Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation

Abstract

Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: THAL-SNS-032 selectively induces degradation of CDK9.
Figure 2: NVP-2 selectively inhibits CDK9.
Figure 3: THAL-SNS-032 exhibits CRBN-dependent anti-proliferative and pro-apoptotic effects.
Figure 4: THAL-SNS-032 exhibits transcriptional effects consistent with a selective CDK9 inhibitor.
Figure 5: THAL-SNS-032 diminishes elongating polymerase II.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. 1

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

    PubMed  PubMed Central  Article  Google Scholar 

  2. 2

    Dorée, M. & Galas, S. The cyclin-dependent protein kinases and the control of cell division. FASEB J. 8, 1114–1121 (1994).

    PubMed  Article  PubMed Central  Google Scholar 

  3. 3

    Sun, T., Co, N.N. & Wong, N. PFTK1 interacts with cyclin Y to activate non-canonical Wnt signaling in hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 449, 163–168 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4

    Liu, Y., Cheng, K., Gong, K., Fu, A.K.Y. & Ip, N.Y. Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis. J. Biol. Chem. 281, 9852–9858 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5

    Peng, J., Marshall, N.F. & Price, D.H. Identification of a cyclin subunit required for the function of Drosophila P-TEFb. J. Biol. Chem. 273, 13855–13860 (1998).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6

    Hirose, Y. & Ohkuma, Y. Phosphorylation of the C-terminal domain of RNA polymerase II plays central roles in the integrated events of eucaryotic gene expression. J. Biochem. 141, 601–608 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. 7

    Wada, T., Takagi, T., Yamaguchi, Y., Watanabe, D. & Handa, H. Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro. EMBO J. 17, 7395–7403 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8

    Yamada, T. et al. P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Mol. Cell 21, 227–237 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9

    Gilchrist, D.A. et al. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. Genes Dev. 22, 1921–1933 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10

    Huang, C.-H. et al. CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma. Genes Dev. 28, 1800–1814 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11

    Lu, H. et al. Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism. eLife 4, e06535 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  12. 12

    Rahl, P.B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13

    Okuda, H., Takahashi, S., Takaori-Kondo, A. & Yokoyama, A. TBP loading by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription. Cell Cycle 15, 2712–2722 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14

    Samarakkody, A. et al. RNA polymerase II pausing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition. Nucleic Acids Res. 43, 3938–3949 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15

    Chao, S.-H. et al. Flavopiridol inhibits P-TEFb and blocks HIV-1 replication. J. Biol. Chem. 275, 28345–28348 (2000).

    CAS  PubMed  Article  Google Scholar 

  16. 16

    König, A., Schwartz, G.K., Mohammad, R.M., Al-Katib, A. & Gabrilove, J.L. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 90, 4307–4312 (1997).

    PubMed  Article  Google Scholar 

  17. 17

    Sedlacek, H. et al. Flavopiridol (L86 8275; NSC 649890), a new kinase inhibitor for tumor therapy. Int. J. Oncol. 9, 1143–1168 (1996).

    CAS  PubMed  Google Scholar 

  18. 18

    Chen, R., Keating, M.J., Gandhi, V. & Plunkett, W. Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death. Blood 106, 2513–2519 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19

    Krystof, V. & Uldrijan, S. Cyclin-dependent kinase inhibitors as anticancer drugs. Curr. Drug Targets 11, 291–302 (2010).

    CAS  PubMed  Article  Google Scholar 

  20. 20

    Misra, R.N. et al. N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent. J. Med. Chem. 47, 1719–1728 (2004).

    CAS  PubMed  Article  Google Scholar 

  21. 21

    Winter, G.E. et al. Drug development. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22

    Lu, J. et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 22, 755–763 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23

    Lai, A.C. et al. Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew. Chem. Int. Ed. Engl. 55, 807–810 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24

    Rodríguez-Molina, J.B., Tseng, S.C., Simonett, S.P., Taunton, J. & Ansari, A.Z. Engineered covalent inactivation of TFIIH-kinase reveals an elongation checkpoint and results in widespread mRNA stabilization. Mol. Cell 63, 433–444 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. 25

    Poss, Z.C. et al. Identification of mediator kinase substrates in human cells using cortistatin A and quantitative phosphoproteomics. Cell Rep. 15, 436–450 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26

    Kanin, E.I. et al. Chemical inhibition of the TFIIH-associated kinase Cdk7/Kin28 does not impair global mRNA synthesis. Proc. Natl. Acad. Sci. USA 104, 5812–5817 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28

    Winter, G.E. et al. BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol. Cell 67, 5–18 e19 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29

    McAlister, G.C. et al. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal. Chem. 84, 7469–7478 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30

    Barsanti, P.A. et al. Pyridine and pyrzaine derivatives as protein kinase modulators. International Patent No. PCT/JP2008/073864 (WO/2011/012661) (2011).

  31. 31

    Zhang, T. et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat. Chem. Biol. 12, 876–884 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33

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

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. 34

    Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35

    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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36

    Mitra, P., Yang, R.M., Sutton, J., Ramsay, R.G. & Gonda, T.J. CDK9 inhibitors selectively target estrogen-receptor-positive breast cancer cells through combined inhibition of MYB and MCL-1 expression. Oncotarget 7, 9069–9083 (2016).

    PubMed  PubMed Central  Google Scholar 

  37. 37

    Bonhoure, N. et al. Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization. Genome Res. 24, 1157–1168 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38

    Wade, J.T. & Struhl, K. The transition from transcriptional initiation to elongation. Curr. Opin. Genet. Dev. 18, 130–136 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39

    Patricelli, M.P. et al. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46, 350–358 (2007).

    CAS  PubMed  Article  Google Scholar 

  40. 40

    Nomanbhoy, T.K. et al. Chemoproteomic evaluation of target engagement by the cyclin-dependent kinase 4 and 6 inhibitor Palbociclib correlates with cancer cell response. Biochemistry 55, 5434–5441 (2016).

    CAS  PubMed  Article  Google Scholar 

  41. 41

    Edwards, A. & Haas, W. in Multiplexed Quantitative Proteomics for High-Throughput Comprehensive Proteome Comparisons of Human Cell Lines. Proteomics in Systems Biology: Methods and Protocols (ed. J. Reinders) 1–13 (Springer New York, New York, NY, 2016).

    Google Scholar 

  42. 42

    McAlister, G.C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86, 7150–7158 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43

    Ting, L., Rad, R., Gygi, S.P. & Haas, W. MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat. Methods 8, 937–940 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44

    R Development Core Team. (R Foundation for Statistical Computing Vienna, Austria, 2013).

  45. 45

    Ritchie, M.E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. 46

    Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47

    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  CAS  Google Scholar 

  48. 48

    Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    CAS  Article  Google Scholar 

  49. 49

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

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. 50

    Orlando, D.A. et al. Quantitative ChIP-Seq normalization reveals global modulation of the epigenome. Cell Rep. 9, 1163–1170 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D. Buckley, E. Wang, J. Paulk, and members of the Gray and Bradner laboratories for helpful discussions. This work was supported by the US National Institutes of Health (CA179483-03 to N.K. and N.S.G., and T32GM007753 to Z.Z.), the Koch Institute and Dana-Farber/Harvard Cancer Center Bridge Grant (N.K., N.S.G., R.A.Y., C.M.O.),

Author information

Affiliations

Authors

Contributions

N.S.G., C.M.O., N.K. and T.Z. conceived the project. N.S.G., Y.L., B.J. and T.Z. conceived and directed chemistry effort. Chemical synthesis and small-molecule structure determination was performed by T.Z., Y.L. and Z.Z. R.A.Y., N.S.G., C.M.O., M.A.E., G.E.W. 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. W.H. and M.B. designed and executed the proteomics efforts. E.S.F. designed and executed statistical analysis of proteomics. T.N. and J.G. designed and executed the Kinativ efforts. M.A.E. designed and performed genomics data analyses. C.M.O., N.K. and N.S.G. wrote the manuscript. All of the authors edited the manuscript.

Corresponding author

Correspondence to Nathanael S Gray.

Ethics declarations

Competing interests

N.S.G. is a Scientific Founder and member of the Scientific Advisory Board of C4 Therapeutics, Syros Pharmaceuticals and Petra Pharmaceuticals and is the inventor on intellectual property licensed to these entities. J.E.B. is a Scientific Founder of Syros Pharmaceuticals, SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics (now Roche) and C4 Therapeutics and is the inventor on intellectual property licensed to these entities. J.E.B. is now an executive and shareholder in Novartis AG. R.A.Y. is a Scientific Founder of Syros Pharmaceuticals and Marauder Therapeutics.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2, Supplementary Figures 1–9 (PDF 2413 kb)

Life Sciences Reporting Summary (PDF 158 kb)

Supplementary Note 1

Chemical Synthesis of THAL-SNS-032, NVP-2, THAL-NVP-2-02-099, THAL-NVP-2- 3 03-069, HAL-NVP-2-03-099, THAL-NVP-2-03-084, THAL-NVP-2-03-105. (PDF 668 kb)

Supplementary Dataset 1

Lysate Kinativ of SNS-032, THAL-SNS-032, and NVP-2 (XLSX 39 kb)

Supplementary Dataset 2

Proteomics of THAL-SNS-032 treatment (XLSX 328 kb)

Supplementary Dataset 3

Selectivity of NVP-2 by Ambit profiling (XLSX 23 kb)

Supplementary Dataset 4

Live cell Kinativ of SNS-032 and THAL-SNS-032 (XLSX 25 kb)

Supplementary Dataset 5

Live cell Kinativ of NVP-2 (XLSX 29 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Olson, C., Jiang, B., Erb, M. et al. Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation. Nat Chem Biol 14, 163–170 (2018). https://doi.org/10.1038/nchembio.2538

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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