Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors

Nature Chemical Biology volume 12, pages 876–884 (2016)Cite this article

Subjects

Abstract

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: THZ531 covalently inhibits CDK12 and CDK13.
Figure 2: THZ531 co-crystal structure with CDK12–cyclin K.
Figure 3: THZ531 induces apoptosis in Jurkat cells.
Figure 4: CDK12 binds regulatory and coding regions of active genes.
Figure 5: THZ531 inhibits transcriptional elongation.
Figure 6: THZ531 reduces DDR and super-enhancer gene expression.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

NCBI Reference Sequence

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Young, R.A. RNA polymerase II. Annu. Rev. Biochem. 60, 689–715 (1991).

    Article  CAS  PubMed  Google Scholar 

  2. Buratowski, S. Progression through the RNA polymerase II CTD cycle. Mol. Cell 36, 541–546 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fuda, N.J., Ardehali, M.B. & Lis, J.T. Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 461, 186–192 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Muñoz, M.J., de la Mata, M. & Kornblihtt, A.R. The carboxy terminal domain of RNA polymerase II and alternative splicing. Trends Biochem. Sci. 35, 497–504 (2010).

    Article  PubMed  Google Scholar 

  5. Sansó, M. & Fisher, R.P. Pause, play, repeat: CDKs push RNAP II's buttons. Transcription 4, 146–152 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Peterlin, B.M. & Price, D.H. Controlling the elongation phase of transcription with P-TEFb. Mol. Cell 23, 297–305 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Bartkowiak, B. et al. CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev. 24, 2303–2316 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cho, E.J., Kobor, M.S., Kim, M., Greenblatt, J. & Buratowski, S. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. Genes Dev. 15, 3319–3329 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chen, H.H., Wang, Y.C. & Fann, M.J. Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation. Mol. Cell. Biol. 26, 2736–2745 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, H.H., Wong, Y.H., Geneviere, A.M. & Fann, M.J. CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing. Biochem. Biophys. Res. Commun. 354, 735–740 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Liang, K. et al. Characterization of human cyclin-dependent kinase 12 (CDK12) and CDK13 complexes in C-terminal domain phosphorylation, gene transcription, and RNA processing. Mol. Cell. Biol. 35, 928–938 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Eifler, T.T. et al. Cyclin-dependent kinase 12 increases 3′ end processing of growth factor-induced c-FOS transcripts. Mol. Cell. Biol. 35, 468–478 (2015).

    Article  PubMed  Google Scholar 

  13. Davidson, L., Muniz, L. & West, S. 3′ end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells. Genes Dev. 28, 342–356 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bartkowiak, B. & Greenleaf, A.L. Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex. J. Biol. Chem. 290, 1786–1795 (2015).

    Article  PubMed  Google Scholar 

  15. Ahn, S.H., Kim, M. & Buratowski, S. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3′ end processing. Mol. Cell 13, 67–76 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Blazek, D. et al. The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes. Genes Dev. 25, 2158–2172 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Winsor, T.S., Bartkowiak, B., Bennett, C.B. & Greenleaf, A.L. A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II. PLoS One 8, e60909 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bennett, C.B. et al. Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction. PLoS One 3, e1448 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Ekumi, K.M. et al. Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via deficient formation and function of the Cdk12/CycK complex. Nucleic Acids Res. 43, 2575–2589 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rottenberg, S. et al. High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc. Natl. Acad. Sci. USA 105, 17079–17084 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bajrami, I. et al. Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity. Cancer Res. 74, 287–297 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Joshi, P.M., Sutor, S.L., Huntoon, C.J. & Karnitz, L.M. Ovarian cancer-associated mutations disable catalytic activity of CDK12, a kinase that promotes homologous recombination repair and resistance to cisplatin and poly(ADP-ribose) polymerase inhibitors. J. Biol. Chem. 289, 9247–9253 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bartkowiak, B., Yan, C. & Greenleaf, A.L. Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas. Biochim. Biophys. Acta 1849, 1179–1187 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bösken, C.A. et al. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505 (2014).

    Article  PubMed  Google Scholar 

  25. Greifenberg, A.K. et al. Structural and functional analysis of the Cdk13/Cyclin K complex. Cell Rep. 14, 320–331 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. Lolli, G., Lowe, E.D., Brown, N.R. & Johnson, L.N. The crystal structure of human CDK7 and its protein recognition properties. Structure 12, 2067–2079 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Kwiatkowski, N. et al. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 511, 616–620 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Patricelli, M.P. et al. In situ kinase profiling reveals functionally relevant properties of native kinases. Chem. Biol. 18, 699–710 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Christensen, C.L. et al. Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor. Cancer Cell 26, 909–922 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chipumuro, E. et al. CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN-driven cancer. Cell 159, 1126–1139 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Larochelle, S. et al. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II. Nat. Struct. Mol. Biol. 19, 1108–1115 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mansour, M.R. et al. Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. Science 346, 1373–1377 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sanda, T. et al. Core transcriptional regulatory circuit controlled by the TAL1 complex in human T cell acute lymphoblastic leukemia. Cancer Cell 22, 209–221 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bowman, E.A. & Kelly, W.G. RNA polymerase II transcription elongation and Pol II CTD Ser2 phosphorylation: A tail of two kinases. Nucleus 5, 224–236 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Ferrando, A.A. et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell 1, 75–87 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Brown, L. et al. Site-specific recombination of the tal-1 gene is a common occurrence in human T cell leukemia. EMBO J. 9, 3343–3351 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Czudnochowski, N., Bösken, C.A. & Geyer, M. Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition. Nat. Commun. 3, 842 (2012).

    Article  PubMed  Google Scholar 

  38. Zhang, T. et al. Discovery of potent and selective covalent inhibitors of JNK. Chem. Biol. 19, 140–154 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhang, Z. & Marshall, A.G. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J. Am. Soc. Mass Spectrom. 9, 225–233 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Ficarro, S.B. et al. Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells. Anal. Chem. 81, 3440–3447 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. Askenazi, M., Parikh, J.R. & Marto, J.A. mzAPI: a new strategy for efficiently sharing mass spectrometry data. Nat. Methods 6, 240–241 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Evans, P.R. & Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. The PyMOL Molecular Graphics System (Version 1.2r3pre, Schrödinger, LLC).

  50. Lovén, J. et al. Revisiting global gene expression analysis. Cell 151, 476–482 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Mootha, V.K. et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267–273 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Huang, W., Sherman, B.T. & Lempicki, R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

    Article  Google Scholar 

  53. Lin, C.Y. et al. Transcriptional amplification in tumor cells with elevated c-Myc. Cell 151, 56–67 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Zhang, Y. et al. Model-based analysis of ChIP–Seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

  56. Pruitt, K.D. et al. RefSeq: an update on mammalian reference sequences. Nucleic Acids Res. 42, D756–D763 (2014).

    Article  CAS  PubMed  Google Scholar 

  57. Lovén, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013).

    PubMed  PubMed Central  Google Scholar 

  58. Whyte, W.A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. George, K. Wong, P. Hammerman, J. Bradner and members of the Gray and Young laboratories for helpful discussions. We thank P. Wisniewski and C. Zollo from the Whitehead FACS facility for help with FACS instruments. We thank J. Love, T. Volkert, and S. Gupta from the Whitehead Genome Core for help with genomics experiments. The authors would like to thank Diamond Light Source for beamtime (proposal mx8421), and the staff of Beamline I03 for assistance with data collection. We thank W. Massefski from the Dana–Farber NMR lab for help in collecting NMR data for the manuscript. M.G. is a member of the DFG excellence cluster ImmunoSensation. This work was supported by the National Institutes of Health (HG002668 and CA109901 to R.A.Y. and 5 R01 CA179483-02 C to N.S.G.), the Koch Institute and Dana–Farber/Harvard Cancer Center Bridge Grant (N.K., N.S.G., R.A.Y.), the DFG (GE 976/9-1 to M.G.), the Hope Funds for Cancer Research Grillo-Marxuach Family Fellowship (B.J.A.), and a NDM Prize Studentship, funded in part by the Medical Research Council (S.E.D.-C.). The SGC is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Genome Canada through Ontario Genomics Institute Grant OGI-055, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and Wellcome Trust Grant 092809/Z/10/Z.

Author information

Author notes

  1. Tinghu Zhang, Nicholas Kwiatkowski and Calla M Olson: These authors contributed equally to this work.

Authors and Affiliations

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

    Tinghu Zhang, Nicholas Kwiatkowski, Calla M Olson, Scott B Ficarro, Yanke Liang, Theresa Manz, Mingfeng Hao, Jarrod A Marto & Nathanael S Gray

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Tinghu Zhang, Nicholas Kwiatkowski, Calla M Olson, Scott B Ficarro, Yanke Liang, Mingfeng Hao, Jarrod A Marto & Nathanael S Gray

  3. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Nicholas Kwiatkowski, Brian J Abraham, Nancy M Hannett & Richard A Young

  4. Structural Genomics Consortium, University of Oxford, Oxford, UK

    Sarah E Dixon-Clarke, Jonathan M Elkins & Alex N Bullock

  5. Department of Structural Immunology, Institute of Innate Immunity, University of Bonn, Bonn, Germany

    Ann K Greifenberg & Matthias Geyer

  6. Center of Advanced European Studies and Research, Bonn, Germany

    Ann K Greifenberg & Matthias Geyer

  7. Blais Proteomics Center, Dana–Farber Cancer Institute, Boston, Massachusetts, USA

    Scott B Ficarro & Jarrod A Marto

  8. Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, Germany

    Theresa Manz

  9. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA

    Bartlomiej Bartkowiak & Arno L Greenleaf

  10. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Richard A Young

Authors
  1. Tinghu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicholas Kwiatkowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Calla M Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sarah E Dixon-Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brian J Abraham
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ann K Greifenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Scott B Ficarro
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jonathan M Elkins
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yanke Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Nancy M Hannett
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Theresa Manz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Mingfeng Hao
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Bartlomiej Bartkowiak
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Arno L Greenleaf
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Jarrod A Marto
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Matthias Geyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Alex N Bullock
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Richard A Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Nathanael S Gray
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.S.G., R.A.Y., N.K., and T.Z. conceived the project. N.S.G. and T.Z. conceived and directed chemistry effort with input from Y.L. Chemical synthesis and small molecule structure determination was performed by T.Z., M.H., and T.M. R.A.Y., N.S.G., B.J.A. and N.K. conceived genomics effort. N.K. and C.M.O. designed and executed cellular biological experimental research with input from N.S.G. and R.A.Y.S.E.D.-C. solved co-crystal structure of CDK12–cyclin K with THZ531 with guidance from J.M.E. and A.N.B. A.K.G. designed and executed CDK in vitro kinase assays with input from M.G. S.B.F. designed and performed protein mass spectrometry on THZ531–CDK12 adducts with input from J.A.M. N.M.H. provided cloning expertise. B.B. and A.L.G. developed and provided CDK12 and CDK13 antibodies. B.J.A. designed and performed genomics data analyses. N.K., T.Z., N.S.G. and R.A.Y. co-wrote the paper. All authors edited the manuscript.

Corresponding authors

Correspondence to Richard A Young or Nathanael S Gray.

Ethics declarations

Competing interests

N.S.G., T.Z., N.K. are inventors on patent applications covering THZ531 (WO 1421 2015/058126, WO/2014/063068, WO 2015/058140), which are licensed to a company Syros co-founded by N.S.G. and R.A.Y.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–11 and Supplementary Tables 1–3. (PDF 33824 kb)

Supplementary Note 1

Synthetic Procedures (PDF 2237 kb)

Supplementary Data Set 1

Mass spectrometry identifies CDK12-cyclin K and CDK13–cyclin K complexes as major targets of bioTHZ531 in Jurkat cell lysates. (XLSX 96 kb)

Supplementary Data Set 2

In vitro Ambit™ binding assay shows THZ531 potently inhibits CDK13. (XLS 74 kb)

Supplementary Data Set 3

Gene expression microarray data of THZ531, Flavopiridol, THZ1-treated cells. (XLSX 8424 kb)

Supplementary Data Set 4

Jurkat enhancers and super–enhancers identified by H3K27Ac ChIP–seq. (XLS 2505 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, T., Kwiatkowski, N., Olson, C. et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat Chem Biol 12, 876–884 (2016). https://doi.org/10.1038/nchembio.2166

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2166

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer