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α-Ketoglutarate links p53 to cell fate during tumour suppression

Nature volume 573, pages 595–599 (2019)Cite this article

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Abstract

The tumour suppressor TP53 is mutated in the majority of human cancers, and in over 70% of pancreatic ductal adenocarcinoma (PDAC)1,2. Wild-type p53 accumulates in response to cellular stress, and regulates gene expression to alter cell fate and prevent tumour development2. Wild-type p53 is also known to modulate cellular metabolic pathways3, although p53-dependent metabolic alterations that constrain cancer progression remain poorly understood. Here we find that p53 remodels cancer-cell metabolism to enforce changes in chromatin and gene expression that favour a premalignant cell fate. Restoring p53 function in cancer cells derived from KRAS-mutant mouse models of PDAC leads to the accumulation of α-ketoglutarate (αKG, also known as 2-oxoglutarate), a metabolite that also serves as an obligate substrate for a subset of chromatin-modifying enzymes. p53 induces transcriptional programs that are characteristic of premalignant differentiation, and this effect can be partially recapitulated by the addition of cell-permeable αKG. Increased levels of the αKG-dependent chromatin modification 5-hydroxymethylcytosine (5hmC) accompany the tumour-cell differentiation that is triggered by p53, whereas decreased 5hmC characterizes the transition from premalignant to de-differentiated malignant lesions that is associated with mutations in Trp53. Enforcing the accumulation of αKG in p53-deficient PDAC cells through the inhibition of oxoglutarate dehydrogenase—an enzyme of the tricarboxylic acid cycle—specifically results in increased 5hmC, tumour-cell differentiation and decreased tumour-cell fitness. Conversely, increasing the intracellular levels of succinate (a competitive inhibitor of αKG-dependent dioxygenases) blunts p53-driven tumour suppression. These data suggest that αKG is an effector of p53-mediated tumour suppression, and that the accumulation of αKG in p53-deficient tumours can drive tumour-cell differentiation and antagonize malignant progression.

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Fig. 1: p53 restoration increases the ratio of cellular αKG to succinate, independently of changes in proliferation.
Fig. 2: αKG recapitulates gene-expression changes that are induced by the restoration of p53.
Fig. 3: p53 restoration and OGDH inhibition both promote tumour-cell differentiation and tumour suppression.
Fig. 4: p53 status and cellular αKG/succinate ratio dictates levels of 5hmC in mouse models of PDAC.

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Data availability

RNA-seq and ATAC-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession codes GSE114263 (ATAC-seq) and GSE114342 (RNA-seq), respectively. ChIP-seq data that were reanalysed for this study are available under the accession code GSE46240. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank members of the Lowe and Finley laboratory. Pancreatic intraepithelial neoplasia and PDAC tissue microarrays were generated by G. Askan and O. Basturk, and were generously provided as a resource from the David M. Rubenstein Center for Pancreatic Cancer Research. J.P.M. IV was supported by an American Cancer Society Postdoctoral Fellowship (126337-PF-14-066-01-TBE). J.J.Y. is supported by a T32 training grant from the NICHD (T32HD060600). F.J.S.-R. is an HHMI Hanna Gray Fellow and was partially supported by an MSKCC Translational Research Oncology Training Fellowship (NIH T32-CA160001). S.D.L. is supported by NIH grant (NIH R01CA204228). R.C. is supported by an American Association for Cancer Research/Pancreatic Cancer Action Network Pathway to Leadership Award. T.B. is supported by the William C. and Joyce C. O'Neil Charitable Trust and Memorial Sloan Kettering Single Cell Sequencing Initiative. C.C.-F is supported by NIH grant (NIH R00CA191021). L.W.S.F. is a Dale F. Frey-William Raveis Charitable Fund Scientist supported by the Damon Runyon Cancer Research Foundation (DFS-23-17) and a Searle Scholar. S.W.L. is an investigator in the Howard Hughes Medical Institute. This work was additionally supported by a Lustgarten Research Investigator Award (S.W.L.), a Program Program Project grant from the National Cancer Institute (S.W.L.), research grants from the Emerson Collective (S.W.L.), the Starr Cancer Consortium (I11-039 and I12-0051 L.W.S.F.), the Concern Foundation (L.W.S.F.), the Anna Fuller Fund (L.W.S.F.) and the Memorial Sloan Kettering Cancer Center Support Grant P30 CA008748.

Author information

Author notes

  1. Steven D. Leach

    Present address: Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA

  2. These authors contributed equally: John P. Morris IV, Jossie J. Yashinskie

Authors and Affiliations

  1. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    John P. Morris IV, Sha Tian, Chi-Chao Chen, Timour Baslan, Francisco J. Sánchez-Rivera, Craig B. Thompson & Scott W. Lowe

  2. Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Jossie J. Yashinskie & Lydia W. S. Finley

  3. Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Jossie J. Yashinskie, Richard Koche, Craig B. Thompson & Lydia W. S. Finley

  4. Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY, USA

    Jossie J. Yashinskie

  5. Department of Surgery, Weill Cornell Medical College, New York, NY, USA

    Rohit Chandwani

  6. Rubinstein Center for Pancreatic Cancer Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Rohit Chandwani & Steven D. Leach

  7. Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Rohit Chandwani & Steven D. Leach

  8. Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY, USA

    Rohit Chandwani

  9. Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY, USA

    Zoran S. Marinkovic & Carlos Carmona-Fontaine

  10. Howard Hughes Medical Insitute, Janelia Research Campus, Ashburn, VA, USA

    Scott W. Lowe

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Contributions

J.P.M., L.W.S.F. and J.J.Y. conceived, designed and performed all studies with additional guidance from S.W.L. R.K. analysed ATAC-seq data. Z.S.M. and C.C.-F. analysed immunohistochemistry images. C.-C.C. and T.B. assisted with the RNA-seq analysis. S.D.L. provided the human pancreatic cancer tissue microarray. F.J.S.-R. cloned IDH overexpression constructs and provided tools for sgRNA expression and aided with the analysis of genome editing. C.B.T., S.T. and R.C. provided additional work in conception, data acquisition and experimental design. J.P.M., L.W.S.F. and S.W.L. wrote the manuscript.

Corresponding authors

Correspondence to Lydia W. S. Finley or Scott W. Lowe.

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Competing interests

S.W.L. is a founder and scientific advisory board member of Blueprint Medicines, Mirimus Inc. and ORIC pharmaceuticals, and on the scientific advisory board of Constellation Pharmaceuticals, Petra Pharmaceuticals and PMV Pharmaceuticals. C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He has also previously served on the board of directors of Merck and Charles River Laboratories.

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Peer review information Nature thanks Kathryn E. Wellen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 KPsh embryonic-stem-cell-based genetically engineered mouse model of PDAC, driven by mutant KRAS and inducible and reversible p53 silencing.

a, KPsh embryonic-stem-cell-based genetically engineered mouse model of PDAC. Embryonic stem cells express PDX1–Cre (transgenic expression of Cre in pancreatic progenitors), LSL–KRAS(G12D) (knock-in, conditional heterozygous expression of mutant KRAS), RIK (knock in, conditional heterozygous expression of rtTA and fluorescent mKate2 from the Rosa26 locus) and Col1a1-TRE-GFP-shp53-shRenilla (a COL1A1 homing cassette targeted with doxycycline-inducible tandem shRNA expressing p53 and Renilla linked to GFP). KPsh mice were generated by blastocyst injection and mothers were enrolled on doxycycline chow at day 5. Cell lines were derived and maintained in doxycycline-containing medium, from tumours that arose in doxycycline-fed mice. All KPsh cells constitutively express mKate2 and rtTA. b, Population doublings of KPsh no. 1, KPsh no. 2 and KPsh no. 3 lines grown on or off doxycycline. c–f, Characterization of levels of p53 (c), BrdU incorporation (d), annexin V staining (e) and senescence-associated β-galactosidase staining (f) in three independent KPsh lines grown on or off doxycycline. D, day. g, Representative gross pathology and epifluorescence images of pancreatic tumours that resulted from orthotopic transplant of KPsh no. 2 cells into doxycycline-fed mice maintained on doxycycline chow (top, n = 3 mice) or withdrawn from doxycycline chow for ten days (bottom, n = 3 mice). KPsh cells uniformly express mKate2 (Kate), wherease GFP expression indicates cells that actively express shRNA targeting p53. h, Representative p21 immunostaining in matched normal host pancreas, or in orthotopic KPsh no. 2 tumours maintained on doxycycline (n = 3) or ten days after doxycycline withdrawal (n = 3). mKate2 indicates injected KPsh no. 2 cells. i, Representative Ki67 immunostaining in orthotopic KPsh no. 2 tumours maintained on doxycycline (n = 3 mice) or ten days after doxycycline withdrawal (n = 3 mice). mKate2 indicates injected KPsh no. 2 cells. j, Small animal ultrasound measurement of tumour volume. KPsh no. 2 cells were injected into doxycycline-fed mice and mice were maintained on doxycycline diet for two weeks. After two weeks (D0), tumour size was measured and mice were randomized into off (n = 6 mice) and on doxycycline chow groups (n = 3 mice). Subsequent tumour size was measured at the indicated time points. n = 3 mice on doxycycline were collected for analysis upon being euthanized; n = 3 mice were analysed after 5 and 10 days of doxycycline withdrawal; n = 3 mice were analysed after 5, 10, and 18 days of doxycycline withdrawal. k, Survival of mice shown in i after randomization into groups maintained on doxycycline chow (n = 3 mice) or after doxycycline withdrawal (n = 6 mice). Experiments in b–f were repeated twice with similar results. Data are presented as representative, independently treated wells (c, f) or mean ± s.d. of n = 3 independently treated wells with individual data points shown (b, d, e). For gel source data of c, see Supplementary Fig. 1. Scale bar, 1 cm (g), 50 μm (h, i).

Source Data

Extended Data Fig. 2 p53 restoration increases the αKG/succinate ratio, independently of changes in proliferation or senescence.

a, Glucose and glutamine consumption, and lactate production, in KPsh no. 1 and KPsh no. 2 cells cultured on or off doxycycline for four or eight days. b, Schematic of the TCA cycle, indicating entry points for glucose- and glutamine-derived carbons. Metabolites in red were assessed by isotope-tracing experiments. c, d, Metabolite fraction containing 13C derived from [U-13C]glucose (13C-Glc) (c) or derived from [U-13C]glutamine (13C-Gln) (d) after four hours of labelling in cells grown off or on doxycycline for six days. e, f, p53 immunoblot (e) or senescence-associated β-galactosidase staining (f) in on-doxycycline KPsh no. 2 cells treated with 3 μM etoposide or 25 nM trametinib for 48 or 96 h. Cells grown in the absence of doxycycline for six days are included as a positive control. g, h, Senescence-associated-β-galactosidase- (g) or BrdU-positive (h) cells treated as in e. i, Western blot of cells that express shRNA targeting Renilla luciferase, p19, Cdkn2a or p21, on or off doxycycline for six days. j, Senescence-associated β-galactosidase staining of cells described in i. k, l, Senescence-associated-β-galactosidase- (k) or BrdU-positive (l) cells treated as in i. m, αKG/succinate ratio in cells that express shRNA targeting Renilla luciferase, p19, Cdkn2a or Cdkn1a, on or off doxycycline for six days. Experiments in a, c, d, f–m were repeated twice with similar results, and the experiment in e was performed once. Data are presented as mean ± s.e.m. of n = 6 independently treated wells (a), mean ± s.d. of n = 3 independently treated wells from a representative experiment with individual data points shown (c, d, f–h, k–m), or are representative of 1 independently treated well (e, f, i, j). For gel source data of e, i, see Supplementary Fig. 1. Significance was assessed in comparison to cells grown with doxycycline by one-way ANOVA with Tukey’s multiple comparison post-test (a) or in the indicated comparisons by two-way ANOVA with Sidak’s post-test (m). Scale bar, 50 μm.

Source Data

Extended Data Fig. 3 Characterization of reversibility of p53-dependent effects in KPsh cells.

a, αKG/succinate ratio in KrasG12D;TRE-shRenilla (KRensh) PDAC cells cultured with or without doxycycline for the indicated number of days. b–d, Western blot for p53 (b), population doublings (c) and αKG/succinate ratio (d) in two KPsh lines cultured with or without doxycycline for six days, or cultured without doxycycline for six days followed by six days of culture with doxycycline. The arrow indicates when doxycycline was re-introduced. Experiments in a, c were performed twice with similar results, and experiments in b, d were performed once. For gel source data of), see Supplementary Fig. 1. Data are presented as mean ± s.d. of n = 3 independently treated wells of a representative experiment with individual data points shown (a, c, d), or are representative of 1 independently treated well (b).

Source Data

Extended Data Fig. 4 Functional p53 transactivation is required to increase the cellular αKG/succinate ratio.

a–c, p53 immunoblot (a), p21 qRT–PCR (b) and αKG/succinate ratio (c) in KPflox RIK-TRE-empty, KPflox RIK-TRE-wild-type (WT)p53 (doxycycline-inducible expression, wild-type p53) and KPflox RIK-TRE-p53(TAD1/2M) (doxycycline-inducible expression, p53 with mutations in both transactivation domain 1 and transactivation domain 2) cells, two days off or on doxycycline. d, p21, Mdm2 and p53 (top), and Idh1 and Pcx (bottom) qRT–PCR in KPsh no. 2 cells off or on doxycycline. Day-0 and day-6 Cdkn1a, Mdm2 and p53 values are also shown in Fig. 1a. e, αKG/succinate ratio in KPsh no. 1, KPsh no. 2 and KPsh no. 3 cells grown on or off doxycycline. f, Idh1 and Pcx qRT–PCR in KPflox RIK-TRE-empty, KPflox RIK-TRE-wild type p53, and KPflox RIK-TRE-p53(TAD1/2M) cells, off or on doxycycline for two days. g, Glucose labelling patterns associated with pyruvate carboxylase (PC) activity. IDH1- and pyruvate-carboxylase-dependent reactions are labelled. h, Fractional m + 3 (top) or m + 5 (bottom) labelling of aspartate and citrate in KPsh no. 1, KPsh no. 2 and KPsh no. 3 cells on or off doxycycline for six days after four hours with [U-13C]glucose. i, Idh1 qRT–PCR in KPsh no. 2 cells that express shRNA targeting Renilla luciferase or Idh1, on or off doxycycline for eight days. j, Levels of IDH1 and p53 in KPsh no. 2 cells that express shRNA targeting Renilla luciferase or Idh1, grown on or off doxycycline for eight days. Arrowhead denotes the specific Idh1 band. k, αKG/succinate ratio in KPsh no. 2 cells that express shRNA targeting Renilla luciferase or Idh1, grown on or off doxycycline. l, m, p53 and IDH1 immunoblot (l) and αKG/succinate ratio (m) in KPflox RIK-TRE-empty, KPflox RIK-TRE-wild-type p53 and KPflox RIK-TRE-p53(TAD1/2M) cells that express shRNA targeting Renilla luciferase or Idh1, grown with doxycycline for two days. n, αKG/succinate ratio in parental KPsh no. 2 cells versus KPsh no. 2 cells that express IDH1 or IDH2 cDNA, grown on doxycycline. Experiments in a–e, h–n were repeated twice with similar results. Data are presented as representative of 1 independently treated well (a, j, l) or as mean ± s.d. of n = 3 independently treated wells of a representative experiment with individual data points shown (b–f, h, i, k, m, n). For gel source data of a, j, l, see Supplementary Fig. 1. Significance was assessed compared to cells grown on doxycycline by one-way ANOVA with Sidak’s multiple comparisons post-test (e) or indicated comparisons (f).

Source Data

Extended Data Fig. 5 p53 binding at Pcx and Idh1.

a. Analysis of ChIP-seq signal at the Pcx locus in primary wild-type p53 and p53-null (KO) mouse embryonic fibroblasts after treatment with doxorubicin. b. Analysis of ChIP-seq signal at the Idh1 locus in primary wild-type p53 and p53-null mouse embryonic fibroblasts after treatment with doxorubicin. p53 binding sites predicted by model-based analysis of ChIP-seq (MACs) comparison of immunoprecipitation samples with input. Response elements predicted by Homer analysis, as described in ‘Epigenomic analysis’ in Methods. ChIP-seq data are from ref. 61.

Extended Data Fig. 6 Increasing the level of intracellular αKG phenocopies effects of p53 reactivation on gene expression.

a, Mean fold change (expressed in log2) of all genes following p53 reactivation or treatment with cell-permeable αKG in n = 2 independently treated wells of KPsh no. 1 cells. All samples treated with equal amounts of DMSO (vehicle). Spearman correlation r = 0.556, P < 1 × 10-15. b, c, qRT–PCR of genes upregulated with both p53 restoration and treatment with αKG in KPsh no. 1 (b) and KPsh no. 2 (c) cells treated for 72 h with vehicle (Veh), dimethyl-αKG (DM-αKG), diethyl-αKG (DE-αKG) or after p53 restoration (without doxycycline for eight days). d, qRT–PCR of genes associated with pancreatic intraepithelial neoplasia cells in KPsh no. 1 cells, grown on or off doxycycline and treated with 4 mM sodium acetate for 72 h. e, OGDH immunoblot in p53-null KPflox RIK or p53-mutant KP(R172H) RIK cells that express shRNA targeting Ogdh for four days. f, Doubling time (days 1 to 4) of KPflox RIK and KP(R172H) RIK cells that express doxycycline-inducible shRNAs targeting Ogdh or Renilla luciferase. g, h, Percentage of senescence-associated β-galactosidase-positive (g) or annexin-V-positive (h) KPflox RIK and KP(R172H) RIK cells that express the indicated shRNA, off or on doxycycline for four days. Etoposide (96 h, 3 μM) was included as a positive control. i, αKG/succinate ratio of KPC(R172H) RIK cells that express doxycycline-inducible shRNAs targeting Ogdh or Renilla luciferase, grown for four days off or on doxycycline. j, qRT–PCR of genes that are co-regulated by p53 and αKG in KP(R172H) RIK cells that express shRNA targeting Ogdh, compared to control cells that express shRNA targeting Renilla luciferase. Experiments in b–d, i, j were repeated twice with similar results and the experiment in e was performed once. Experiments in f–h were repeated in two additional lines. For gel source data of e, see Supplementary Fig. 1. Data are presented as individual data points (b, c, j), mean ± s.d. (d, f, g, h), mean ± s.e.m. (i) or as a representative image (e) of n = 3 independently treated wells of a representative experiment, with individual data points shown. Significance was assessed in the indicated comparisons by two-way ANOVA with Sidak’s multiple comparison post test (i) or compared to cells that express shRNA targeting Renilla luciferase by one-way ANOVA with Tukey’s multiple comparison post-test (g, h).

Source Data

Extended Data Fig. 7 Depletion of OGDH induces differentiation and decreases tumour growth.

a, Representative CK19 staining of orthotopic tumours derived from KPsh no. 2 cells, injected in mice on doxycycline (n = 3 mice) or ten days after doxycycline withdrawal (n = 3 mice). mKate2 marks the injected KPsh no. 2 cells. b, Representative gross images of orthotopic tumours derived from KPflox cells, 12 days after injection of cells that express shRNA targeting Renilla luciferase (n = 5 mice), Ogdh (n = 4 mice) or Sdha (n = 4 mice) into doxycycline-fed mice. GFP indicates cells expressing shRNA. c, Small animal ultrasound measurement of tumours derived from KPflox cells expressing doxycycline-inducible shRNA targeting Renilla luciferase (n = 4 mice), Ogdh (Ogdh shRNA no. 1, n = 4 mice, Ogdh shRNA no. 2, n = 3 mice),or Sdha (n = 4 mice), after enrolling on doxycycline chow. d, Representative H & E staining of orthotopic tumours derived from KPflox cells, nine days after expression of doxycycline-inducible shRNA targeting Renilla luciferase, Ogdh or Sdha in established tumours from c. GFP marks the injected KPflox cells that express the indicated shRNA. e, Representative CK19 staining of orthotopic tumours derived from KPflox cells, expressing doxycycline-inducible shRNA targeting Renilla luciferase, Ogdh or Sdha from Fig. 3b. GFP marks the injected KPflox cells that express the indicated shRNA. f, Representative CK19 staining of orthotopic tumours derived from KPflox cells, nine days after the expression of doxycycline-inducible shRNA targeting Renilla luciferase, Ogdh or Sdha in established tumours from c. GFP marks the cells that express the indicated shRNA. g, Western blot (top) of SDHA in KPflox cells that express doxycycline-inducible shRNA targeting Renilla luciferase or Sdha, grown with doxycycline for four days. The αKG/succinate ratio (bottom) in KPflox cells that express doxycycline-inducible shRNA targeting Renilla luciferase or Sdha, grown with doxycycline for four days. For c, data are presented as mean ± s.d.; individual longitudinally tracked tumour volumes are presented in the Source Data. For gel source data of g, see Supplementary Fig. 1. The experiment in g was performed once, and gas chromatography mass spectrometry (GCMS) is presented as mean ± s.d. of n = 3 independently treated wells with individual data points shown. Scale bar, 50 μm (a, d, e, f), 1 cm (b).

Source Data

Extended Data Fig. 8 Inhibition of OGDH reduces tumour-cell competitive fitness in vivo.

a, αKG/succinate ratio of KPCflox RIK and KPC(R172H) RIK cells that express doxycycline-inducible shRNAs targeting Renilla, Ogdh and Sdha, grown for four days with or without doxycycline. b, Schematic of the in vivo competition assay. mKate2-positive KPflox RIK and KP(R172H) RIK cells were infected with retroviruses that encode doxycycline-inducible, GFP-linked shRNAs targeting Renilla, Ogdh or Sdha. Cells were selected for viral integration, induced with doxycycline for 2 days, mixed with uninfected parental cells at a ratio of 8:2 and analysed by flow cytometry to determine initial ratio of cells that express shRNA to uninfected cells. This cell mixture was injected orthotopically into doxycycline-fed recipient mice. After three weeks of tumour growth, pancreatic tumours were removed, weighed, dissociated and analysed by flow cytometry to measure final ratio of cells that express shRNA to uninfected cells. Data are presented in Fig. 3c. c, d, Tumour mass of orthotopically injected KPflox RIK (c) or KP(R172H) RIK (d) cells that express a mixture of shRNAs targeting Renilla, Ogdh and Sdha mixed with uninfected cells from Fig. 3c. KPflox RIK cells: n = 5 mice (shRenilla), n = 4 mice (shOgdh no. 1, shOgdh no. 2, shSdha no. 1, shSdha no. 2 and shSdha no. 3). KP(R172K) RIK cells: n = 5 mice (shRenilla), n = 4 mice (shOgdh no. 1, shOgdh no. 2, shSdha no. 1 and shSdha no. 2), n = 3 mice (shSdha no. 3). e, Representative gross images of pancreatic tumours arising in doxycycline-fed mice three weeks after orthotopic transplant of KPCflox RIK (top) and KPC(R172H) RIK (bottom) cells that express doxycycline-inducible shRNAs targeting Renilla, Ogdh and Sdha mixed at an 8:2 ratio with uninfected cells (from Fig. 3c). The experiment in a was performed once. Data are presented as mean ± s.d. of n = 3 independently treated wells of a representative experiment with individual data points shown. Significance was assessed compared to control cells that express shRNA targeting Renilla luciferase by one-way ANOVA with Dunnett’s multiple comparison post-test (c). Scale bar, 1 cm.

Source Data

Extended Data Fig. 9 p53 reactivation and OGDH inhibition induce 5hmC accumulation in PDAC cells.

a, Median fluorescence intensity of 5hmC in KPsh no. 2 cells grown with or without doxycycline for eight days. b, qRT–PCR of Tet1, Tet2 and Tet3 expression in KPsh no. 2 cells grown with or without doxycycline for the indicated number of days. c, Sequence analysis of CRISPR–Cas9 editing. The percentage of amplicons flanking the sgRNA target sequence is shown, with the indicated genotype amplified from KPsh no. 2 cells that express sgRNAs targeting Tet1, Tet2 and Tet3. d, 5hmC median fluorescence intensity (MFI) in KPsh no. 2 cells that express sgRNAs targeting Tet1, Tet2 and Tet3, grown with or without doxycycline for eight days. e, 5hmC median fluorescence intensity in KPsh no. 2 cells grown with 4 mM DM-αKG for 72 h. f, 5hmC median fluorescence intensity in 8988 and Panc1 cells, grown with 4 mM DM-αKG for 72 h. g, 5hmC median fluorescence intensity in KPCflox RIK and KPC(R172H) RIK cells that express doxycycline-inducible shRNAs targeting Renilla luciferase or Ogdh, grown for four days with or without doxycycline. h, Representative 5hmC staining in orthotopic tumours derived from KPflox cells that express doxycycline-inducible shRNAs targeting Renilla luciferase (n = 5 mice), Ogdh (n = 4 mice) or Sdha (n = 4 mice) two weeks after injection in mice maintained on doxycycline. i, Representative 5hmC staining of orthotopic tumours derived from KPflox cells nine days after activation of doxycycline-inducible shRNAs targeting Renilla luciferase (n = 4 mice), Ogdh (Ogdh shRNA no. 1, n = 4 mice, Ogdh shRNA no. 2, n = 3 mice), or Sdha shRNA no. 1, Sdha shRNA no. 2 and Sdha shRNA no. 3 (n = 4) in established tumours. GFP marks cells that express the indicated shRNA. Experiments in a, d–g were repeated twice with similar results, and the experiment in b was repeated in an additional line. Data are presented as mean ± s.d. of n = 3 independently treated wells of a representative experiment, with individual data points shown. Significance was assessed by two-tailed Student’s t-test (a, e, f) or compared to control cells expressing shRNA targeting Renilla luciferase by one-way ANOVA with Tukey’s multiple comparison post-test (g). Scale bar, 50 μm.

Source Data

Extended Data Fig. 10 Increase in the cellular αKG/succinate ratio enforces p53-driven tumour suppression.

a–c, SDHA and p53 immunoblot (a), αKG/succinate ratio (b) and 5hmC median fluorescence intensity (c) in KPsh no. 2 cells that express constitutive shRNAs targeting Sdha or Renilla luciferase, grown with or without doxycycline for eight days. d, Representative 5hmC staining in orthotopic tumours derived from KPsh no. 2 cells that express constitutive shRNAs targeting Sdha or Renilla luciferase, in mice maintained on doxycycline (n = 3 mice) or ten days after doxycycline withdrawal (n = 3 mice). GFP denotes cells that express shRNA targeting p53. 5hmC staining per nucleus is quantified in Fig. 4g. e, f, Small animal ultrasound measurement of tumours derived from KPsh no. 2 cells that express constitutive shRNAs targeting Renilla luciferase (left) or Sdha (right) orthotopically injected into doxycycline-fed mice maintained on a doxycycline diet for two weeks. After two weeks (day 0), tumour size was measured and mice were randomized into off- and on-doxycycline chow groups (shRenilla, n = 5 mice, Sdha shRNA no. 1, Sdha shRNA no. 2 and Sdha shRNA no. 3, n = 4 mice). Subsequent tumour size and mouse survival was monitored up to ten days after doxycycline withdrawal. g, Fold change in tumour size from day 5 to day 10 after withdrawal of doxycycline chow (day 0) from mice that bear orthotopic tumours derived from KPsh no. 2 cells that express constitutive shRNAs targeting Sdha or Renilla luciferase. h, Representative H & E staining of orthotopic tumours derived from KPsh no. 2 cells that express constitutive shRNAs targeting Sdha or Renilla luciferase maintained on doxycycline or ten days after doxycycline withdrawal. Experiments in a, b were performed once, and the experiment in c was repeated twice with similar results. For gel source data of a, see Supplementary Fig. 1. Data are presented as a representative, independently treated well (a) or as mean ± s.d. of n = 3 independently treated wells with individual data points shown (b, c). Significance was assessed by two-tailed, unpaired t-test (g). Scale bar, 50 μm.

Source Data

Supplementary information

Supplementary Figure 1

Uncropped scans of source data for immunoblots.

Reporting Summary

Supplementary Figure 2

Gating strategies for flow cytometry experiments. a, BrdU experiment gating strategy. Left, cells were identified by forward and side scatter. Middle, forward scatter area and forward scatter height used to discriminate doublets. Right, histogram indicating BrdU positive cells. b, Annexin-V experiment gating strategy. Left, cells were identified by forward and side scatter. Right, Annexin-V and DAPI staining. Percentage of AnnexinV+ DAPI+ and Annexin V+ DAPI- cells were added to report Annexin V percentage in all experiments. c,d, In vivo shRNA competition assay gating strategy. c, Strategy to determine initial shRNA (GFP+) percentage at injection. From left to right: cells were identified by forward and side scatter. Forward scatter area and forward scatter height used to discriminate doublets. Viable cells identified by negative DAPI staining. Percent GFP+ cells determined out of total mKate positive cells. d, Strategy to determine final shRNA (GFP+) percentage after 3 weeks of tumour growth. From left to right: cells were identified by forward and side scatter. Forward scatter area and forward scatter height used to discriminate doublets. Viable cells identified by negative DAPI staining. Percent GFP+ cells determined out of total mKate positive cells. e, Strategy to determine 5hmC median fluorsescence intensity (MFI) in mouse and human PDAC cells. From left to right: cells were identified by forward and side scatter. Forward scatter area and forward scatter height used to discriminate doublets. 5hmC MFI was determined from resulting singlet histogram. Right panel, representative histograms of KPsh cells grown on or off dox for 8 days.

Supplementary Figure 3

Unmerged 5hmC immunofluorescence.

Supplementary Table 1

GCMS data.

Supplementary Table 2

GSEA gene lists.

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Morris, J.P., Yashinskie, J.J., Koche, R. et al. α-Ketoglutarate links p53 to cell fate during tumour suppression. Nature 573, 595–599 (2019). https://doi.org/10.1038/s41586-019-1577-5

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