Abstract
The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.
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Data availability
RNA-seq data supporting the findings of this study have been deposited in the Gene Expression Omnibus under the accession code GSE183434. Alignment was performed against the mouse mm10 genome assembly. Gene essentiality data and NSCLC gene expression data are available from the DepMap Portal (https://depmap.org/portal/). Isotopologue distributions from all MS isotope tracing experiments are provided in Supplementary Table 4. Source data are provided with this paper.
Code availability
The code used to perform gene essentiality correlation and network modelling is provided at GitHub (https://github.com/finley-lab/coessentiality-network).
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Acknowledgements
We thank the members of the Finley laboratory for discussion, and A. Boire, A. Intlekofer, S. Vardhana, E. Reznik and K. S. Tan for feedback. Rex1::GFPd2 cells were a gift from A. Smith. P.K.A. is an NICHD Ruth L. Kirschstein Predoctoral fellow (F31HD098824). B.T.J. is a Gerstner Sloan Kettering Grayer Fellow and is supported by a Medical Scientist Training Program grant from the NIGMS of the National Institutes of Health under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. K.I.P. is supported by a Bruce Charles Forbes Pre-Doctoral Fellowship (MSKCC). J.S.B. is supported by a Human Frontier Science Program Fellowship (LT000200/2021-L). L.B.S. is supported by R00CA218679 and P30CA015704. L.W.S.F. is a Searle Scholar. This research was also supported by grants to L.W.S.F. from the Pershing Square Sohn Prize for Cancer Research, the Starr Foundation (I12-0051), the NIH/NCI (R37 CA252305) and the Anna Fuller Fund as well as the Memorial Sloan Kettering Cancer Center Support Grant P30CA008748.
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P.K.A. and L.W.S.F. conceived the study. P.K.A. and B.T.J. performed all of the experiments with assistance from K.I.P., J.S.B. and J.E.; M.L.H., O.J.N. and S.P.A. performed LC–MS experiments under the guidance of L.B.S.; B.T.J. performed genetic co-essentiality mapping and network modelling with guidance from E.D. K.I.P. performed RNA-seq. L.B.S. provided additional study guidance. L.W.S.F. supervised the project. P.K.A. and L.W.S.F. wrote the manuscript with input from all of the authors.
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P.K.A., B.T.J. and L.W.S.F. are listed as inventors on a provisional patent application (US provisional application no. 63/272,940) filed by the Memorial Sloan Kettering Cancer Center. The patent application covers the use of ACL inhibitors to modify the self-renewal potential of ES cells. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Metabolic gene essentiality correlations across cancer cell lines.
Heatmap depicting hierarchical clustering of pairwise gene essentiality score correlations of core metabolic pathway genes derived from four GO terms: tricarboxylic acid (TCA) cycle, canonical glycolysis, one-carbon metabolic process and fatty-acyl-CoA metabolic process. Genes are colour coded to the left of the heatmap according to the GO term. TCA cycle genes are highlighted (red) in the dendrogram. Gene names and correlation scores can be found in Supplementary Table 1.
Extended Data Fig. 2 Effect of ACL inhibition on 13C labelling of TCA cycle metabolites.
a, Two-dimensional network diagram representing gene essentiality score correlations between TCA cycle genes and their top co-dependencies. The strength of the correlation between genes is represented by both the length and thickness of the connecting edge. Correlation scores are shown in Supplementary Table 1. b, c, Fractional enrichment of citrate (left) and malate (right) in three NSCLC cell lines cultured in medium containing [U-13C]glucose (b) or [U-13C]glutamine (c) and treated with vehicle or 50 μM BMS-303141(ACLi) for 24 h. d, Schematic depicting [U-13C]asparagine labelling of aspartate and citrate in cells expressing guinea pig asparaginase (ASNase). Asparagine-derived aspartate will generate M+4-labelled citrate. Top, M+4-labelled citrate metabolized via the canonical TCA cycle will lose two labelled carbons as CO2, ultimately regenerating citrate that retains two labelled carbons (M+2). Bottom, M+4-labelled citrate metabolized by ACL will yield M+4 labelled oxaloacetate that will ultimately regenerate M+4-labelled citrate. e, f, Fractional labelling of aspartate (left) and citrate (right) (e) or citrate M+2 relative to citrate M+4 (cit+2/cit+4) (f) in ASNase-expressing 143B human osteosarcoma cells cultured in medium containing [U-13C]asparagine and treated with vehicle or 50 μM ACLi for 24 h. Data are mean ± SD, n = 3 independent replicates. Significance was assessed in comparison to vehicle treatment by two-way ANOVA with Sidak’s multiple comparisons post-test (b-c, e) or using unpaired two-tailed Student’s t-test (f).
Extended Data Fig. 3 ACO2 and ACL disruption in embryonic stem cells.
a, Fractional M+2 enrichment of citrate and malate in mouse ES cells cultured in medium containing [U-13C]glucose. b, Fractional enrichment of malate M+2 relative to citrate M+2 (mal+2/cit+2) derived from [U-13C]glucose in ES cells following treatment with vehicle or 50 μM BMS-303141 (ACLi) for 24 h. c, d, Immunoblot of clonal mouse ES cells in which CRISPR/Cas9-mediated editing was used to target either a non-genic region of chromosome 8 (Ctrl) and Acly (ACLY-1 and ACLY-2) (c) or Aco2 (ACO2-1 and ACO2-2) (d). e, f, Assessment of the [U-13C]glucose-derived mal+2/cit+2 ratio (e) or steady-state levels of TCA cycle metabolites represented as the fold change (expressed in log2) relative to Ctrl (f) in control and Aco2-edited ES cells. g, h, j, k, Fractional M+1 enrichment of NADH (g), lactate (h), fumarate (j) and succinate (k) in control and Acly-edited ES cells cultured in medium containing [4-2H]glucose. i, Schematic depicting deuterium transfer from [4-2H]glucose first onto malate in the cytoplasm then onto TCA cycle metabolites in the mitochondria. l, Quantification of the lactate over pyruvate ratio in control and Acly-edited ES cells. m, The baseline oxygen consumption rate (OCR) in control and Acly-edited ES cells normalized to protein content. Twelve technical replicates were averaged for each of three independent experiments. Data are mean ± SD, n = 3 independent replicates unless otherwise noted. Significance was assessed using unpaired two-tailed Student’s t-test (a, b) or in comparison to control cells by one-way ANOVA with Sidak’s multiple comparisons post-test for all other panels.
Extended Data Fig. 4 SLC25A1 and MDH1 contribute to TCA cycle metabolism in embryonic stem cells.
a, b, Immunoblot of clonal mouse ES cells in which CRISPR/Cas9-mediated editing was used to target either a non-genic region of chromosome 8 (Ctrl) and Slc25a1 (SLC25A1-1 and SLC25A1-2) (a) or Mdh1 (MDH1-1 and MDH1-2) (b). c, d, Fractional M+1 enrichment of malate (Mal), fumarate (Fum), aspartate (Asp) and citrate (Cit) in control (Ctrl) and Slc25a1-edited ES cells (c) or Mdh1-edited ES cells (d) cultured in medium containing [4-2H]glucose. e, f, Fractional M+2 enrichment of citrate, fumarate, malate and aspartate derived from [U-13C]glucose in control and Slc25a1-edited (e) or Mdh1-edited (f) ES cells. g, Steady-state levels of TCA cycle metabolites in Slc25a1-edited or Mdh1-edited ES cells. Levels are represented as the fold change (expressed in log2) relative to chromosome 8-targeted control cells. Data are mean ± SD, n = 3 independent replicates. Significance was assessed in comparison to control cells by two-way ANOVA (c-f) with Sidak’s multiple comparisons post-test.
Extended Data Fig. 5 Effect of myogenic differentiation on 13C-glucose labelling of TCA cycle intermediates.
a, Immunoblot comparing expression of myogenesis markers MYOG and MYH3 between proliferating (Prolif) and 100% confluent (Conf) myoblasts and myotubes differentiated for 3, 5 or 7 days. b, Fractional labelling of citrate (left) and malate (right) in proliferating and confluent myoblasts and myotubes differentiated for 3, 5 or 7 days cultured in medium containing [U-13C]glucose. c, Fractional M+1 enrichment from [4-2H]glucose of malate (Mal), fumarate (Fum), aspartate (Asp) and citrate (Cit) in myoblasts and myotubes differentiated for 5 days. d, Immunoblot comparing expression of ACL and ACO2 in C2C12 cells expressing doxycycline-inducible shRNAs targeting Acly (shAcly-1 and shAcly-2), Aco2 (shAco2-1 and shAco2-2) or Renilla luciferase (shRen, used as a control). Cells were cultured on doxycycline for two days to induce shRNA expression. e-h, Fractional M+2 enrichment of citrate (left) and malate (right) or malate M+2 relative to citrate M+2 (mal+2/cit+2) in myoblasts (e, f) or myotubes (g, h) expressing doxycycline-inducible shRNAs targeting Acly, Aco2 or Renilla luciferase cultured in medium containing [U-13C]glucose. Myoblasts and myotubes were cultured on doxycycline for 2 or 4 days, respectively, to induce shRNA expression. Data are mean ± SD, n = 3 independent replicates. In b, significance was assessed using one-way ANOVA with Sidak’s multiple comparisons post-test to compare total metabolite fraction labelled relative to proliferating myoblasts. In remaining panels, significance was assessed by two-way ANOVA in comparison to myoblasts (c) or by one-way ANOVA in comparison to shRen-expressing myoblasts (e-f) or myotubes (g-h) with Sidak’s multiple comparisons post-test.
Extended Data Fig. 6 Transcriptional profiles associated with TCA cycle choice.
a, Gene set enrichment analysis showing that genes positively correlated with fractional enrichment of malate M+2 relative to citrate M+2 (mal+2/cit+2) derived from [U-13C]glucose in 68 NSCLC cell lines are enriched for KEGG citric acid (TCA) cycle-associated genes. b, c, Fractional M+2 enrichment of citrate (Cit), fumarate (Fum), malate (Mal) and aspartate (Asp) (b) or mal+2/cit+2 (c) derived from [U-13C]glucose in mouse ES cells following treatment with vehicle, 5 mM DCA or 10 µM MPCi for 24 h. Data are mean ± SD, n = 3 independent samples. In b-c, significance was assessed in comparison to vehicle treatment by two-way ANOVA (b) or one-way ANOVA (c) with Sidak’s multiple comparisons post-test.
Extended Data Fig. 7 ACL loss blunts exit from naive pluripotency.
a, Experimental setup for cell fate transitions. Mouse ES cells cultured in serum and leukemia inhibitory factor (LIF) are a heterogenous population that can be converted to the naive, ground state of pluripotency by addition of MEK and GSK3β inhibitors (2i) in either serum replete (serum/LIF+2i, d-f) or serum-free (2i/LIF, g-i) media formulations. Transition to serum-free medium lacking 2i/LIF (−2i/LIF) induces exit from the naive pluripotent state, enabling ES cells to gain differentiation competence. b, RT–qPCR of pluripotency-associated (Nanog, Esrrb, Klf2, Rex1 and Oct4) and early differentiation-associated (Fgf5, Otx2 and Sox1) genes in 2i/LIF-cultured ES cells subjected to 2i/LIF withdrawal for 12, 24 or 40 h. Levels are represented as the fold change (expressed in log2) relative to naive, 2i/LIF-cultured ES cells (0 h). c, Quantification of alkaline phosphatase (AP)-positive colonies representing ES cells that failed to exit from the pluripotent state. 2i/LIF-cultured ES cells were subjected to 2i/LIF withdrawal for 0, 12, 24 or 40 h and then reseeded at clonal density into medium containing 2i and LIF. d-f, Fractional labelling of citrate (Cit), malate (Mal) and aspartate (Asp) in serum/LIF+2i-cultured ES cells incubated with [U-13C]glucose (d), [U-13C]glutamine (e) or [4-2H]glucose (f) subjected to exit from pluripotency for the indicated times. g, Fractional enrichment of glucose-derived malate M+2 relative to citrate M+2 (mal+2/cit+2) in 2i/LIF-cultured ES cells subjected to 2i/LIF withdrawal for the indicated times. h, i, Fractional labelling of citrate, malate and aspartate in 2i/LIF-cultured ES cells cultured in medium containing [U-13C]glucose (h) or [U-13C]glutamine (i) subjected to 2i/LIF withdrawal for the indicated times. j, Immunoblot of polyclonal ES cells in which CRISPR/Cas9-mediated editing was used to target either a non-genic region of chromosome 8 (sgChr8) or Tcf7l1 (sgTcf7l1). k, RT–qPCR of pluripotency-associated (Nanog,Esrrb, and Rex1) and early differentiation-associated (Sox1) genes in control and Tcf7l1-edited ES cells adapted to the naive, ground state and subjected to 2i/LIF withdrawal for the indicated times. Levels are represented as the fold change (expressed in log2) relative to chromosome 8-targeted control cells in the naive, ground state (0 h). l, m, Fractional labelling of citrate (left) and malate (right) (l) and glucose-derived mal+2/cit+2 ratio (m) in chromosome 8-targeted control or Tcf7l1-edited ES cells cultured in medium containing [U-13C]glucose subjected to 2i/LIF withdrawal for the indicated times. Data are mean ± SD, n = 3 independent replicates. In d-e, h-i, and l, significance was assessed using one-way ANOVA (d-e, h-i) or two-way ANOVA (l) with Sidak’s multiple comparisons post-test to compare total metabolite fraction labelled relative to the 0 h timepoint (d-e, h-i) or control cells (l). In remaining panels, significance was assessed relative to the 0 h timepoint using one-way ANOVA (c, f-g) or chromosome 8-targeted control cells at each time point using two-way ANOVA (k, m) with Sidak’s multiple comparisons post-test.
Extended Data Fig. 8 Acetate does not reverse the effects of ACL loss on exit from pluripotency.
a, b, Fractional labelling of citrate (Cit), malate (Mal) and aspartate (Asp) in control and Acly-edited ES cells cultured in medium containing [U-13C]glucose (a) or [U-13C]glutamine (b) following 40 h of 2i/LIF withdrawal. c, d, Fractional enrichment of malate M+2 relative to citrate M+2 (mal+2/cit+2) derived from [U-13C]glucose (c) or steady-state levels of TCA cycle metabolites (d) in naive, 2i-adapted control (Ctrl) and Acly-edited (ACLY-1 and ACLY-2) ES cells. Steady-state levels are represented as the fold change (expressed in log2) relative to control cells. e, f, Assessment of the [U-13C]glucose-derived mal+2/cit+2 ratio (e) and steady-state levels of TCA cycle metabolites (f) in control and Acly-edited ES cells subjected to 2i/LIF withdrawal for 40 h. g, Relative viability (measured by PI exclusion) of control and Acly-edited ES cells maintained in the naive pluripotent state (+2i/LIF, left) or subjected to 2i/LIF withdrawal for 40 h (−2i/LIF, right). h, Immunoblot showing expression of ACSS2, the enzyme that converts acetate to acetyl-CoA in the cytosol, in naive, ground state ES cells subjected to 2i/LIF withdrawal for the indicated times. i, Fractional labelling of palmitate in control and Acly-edited ES cells cultured in medium containing [U-13C]acetate following 40 h of 2i/LIF withdrawal. Each bar represents one independent sample. j, Immunoblot comparing levels of acetylation (ac) at indicated histone lysine residues in control and Acly-edited ES cells subjected to 2i/LIF withdrawal for 40 h in the presence of vehicle or 5 mM sodium acetate. k, Relative viability of control and Acly-edited ES cells subjected to 2i/LIF withdrawal for 40 h in the presence of vehicle or 5 mM sodium acetate. l, Quantification of GFP mean fluorescence intensity (MFI) encoded by the Rex1::GFPd2 reporter in ES cells subjected to 2i/LIF withdrawal for 40 h in the presence of vehicle or 50 μM BMS-303141 (ACLi). Naïve ES cells (+2i/LIF) are included as a control. Representative histograms are shown in Fig. 4d. m, RT–qPCR of pluripotency-associated (Nanog, Esrrb and Rex1) and early differentiation-associated (Sox1) genes in control and Acly-edited ES cells subjected to 2i/LIF withdrawal for 40 h. Levels are represented as the fold change (expressed in log2) relative to chromosome 8-targeted control cells. n, Alkaline phosphatase (AP) staining of colony formation assay representing control and Acly-edited ES cells that failed to exit the naive pluripotent state. 2i-adapted ES cells were subjected to 2i/LIF withdrawal for 40 h and then reseeded at clonal density into medium containing 2i/LIF. Quantification is shown in Fig. 4e. o, RT–qPCR of pluripotency-associated genes in control and Acly-edited ES cells subjected to 2i/LIF withdrawal for 40 h in the presence of vehicle or 5 mM sodium acetate. p, Quantification of GFP MFI encoded by the Rex1::GFPd2 reporter in ES cells subjected to 2i/LIF withdrawal for 40 h in the presence of DMSO or 50 μM BMS-303141(ACLi) and vehicle or 5 mM sodium acetate. q, Quantification of AP-positive colonies representing control and Acly-edited ES cells that failed to exit from the pluripotent state. ES cells were subjected to 2i/LIF withdrawal for 40 h in the presence of vehicle or 5 mM sodium acetate prior to reseeding at clonal density into medium containing 2i and LIF. Data are mean ± SD, n = 5 (p), n = 4 (g, k, l) or n = 3 (all other experiments) independent replicates. In a-b, significance was assessed using one-way ANOVA with Sidak’s multiple comparisons post-test to compare total metabolite fraction labelled relative to control cells. In remaining panels, significance was assessed by two-way ANOVA relative to control cells (k, q) or DMSO treatment (p) with Sidak’s multiple comparisons post-test, or by one-way ANOVA in comparison to control cells (c, e, g) with Sidak’s multiple comparisons post-test or in the indicated comparisons (l) with Tukey’s multiple comparisons post-test.
Extended Data Fig. 9 Effect of SLC25A1 and MDH1 loss in exit from naive pluripotency.
a, b, Relative viability (measured by PI exclusion) of control and Slc25a1-edited (left) and Mdh1-edited (right) ES cells maintained in the naive pluripotent state (+2i/LIF, a) or subjected to 2i/LIF withdrawal for 40 h (−2i/LIF, b). c, Steady-state levels of TCA cycle metabolites in control and Slc25a1-edited (left) and Mdh1-edited (right) ES cells subjected to 2i/LIF withdrawal for 40 h. Steady-state levels are represented as the fold change (expressed in log2) relative to control cells. d, e, Relative O-propargyl-puromycin (OP-puro) mean fluorescence intensity (MFI) in control, Slc25a1-edited, Acly-edited and Mdh1-edited ES cells that have been maintained in the naive pluripotent state (d) or subjected to 2i/LIF withdrawal for 40 h (e). Dotted line represents OP-puro MFI following cycloheximide (CHX) treatment as a control. f, g, Population doublings of control, Slc25a1-edited, Acly-edited and Mdh1-edited ES cells that have been maintained in the naive pluripotent state (f) or subjected to 2i/LIF withdrawal for 40 h (g). h, i, RT–qPCR of pluripotency-associated (Nanog, Esrrb and Rex1) and early differentiation-associated (Sox1) genes in control and Slc25a1-edited (h) and Mdh1-edited (i) ES cells subjected to 2i/LIF withdrawal for 40 h. Data are mean ± SD, n = 3 independent samples. Significance was assessed in comparison to control cells by one-way ANOVA with Sidak’s multiple comparisons post-test.
Extended Data Fig. 10 Mode of TCA cycle engagement regulates cell fate.
a, Population doublings of control and Aco2-edited ES cells cultured in metastable (serum/LIF) conditions. b, Cumulative population doublings over the indicated passages of control and Aco2-edited ES cells upon conversion to the naive, ground state of pluripotency via addition of MEK and GSK3β inhibitors (+2i). c, RT–qPCR of pluripotency-associated genes at the indicated passages in control and Aco2-edited ES cells following addition of 2i. Gene expression at every passage was normalized to passage 0 (p0). Data are mean ± SD, n = 1 (b) or n = 3 (a, c) independent replicates. Significance was assessed in comparison to control cells by one-way ANOVA with Sidak’s multiple comparisons post-test (a) or relative to control cells at each timepoint with P values coloured according to comparison by two-way ANOVA with Sidak’s multiple comparisons post-test (c).
Supplementary information
Supplementary Information
Supplementary Fig. 1: uncropped scans of source data for immunoblots. Supplementary Fig. 2: gating strategy for FACS analysis.
Supplementary Table 1
Source data for Fig. 1a and Extended Data Figs. 1 and 2a. Correlations derived from gene co-essentiality analysis. Data underlie the 2D network diagrams shown in Fig. 1a and Extended Data Fig. 2a. and the heat map shown in Extended Data Fig. 1.
Supplementary Table 2
All sgRNA, shRNA and RT–qPCR primer sequences. Primer sequences for sgRNAs used to perform CRISPR–Cas9-mediated editing in mouse ES cells to generate clonal lines, shRNAs used to infect C2C12 mouse cells and gene expression analysis using RT–qPCR.
Supplementary Table 3
CRISPR-sequencing primers and derived amplicons. The primer sequences used to amplify genomic fragments containing edited loci of interest for sgRNA editing analysis in clonal mouse ES cell lines and the associated amplicons derived from this analysis. Amplicons are annotated to show editing of each allele for all clonal ES cell lines.
Supplementary Table 4
Source data for all isotope tracing MS experiments. Full isotopologue distributions generated by isotope-tracing MS experiments.
Source data
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Arnold, P.K., Jackson, B.T., Paras, K.I. et al. A non-canonical tricarboxylic acid cycle underlies cellular identity. Nature 603, 477–481 (2022). https://doi.org/10.1038/s41586-022-04475-w
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DOI: https://doi.org/10.1038/s41586-022-04475-w
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