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Hexokinase detachment from mitochondria drives the Warburg effect to support compartmentalized ATP production

Nature Metabolism volume 8, pages 215–236 (2026)Cite this article

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Abstract

Hexokinase (HK) catalyses the phosphorylation of glucose to glucose 6-phosphate, marking the first step of glucose metabolism. Most cancer cells co-express two homologous HK isoforms, HK1 and HK2, which can each bind the outer mitochondrial membrane (OMM). CRISPR screens performed across hundreds of cancer cell lines indicate that both isoforms are dispensable for growth in conventional culture media. By contrast, HK2 deletion impaired cell growth in human plasma-like medium. Here we show that this conditional HK2 dependence can be traced to the subcellular distribution of HK1. Notably, OMM-detached (cytosolic) rather than OMM-docked HK supports cell growth and aerobic glycolysis (the Warburg effect), an enigmatic phenotype of most proliferating cells. We show that under conditions promoting increased translocation of HK1 to the OMM, HK2 is required for cytosolic HK activity to sustain this phenotype, thereby driving sufficient glycolytic ATP production. Our results reveal a basis for conditional HK2 essentiality and suggest that demand for compartmentalized ATP synthesis explains why cells engage in aerobic glycolysis.

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Fig. 1: HK2 deletion impairs cell growth in HPLM and reduces in vivo tumour burden.
Fig. 2: HK2-mediated catalytic activity is necessary to support cell growth in HPLM.
Fig. 3: Relative HK1–OMM binding varies with nutrient conditions, and HK detachment from the OMM promotes cell growth.
Fig. 4: HK detachment from the OMM promotes aerobic glycolysis.
Fig. 5: HK2 dependence is not linked to a direct gene–nutrient interaction.
Fig. 6: HK detachment from the OMM drives glycolytic ATP production.
Fig. 7: Conditional HK2 dependence varies with cell-intrinsic factors.

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Data availability

All data needed to evaluate the findings of this study can be found within the Article, Extended Data or Supplementary Information. The individual plasmids generated in this study have been deposited in Addgene (identifiers found in Supplementary Table 5). Unique reagents generated in this study are available upon reasonable request from the corresponding author. Identifiers for deposited datasets accessed in this study are found in Supplementary Table 5. Source data are provided with this paper.

Code availability

This paper does not report original code.

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Acknowledgements

We thank members of the Cantor Lab for upkeep of both the LC–MS system and cell sorter and for helpful discussions, and members of the J. Fan Lab for training and helpful tips on using the Seahorse XFe96 Analyzer. We also thank M. Stefely for organelle figure assets, K. Tharp and D. M. Sabatini for helpful comments on the manuscript and the University of Wisconsin Carbone Cancer Center Small Animal Imaging and Radiotherapy Facility (supported in part through NCI/NIH P30CA014520) for use of its facilities and services. This work was supported by grants from the American Cancer Society (RSG-21-170-01-TBE to J.R.C.), the St. Baldrick’s Foundation Empowering Pediatric Immunotherapy for Childhood Cancers Team Grant (to C.M.C.) and the Midwest Athletes Against Childhood Cancer (MACC) Fund (to M.H.F. and C.M.C.). Fellowship support was provided by the NIH (T32HG002760 to K.S.H.) and the University of Wisconsin–Madison Department of Biochemistry to K.S.H., K.M.F. and G.R.C. J.R.C. is a Hartwell Foundation Investigator.

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Authors and Affiliations

  1. Morgridge Institute for Research, Madison, WI, USA

    Kimberly S. Huggler, Kyle M. Flickinger, Carlos A. Mellado Fritz & Jason R. Cantor

  2. Department of Biochemistry, University of Wisconsin–Madison, Madison, WI, USA

    Kimberly S. Huggler, Kyle M. Flickinger, Carlos A. Mellado Fritz, Gavin R. Chang, Meghan F. McGuire & Jason R. Cantor

  3. Department of Pediatrics, University of Wisconsin–Madison, Madison, WI, USA

    Matthew H. Forsberg & Christian M. Capitini

  4. Carbone Cancer Center, University of Wisconsin–Madison, Madison, WI, USA

    Christian M. Capitini & Jason R. Cantor

  5. Department of Biomedical Engineering, University of Wisconsin–Madison, Madison, WI, USA

    Jason R. Cantor

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Contributions

K.S.H. and J.R.C. initiated the project and designed the research plan. K.S.H. performed most of the experiments, with assistance from C.A.M.F., G.R.C. and M.F.M. K.M.F. performed coupled HK-G6PD activity and differential centrifugation experiments. J.R.C. performed a subset of the growth assay, growth curve and metabolomics experiments. M.H.F. performed the xenograft and bioluminescence imaging experiments with supervision from C.M.C. K.S.H. and J.R.C. analysed and interpreted the experimental data, with assistance from K.M.F. J.R.C. wrote the manuscript with assistance from K.S.H. All authors discussed the manuscript. J.R.C. supervised the studies.

Corresponding author

Correspondence to Jason R. Cantor.

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Competing interests

J.R.C. is an inventor on an issued patent for HPLM assigned to the Whitehead Institute (Patent number US11453858). C.M.C. reports honorarium from Bayer and Novartis, and equity interest from Elephas, for advisory board memberships. These entities had no input in the study design, analysis, manuscript preparation or decision to submit for publication. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Related to HK2 deletion impairs cell growth in HPLM and reduces in vivo tumor burden.

(a) Human cell lines ranked by probability of dependency values for HK2 across CRISPR screen data cataloged in the DepMap9. Probability > 0.5 is the reference threshold for essentiality. (b) Dependency phenotypes for HK3, GCK, and HKDC1 from conditional essentiality profiling in K562 cells14 (top) and the DepMap project (bottom)9. c) Immunoblots for expression of HK1 and HK2 in K562 cells. GAPDH served as the loading control in both cases. (d) Images depicting leukemia tumor burden based on Akaluciferase flux in mice engrafted with HK2-knockout K562 cells that either harbored empty vector (EV) (left) or expressed an HK2 cDNA (right). Data are represented colorimetrically with the scale bars depicted.

Source data

Extended Data Fig. 2 Related to HK2-mediated catalytic activity is necessary to support cell growth in HPLM.

(a) Pseudocolor Coomassie-stained gel imaged using a LI-COR Odyssey FC. 1: M.W. standards; 2: wild-type HK2-3xFLAG; 3: HK2 (D209A, D657A)-3xFLAG. (b) Schematic for a method to evaluate HK activity based on glucose 6-phosphate (G6P) production from reactions containing recombinant HK2.

Extended Data Fig. 3 Related to relative HK1-OMM binding varies with nutrient conditions and HK detachment from the OMM promotes cell growth.

(a) Immunoblots for expression of HK2 in whole-cell lysates (WC) and mitochondria isolated (IP:HA) from K562 cells that expressed 3xMyc-eGFP-OMP25 (Control-MITO) or 3xHA-eGFP-OMP25 (HA-MITO). COXIV served as a mitochondrial loading control for HK1 (immunoblot displayed in Fig. 3a) and HK2 (immunoblot displayed in this panel) from the same samples. Immunoblot for expression of HK2 is identical to that displayed in Fig. 3a. (b) HK signal normalized by COXIV signal in IP:HA versus WC from the same respective cells. nd, not detected. Comparison of normalized signals between HK1 and HK2 is derived by using the same experimental samples run across different blots and processed in parallel. (c, i, m) Immunoblots for expression of HK1 and HK2 (c) or Flag (i, m) in dounce homogenates (DH) and corresponding cytosolic (Cyt) and organellar (Org) fractions isolated from HK2-knockout cells. GAPDH served as cytosolic control marker. COXIV served as a mitochondrial control marker. (d, j) HK (d) or Flag (j) signal normalized by COXIV signal in Org versus DH from the same respective cells. Comparisons of normalized signal between HK1 and HK2 are derived using the same experimental samples run across different blots and processed in parallel (d). Comparisons of normalized signal are derived from samples run on the same blot (j). (e, f, k, l) Immunoblots for expression of Flag (e, g), HK1 (e), or HK2 (f, k, l) in HK2-knockout and control cells. GAPDH served as the loading control in each case. (g) Immunoblot for expression of Flag in WC and IP:HA from HK2-knockout cells that expressed HA-MITO. COXIV served as a mitochondrial control marker. S6K served as a non-mitochondrial control marker. (h) HK1 (mean ± s.d., n = 3) or Flag signal normalized by COXIV signal in IP:HA versus WC from the same respective cells. P value, Two-tailed Welch’s t-test between the indicated bars identical to that displayed in Fig. 3c. For comparisons involving normalized Flag signal, MBD-deficient HK1 and MBD-deficient HK2 samples were run on the same blot. Wild-type HK1 samples were run on a different blot and were processed on a separate day. (n) Pseudocolor Coomassie-stained gel imaged using a LI-COR Odyssey FC. 1: TOM20-HK2-3xFLAG; 2: M.W. standards. HK1 signal normalized by COXIV signal in IP:HA versus WC from the same respective cells (mean ± s.e.m., n = 3 biologically independent samples). (o) G6P levels measured from reactions of recombinant TOM20-HK2 with glucose and ATP (mean ± s.d., n = 3 independent reactions). (p) Immunoblots for expression of HK1 and HK2 from HK2-knockout and control cells used for lysate-based HK activity assays. GAPDH served as the loading control in both cases. (q) Relative growth of HK2-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. In a and c, L.B., low brightness; H.B., high brightness.

Source data

Extended Data Fig. 4 Related to HK detachment from the OMM promotes aerobic glycolysis.

(a) Specific growth rates for control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test. (b) Flow cytometric analysis of HK2-knockout and control cells stained with 7-Aminoactinomycin D (7-AAD), which is excluded from live cells. (c) Relative growth rates for HK2-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. (d) Net rates of lactate secretion versus glucose uptake in HK2-knockout cells (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test. (e, f, g, h, j) Fractional labeling of pyruvate (e, f), serine (g), citrate (h), and malate (j) in HK2-knockout cells (mean ± s.d., n = 3 biologically independent samples) grown in conditions with [U-13C-glucose]. Defined lactate levels in HPLM and RPMI (f, bottom right). (i, k) Total 13C labeling of citrate (i) and malate (k) in HK2-knockout cells (mean ± s.d., n = 3 biologically independent samples) grown in conditions with [U-13C-glucose]. (l) Schematic depicting the incorporation of 13C from [1,2-13C] glucose into 3-phosphoglycerate (3PG) and lactate. Orange-outlined box, labeling generated when [1,2-13C] glucose is metabolized via the oxidative pentose phosphate pathway (ox-PPP). Green-outlined box, labeling generated when [1,2-13C] glucose is metabolized via glycolysis. (m, n) Fractional labeling of 3PG (m) and lactate (n) in HK2-knockout cells (mean ± s.d., n = 3 biologically independent samples) grown in conditions with [1,2-13C-glucose]. In e, f, g, h, i, j, k, m, and n, Values above brackets indicate differences in fractional labeling between bars. Two-tailed Welch’s t-test.

Source data

Extended Data Fig. 5 Related to HK2 dependence is not linked to a direct gene-nutrient interaction.

(a, c, e, g, j, k) Rates of net exchange for glucose (a), glutamine (c), lactate (e), pyruvate (g), and alanine (j, k) for HK2-knockout cells (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test between bars. Blue box (k, bottom right), exogenous alanine levels at 72 h vs 24 h (mean ± s.d., n = 3 biologically independent samples). No exchange (ne) was defined as a change in exogenous levels less than 5%. (b, d, f, h, i) Exogenous levels of glucose (b), glutamine (d), lactate (f), pyruvate (h), and alanine (i) during log growth of HK2-knockout cells (mean ± s.e.m., n = 3 biologically independent samples). Blue box (i, right), exogenous alanine levels at 72 h vs 24 h (mean ± s.d., n = 3 biologically independent samples). (l) Immunoblot for expression of HK1 in WC and IP:HA from HK2-knockout cells that expressed HA-MITO. COXIV served as a mitochondrial control marker. S6K served as a non-mitochondrial control marker. (m) HK1 signal normalized by COXIV signal in IP:HA versus WC from the same respective cells. (n) Fractional labeling of lactate in HK2-knockout cells (mean ± s.d., n = 3 biologically independent samples). Values above brackets indicate differences in fractional labeling between bars. Two-tailed Welch’s t-test. (o, p, q, r) Relative growth of HK2-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. For o, p, and q, metabolites that comprised each respective pool of HPLM-specific metabolites can be found in Fig. 5i.

Source data

Extended Data Fig. 6 Related to HK detachment from the OMM drives glycolytic ATP production.

(a) Relative growth of HK2-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. (b, c, d, e, f) Relative levels of the indicated metabolites in HK2-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. Hypoxanthine (Hx), adenosine (Ado), and guanosine (Guo) can serve as salvage substrates for purine synthesis. Cytidine (Cyt) and uridine (Urd) can serve as salvage substrates for pyrimidine synthesis. Thymidine (Thy) is a salvage substrate for thymidylate (b, right). N-acetylglucosamine (GlcNAc) can act as a salvage substrate for UDP-GlcNAc (c, right). Glycerol-3-phosphate (Glycerol-3P) and acetyl-CoA are key intermediates involved in palmitate synthesis (d, right). Dimethyl α-KG (DM-α-KG) may deliver an additional carbon source to the TCA cycle (e, right). NADPH is a key product of the oxidative pentose phosphate (ox-PPP) pathway (f, right). (g) Pyruvate-to-lactate ratios in HK2-knockout and control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test. (h, i) Relative growth of HK2-knockout and control cells treated with duroquinone (DQ) (h) or UK5099 (i) versus vehicle-treated control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. DQ can serve as an electron acceptor for the NAD+-generating reaction catalyzed by NAD(P)H dehydrogenase [quinone] 1 (NQO). DHQ, durohydroquinone. (h, right). UK5099 is a small molecule inhibitor of the mitochondrial pyruvate carrier (MPC) (i, right). (j) Oxygen consumption rates (OCR) and proton efflux rates (PER) for HK2-knockout and control cells (mean ± s.e.m., n = 6 biologically independent replicates). OA, oligomycin A; Rot, rotenone; AA, antimycin A. (k) Basal OCR (left) and OCR coupled to ATP production (right) for HK2-knockout versus control cells (mean ± s.d., n = 6 biologically independent replicates). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells). (l) Total population doublings for control cells (mean ± s.d., n = 3 biologically independent samples). Value above bracket indicates difference in total doublings.

Source data

Extended Data Fig. 7 Related to conditional HK2 dependence varies with cell-intrinsic factors.

(a) HK signal normalized by COXIV signal in IP:HA versus WC from the same respective cells. nd, not detected. (b) Immunoblots for expression of HK1 and HK2 (c) in dounce homogenates (DH) and corresponding cytosolic (Cyt) and organellar (Org) fractions isolated from NOMO1 cells. S6K served as cytosolic control marker. COXIV served as a mitochondrial control marker. L.B., low brightness; H.B., high brightness. (c) HK signal normalized by COXIV signal in Org versus DH from the same respective cells. Comparisons of normalized signal between HK1 and HK2 are derived using the same experimental samples run across different blots and processed in parallel. (d) Relative growth of indicated cell lines transduced with sgHK2 versus sgAAVS1 (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. (e) Relative growth rates of indicated cell lines transduced with sgHK2 versus sgAAVS1 (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. (f) Distribution of mRNA transcript levels for indicated monocarboxylate transporter (MCT)-encoding SLC16 isoforms across human cancer lines from cataloged RNA-Seq data23. Displayed percentage, cell lines with log2(TPM + 1) greater than 1. Bolded line, median. TPM, transcripts per million. (g) mRNA transcript levels for SLC16A7 (top) and SLC16A8 (bottom) across the indicated cell lines from analyzed RNA-seq data80. SLC16A7 encodes MCT2. SLC16A8 encodes MCT3. nTPM, normalized TPM. (h) Human cancer lines ranked by SLC16A3 mRNA transcript levels from cataloged RNA-Seq data23. Labeled points indicate cell lines that exhibited conditional HK2 dependence in this study.

Source data

Extended Data Fig. 8 Expression of HK1 and HK2 in non-diseased tissue sites.

(a) Comparison between HK1 and HK2 mRNA transcript levels from the GTEx85. Dotted lines are at transcripts per million (TPM) = 20. (b) Annotated sites for which the TPM value for HK2 is greater than 20.

Source data

Extended Data Fig. 9 Conditional gene essentiality identifies regulatory nodes of the Warburg effect.

(a) Schematic depicting all steps of aerobic glycolysis from glucose uptake to lactate secretion and others involved in mitochondrial pyruvate metabolism (left). Dependency phenotypes for genes expressed in K562 cells according to cataloged RNA-seq data23. Essentiality is defined as a probability of dependency > 0.5. See Supplementary Table 4 (right). TIGAR is not expressed in K562 cells23. (b) Dependency phenotypes for PFKP and SLC16A3 from the DepMap9.

Supplementary information

Reporting Summary

Supplementary Table 1

Synthetic media construction.

Supplementary Table 2

Datasets related to cellular metabolomics.

Supplementary Table 3

Datasets related to Seahorse assays.

Supplementary Table 4

Fermentation-related gene panel.

Supplementary Table 5

Reagents and resources.

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Gating strategy for cell death analysis.

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Huggler, K.S., Flickinger, K.M., Forsberg, M.H. et al. Hexokinase detachment from mitochondria drives the Warburg effect to support compartmentalized ATP production. Nat Metab 8, 215–236 (2026). https://doi.org/10.1038/s42255-025-01428-1

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