high and low glucose. i,j, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with etoposide (1.5 μM), doxorubicin (100 nM), cisplatin (5 μg/ml) for 24 hr (i) or aphidicolin for 48 h (j) in high and low glucose. Statistical significance is determined by two-sided Student’s t-test (d). ns: not significant.

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Extended Data Fig. 4 Characterization of Jurkat-RPS3-Keima and Jurkat-UCK tKO cell lines.

a. PCR confirmation of RPS3-Keima allele (left) and immunoblot of FLAG-tagged RPS3-Keima protein (right). b, Immunoblot analysis of Keima processing in Jurkat-RPS3-Keima cells treated with brequinar (300 nM), RTX (10 nM) or Torin1 (150 nM). *RPS3-Keima, **processed Keima. c, Proliferation of UCK2 knockout or UCK1/2 double knockout Jurkat cells treated with brequinar ± uridine (U, 50 μg/ml), n = 3 biological replicates. d, Proliferation of Jurkat-UCK tKO clones treated with PALA (150 μM) ± uridine (50 μg/ml), n = 3 biological replicates. Data are mean ± SD.

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Extended Data Fig. 5 The phenotypes of UGP2 deficiency are reversed by reintroduction of UGP2 cDNA.

Population doubling of Jurkat UGP2 KO and UGP2 cDNA rescue (coUGP2) cells. Data are mean ± SEM.

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Extended Data Fig. 6 Assessment of apoptotic pathways activated by pyrimidine synthesis inhibitors.

a. Immunoblot analysis of PARP, cleaved caspases-8/−9 and BID in Jurkat cells treated brequinar, PALA or RTX ± thymidine (dT, 10 μM) or uridine (50 μg/ml). b, Immunoblot analysis of replication stress and apoptosis in Jurkat cells upon FAS activation (anti-Fas antibody 50 ng/ml) ± Z-VAD (50 μM) in high and low glucose. c, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with staurosporine (STS) ± Z-VAD (50 μM) for 12 hr in high and low glucose. d. Immunoblot analysis of replication stress and apoptosis in Jurkat cells cultured in conditioned media collected from cells treated with the indicated inhibitors or fresh media containing brequinar (B), RTX (R) or anti-Fas antibody (F) for 24 hr. Uridine (50 μg/ml) or thymidine (dT, 10 μM) were added following media collection. e, Immunoblot analysis of cFLIP, caspase-8 and apoptosis in Jurkat-FLAG-cFLIP cells treated with anti-Fas antibody (50 ng/ml). f, Immunoblot analysis of cFLIP, caspase-8, replication stress and apoptosis in Jurkat-FLAG-cFLIP cells treated with brequinar (B, 400 nM) or RTX (R, 10 nM) for 48 hr in high and low glucose. g, Immunoprecipitation of FLAG-cFLIP::procaspase-8::FADD complexes in Jurkat-FLAG-cFLIP cells treated with RTX (25 nM) for 48 hr. Z-VAD (50 μM) was added to stabilize FLAG-cFLIP::procaspase-8::FADD complexes. h, Flow cytometric analysis of DNA fragmentation (DAPI, upper) and TOM20 (lower) in Jurkat cells treated with RTX (25 nM) for 48 hr in high and low glucose. i, Flow cytometric analysis of cytochrome c (CYCS) release (left) and immunoblot analysis of BAK, replication stress and apoptosis (right) in Jurkat BAK KO cells treated with brequinar (B, 400 nM), RTX (R, 25 nM for flow cytometry; 10 nM for immunoblot) or aphidicolin (A, 4 μg/ml for flow cytometry; 5 μg/ml for immunoblot) for 48 hr. j, Immunoblot analysis of tBID, replication stress and apoptosis in Jurkat cells with DOX-inducible expression of tBID in high and low glucose. Cells were treated with 500 ng/ml doxycycline for the indicated time. k, Immunoblot analysis of BAK1, tBID and apoptosis in Jurkat BAK1 KO cells treated with ABT-263 (5 μM for 24 hr), staurosporine (250 nM for 4 hr) or expressing DOX-inducible tBID (0.5 μg/ml DOX for 6 hr). l,m, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with 200 nM brequinar ± AZD6738 (ATRi, l) or MK8776 (Chk1i, m) in high and low glucose for 48 hr.

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Nam, M., Xia, W., Mir, A.H. et al. Glucose limitation protects cancer cells from apoptosis induced by pyrimidine restriction and replication inhibition. Nat Metab 6, 2338–2353 (2024). https://doi.org/10.1038/s42255-024-01166-w

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