D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation1,2. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results2. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3–CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3–CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3–CDK6 complexes. We propose that measuring the levels of cyclin D3–CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3–CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Cyclin-dependent kinases. Genome Biol. 15, 122 (2014)

  2. 2.

    , & Targeting CDK4 and CDK6: from discovery to therapy. Cancer Discov. 6, 353–367 (2016)

  3. 3.

    , & Cyclins and CDKs in development and cancer: a perspective. Oncogene 24, 2909–2915 (2005)

  4. 4.

    , , , & Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure. Oncogene 29, 4018–4032 (2010)

  5. 5.

    et al. A synthetic lethal interaction between K-Ras oncogenes and Cdk4 unveils a therapeutic strategy for non-small cell lung carcinoma. Cancer Cell 18, 63–73 (2010)

  6. 6.

    et al. Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors. Cell Cycle 11, 2756–2761 (2012)

  7. 7.

    et al. The requirement for cyclin D function in tumor maintenance. Cancer Cell 22, 438–451 (2012)

  8. 8.

    et al. Therapeutic targeting of the cyclin D3:CDK4/6 complex in T cell leukemia. Cancer Cell 22, 452–465 (2012)

  9. 9.

    , & Regulation of cancer cell metabolism. Nat. Rev. Cancer 11, 85–95 (2011)

  10. 10.

    et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230–233 (2008)

  11. 11.

    , & The molecular determinants of de novo nucleotide biosynthesis in cancer cells. Curr. Opin. Genet. Dev. 19, 32–37 (2009)

  12. 12.

    et al. The crystal structures of eukaryotic phosphofructokinases from baker’s yeast and rabbit skeletal muscle. J. Mol. Biol. 407, 284–297 (2011)

  13. 13.

    , , , & Functional linkage of adenine nucleotide binding sites in mammalian muscle 6-phosphofructokinase. J. Biol. Chem. 287, 17546–17553 (2012)

  14. 14.

    , , , & Structure and allosteric regulation of eukaryotic 6-phosphofructokinases. Biol. Chem. 394, 977–993 (2013)

  15. 15.

    Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumor cells. Int. J. Biochem. Cell Biol. 43, 969–980 (2011)

  16. 16.

    et al. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 334, 1278–1283 (2011)

  17. 17.

    et al. Cells overexpressing fructose-2,6-bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress. FEBS Lett. 480, 261–264 (2000)

  18. 18.

    et al. Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature 491, 458–462 (2012)

  19. 19.

    et al. Phosphofructokinase 1 glycosylation regulates cell growth and metabolism. Science 337, 975–980 (2012)

  20. 20.

    et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510, 298–302 (2014)

  21. 21.

    et al. Characterization of phosphofructokinase activity in Mycobacterium tuberculosis reveals that a functional glycolytic carbon flow is necessary to limit the accumulation of toxic metabolic intermediates under hypoxia. PLoS One 8, e56037 (2013)

  22. 22.

    & Phosphofructokinases from Escherichia coli. Methods Enzymol. 90, 60–70 (1982)

  23. 23.

    , , & Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro. Reproduction 124, 675–681 (2002)

  24. 24.

    , , & Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer. J. Biol. Chem. 268, 13178–13186 (1993)

  25. 25.

    et al. The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis. J. Biol. Chem. 271, 17875–17880 (1996)

  26. 26.

    et al. Loss of RBF1 changes glutamine catabolism. Genes Dev. 27, 182–196 (2013)

  27. 27.

    , & Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab. Eng. 8, 324–337 (2006)

  28. 28.

    , & Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions. Metab. Eng. 9, 68–86 (2007)

  29. 29.

    , , & Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line. J. Biol. Chem. 283, 20621–20627 (2008)

  30. 30.

    , , , & An elementary metabolite unit (EMU) based method of isotopically nonstationary flux analysis. Biotechnol. Bioeng. 99, 686–699 (2008)

  31. 31.

    et al. Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors. NMR Biomed. 25, 1177–1186 (2012)

  32. 32.

    et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010)

  33. 33.

    , & An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994)

  34. 34.

    & Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007)

  35. 35.

    , , , & A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006)

  36. 36.

    et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat. Med. 21, 1318–1325 (2015)

Download references


Supported by R01 CA083688, R01 CA202634, P01 CA080111 (P.S.), R01 CA163698 (N.J.D.) and F32 CA165856 (B.N.N.). N.J.D. is a James and Shirley Curvey MGH Research Scholar. X.G. was supported by an NIH post-doc training grant (T32CA009361) and NIH grant P50 CA090381-14 (DF/HCC SPORE in Prostate Cancer). J.M.S. was supported by a Mobilnos´c´ Plus fellowship. We thank M. Eck, J. Daly, P. Hydbring, T. Otto, W. Michowski and I. Harris for help.

Author information


  1. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Haizhen Wang
    • , Xueliang Gao
    • , Yan Geng
    • , Hong Ren
    • , Jan M. Suski
    • , Thomas M. Roberts
    •  & Piotr Sicinski
  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Haizhen Wang
    • , Yan Geng
    • , Hong Ren
    • , Jan M. Suski
    •  & Piotr Sicinski
  3. Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA

    • Brandon N. Nicolay
    •  & Nicholas J. Dyson
  4. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Joel M. Chick
    •  & Steven P. Gygi
  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Xueliang Gao
    •  & Thomas M. Roberts
  6. Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA

    • Hui Gao
    • , Guizhi Yang
    •  & Juliet A. Williams
  7. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 USA

    • Mark A. Keibler
  8. Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Ewa Sicinska
  9. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Ulrike Gerdemann
    •  & W. Nicholas Haining
  10. Division of Pediatric Hematology and Oncology, Children’s Hospital, Boston, Massachusetts 02115, USA

    • W. Nicholas Haining
  11. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • W. Nicholas Haining
  12. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Kornelia Polyak


  1. Search for Haizhen Wang in:

  2. Search for Brandon N. Nicolay in:

  3. Search for Joel M. Chick in:

  4. Search for Xueliang Gao in:

  5. Search for Yan Geng in:

  6. Search for Hong Ren in:

  7. Search for Hui Gao in:

  8. Search for Guizhi Yang in:

  9. Search for Juliet A. Williams in:

  10. Search for Jan M. Suski in:

  11. Search for Mark A. Keibler in:

  12. Search for Ewa Sicinska in:

  13. Search for Ulrike Gerdemann in:

  14. Search for W. Nicholas Haining in:

  15. Search for Thomas M. Roberts in:

  16. Search for Kornelia Polyak in:

  17. Search for Steven P. Gygi in:

  18. Search for Nicholas J. Dyson in:

  19. Search for Piotr Sicinski in:


H.W. and P.S. designed the study. H.W. performed all experiments with the help of collaborators as follows. B.N.N. and N.J.D. performed and interpreted isotopic enrichment analyses. J.M.C. and S.P.G. contributed mass spectrometric and biocomputational analyses. X.G. helped with viral transductions and kinase assays. Y.G. helped with T-ALL xenografts and kinase assays. H.R. helped with analyses of cyclin levels and tissue culture. J.M.S. helped with design and construction of expression vectors. T.M.R. helped with supervision. H.G., G.Y. and J.A.W. contributed ribociclib xenograft studies. M.A.K. contributed some isotopic-enrichment analyses. E.S. carried out pathological analyses. U.G. and W.N.H. isolated human T cells. K.P. helped with breast cancer studies. H.W. and P.S. wrote the paper. P.S. directed the study.

Competing interests

P.S., T.M.R. and K.P. are consultants and recipients of research grants from Novartis. H.G., G.Y. and J.A.W. are employees of Novartis Institutes for BioMedical Research.

Corresponding author

Correspondence to Piotr Sicinski.

Reviewer Information Nature thanks J. Bartek, H. Christofk and A. Schulze for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure

    This file contains the uncropped blots.

Excel files

  1. 1.

    Supplementary Table 1

    This tablecontains affinity purification tandem mass spectrometry analysis of CDK6 interactome in T-ALL cells.

  2. 2.

    Supplementary Table 2

    This table contains gene set enrichment analysis of common CDK6 interactors.

  3. 3.

    Supplementary Table 3

    This table contains CDK6-associated glycolytic enzymes that contain potential CDK phosphorylation residues.

  4. 4.

    Supplementary Table 4

    This table contains LC-MS/MS analysis of cyclin D3-CDK6 dependent phosphorylation sites on PKM2 and PFKP.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Newsletter Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing