Abstract
There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex–associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway–regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.
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References
Eggert, U.S. The why and how of phenotypic small-molecule screens. Nat. Chem. Biol. 9, 206–209 (2013).
Blagg, J. & Workman, P. Chemical biology approaches to target validation in cancer. Curr. Opin. Pharmacol. 17, 87–100 (2014).
Fisher, M. & Nelson, A. The chemical genetic approach: the interrogation of biological mechanisms with small molecule probes. in New Frontiers in Chemical Biology: Enabling Drug Discovery (ed. Bunnage, M.E.) 1–28 (Royal Society of Chemistry, 2011).
Bantscheff, M. & Drewes, G. Chemoproteomic approaches to drug target identification and drug profiling. Bioorg. Med. Chem. 20, 1973–1978 (2012).
Ong, S.E. et al. Identifying the proteins to which small-molecule probes and drugs bind in cells. Proc. Natl. Acad. Sci. USA 106, 4617–4622 (2009).
Swinney, D.C. & Anthony, J. How were new medicines discovered? Nat. Rev. Drug Discov. 10, 507–519 (2011).
Huang, S.M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).
Liu, J. et al. Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974. Proc. Natl. Acad. Sci. USA 110, 20224–20229 (2013).
Kahn, M. Can we safely target the WNT pathway? Nat. Rev. Drug Discov. 13, 513–532 (2014).
Mallinger, A. et al. Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen. J. Med. Chem. 58, 1717–1735 (2015).
Clevers, H. Wnt/beta-catenin signaling in development and disease. Cell 127, 469–480 (2006).
Angers, S. & Moon, R.T. Proximal events in Wnt signal transduction. Nat. Rev. Mol. Cell Biol. 10, 468–477 (2009).
Carlsten, J.O., Zhu, X. & Gustafsson, C.M. The multitalented Mediator complex. Trends Biochem. Sci. 38, 531–537 (2013).
Kim, S., Xu, X., Hecht, A. & Boyer, T.G. Mediator is a transducer of Wnt/beta-catenin signaling. J. Biol. Chem. 281, 14066–14075 (2006).
Allen, B.L. & Taatjes, D.J. The Mediator complex: a central integrator of transcription. Nat. Rev. Mol. Cell Biol. 16, 155–166 (2015).
Schiano, C., Casamassimi, A., Vietri, M.T., Rienzo, M. & Napoli, C. The roles of mediator complex in cardiovascular diseases. Biochim. Biophys. Acta 1839, 444–451 (2014).
Schiano, C. et al. Involvement of Mediator complex in malignancy. Biochim. Biophys. Acta 1845, 66–83 (2014).
Sharma, K. et al. Proteomics strategy for quantitative protein interaction profiling in cell extracts. Nat. Methods 6, 741–744 (2009).
Nemet, J., Jelicic, B., Rubelj, I. & Sopta, M. The two faces of Cdk8, a positive/negative regulator of transcription. Biochimie 97, 22–27 (2014).
Conaway, R.C. & Conaway, J.W. Function and regulation of the Mediator complex. Curr. Opin. Genet. Dev. 21, 225–230 (2011).
Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).
Schneider, E.V. et al. The structure of CDK8/CycC implicates specificity in the CDK/cyclin family and reveals interaction with a deep pocket binder. J. Mol. Biol. 412, 251–266 (2011).
Lipton, J.H. et al. Comparative efficacy of tyrosine kinase inhibitor treatments in the third-line setting, for chronic-phase chronic myelogenous leukemia after failure of second-generation tyrosine kinase inhibitors. Leuk. Res. 39, 58–64 (2015).
Cainap, C. et al. Linifanib versus sorafenib in patients with advanced hepatocellular carcinoma: results of a randomized phase III trial. J. Clin. Oncol. 33, 172–179 (2015).
Gozgit, J.M. et al. Ponatinib (AP24534), a multitargeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models. Mol. Cancer Ther. 11, 690–699 (2012).
Shankar, D.B. et al. ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia. Blood 109, 3400–3408 (2007).
Garuti, L., Roberti, M. & Bottegoni, G. Non-ATP competitive protein kinase inhibitors. Curr. Med. Chem. 17, 2804–2821 (2010).
Firestein, R. et al. CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity. Nature 455, 547–551 (2008).
Jardé, T. et al. In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-OΔN89beta-catenin system. Oncogene 32, 883–893 (2013).
Krasley, E., Cooper, K.F., Mallory, M.J., Dunbrack, R. & Strich, R. Regulation of the oxidative stress response through Slt2p-dependent destruction of cyclin C in Saccharomyces cerevisiae. Genetics 172, 1477–1486 (2006).
Mokry, M. et al. Integrated genome-wide analysis of transcription factor occupancy, RNA polymerase II binding and steady-state RNA levels identify differentially regulated functional gene classes. Nucleic Acids Res. 40, 148–158 (2012).
Bancerek, J. et al. CDK8 kinase phosphorylates transcription factor STAT1 to selectively regulate the interferon response. Immunity 38, 250–262 (2013).
Galbraith, M.D. et al. HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia. Cell 153, 1327–1339 (2013).
Donner, A.J., Ebmeier, C.C., Taatjes, D.J. & Espinosa, J.M. CDK8 is a positive regulator of transcriptional elongation within the serum response network. Nat. Struct. Mol. Biol. 17, 194–201 (2010).
Galbraith, M.D., Donner, A.J. & Espinosa, J.M. CDK8: a positive regulator of transcription. Transcription 1, 4–12 (2010).
Alarcón, C. et al. Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways. Cell 139, 757–769 (2009).
Rickert, P., Seghezzi, W., Shanahan, F., Cho, H. & Lees, E. Cyclin C/CDK8 is a novel CTD kinase associated with RNA polymerase II. Oncogene 12, 2631–2640 (1996).
Morris, E.J. et al. E2F1 represses beta-catenin transcription and is antagonized by both pRB and CDK8. Nature 455, 552–556 (2008).
Rieger, M.E., Sims, A.H., Coats, E.R., Clarke, R.B. & Briegel, K.J. The embryonic transcription cofactor LBH is a direct target of the Wnt signaling pathway in epithelial development and in aggressive basal subtype breast cancers. Mol. Cell. Biol. 30, 4267–4279 (2010).
Liu, B.Y. et al. Mammary tumor regression elicited by Wnt signaling inhibitor requires IGFBP5. Cancer Res. 72, 1568–1578 (2012).
Adler, A.S. et al. CDK8 maintains tumor dedifferentiation and embryonic stem cell pluripotency. Cancer Res. 72, 2129–2139 (2012).
Firestein, R. et al. CDK8 expression in 470 colorectal cancers in relation to beta-catenin activation, other molecular alterations and patient survival. Int. J. Cancer 126, 2863–2873 (2010).
Kim, M.Y., Han, S.I. & Lim, S.C. Roles of cyclin-dependent kinase 8 and beta-catenin in the oncogenesis and progression of gastric adenocarcinoma. Int. J. Oncol. 38, 1375–1383 (2011).
Porter, D.C. et al. Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities. Proc. Natl. Acad. Sci. USA 109, 13799–13804 (2012).
Kapoor, A. et al. The histone variant macroH2A suppresses melanoma progression through regulation of CDK8. Nature 468, 1105–1109 (2010).
Frye, S.V. The art of the chemical probe. Nat. Chem. Biol. 6, 159–161 (2010).
Workman, P. & Collins, I. Probing the probes: fitness factors for small molecule tools. Chem. Biol. 17, 561–577 (2010).
Cee, V.J., Chen, D.Y., Lee, M.R. & Nicolaou, K.C. Cortistatin A is a high-affinity ligand of protein kinases ROCK, CDK8, and CDK11. Angew. Chem. Int. Ed. Engl. 48, 8952–8957 (2009).
Schneider, E.V., Bottcher, J., Huber, R., Maskos, K. & Neumann, L. Structure-kinetic relationship study of CDK8/CycC specific compounds. Proc. Natl. Acad. Sci. USA 110, 8081–8086 (2013).
Huang, D., Zhou, T., Lafleur, K., Nevado, C. & Caflisch, A. Kinase selectivity potential for inhibitors targeting the ATP binding site: a network analysis. Bioinformatics 26, 198–204 (2010).
Neumann, L., von Konig, K. & Ullmann, D. HTS reporter displacement assay for fragment screening and fragment evolution toward leads with optimized binding kinetics, binding selectivity, and thermodynamic signature. Methods Enzymol. 493, 299–320 (2011).
Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method. Methods 25, 402–408 (2001).
Simon, R. et al. Design and Analysis of DNA Microarray Investigations (Springer, New York, 2003).
Korn, E.L. et al. Controlling the number of false discoveries: application to high-dimensional genomic data. J. Stat. Plan. Inference 75, 447–460 (2012).
Sansom, O.J. et al. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev. 18, 1385–1390 (2004).
Li, Y.Q. et al. Target cells for the cytotoxic effects of carcinogens in the murine small intestine. Carcinogenesis 13, 361–368 (1992).
Hamburger, A.W. & Salmon, S.E. Primary bioassay of human tumor stem cells. Science 197, 461–463 (1977).
Workman, P. et al. Guidelines for the welfare and use of animals in cancer research. Br. J. Cancer 102, 1555–1577 (2010).
Acknowledgements
This work was supported by Cancer Research UK (grant numbers C309/A11566, C368/A6743 and A368/A7990). We acknowledge Cancer Research UK funding to the Cancer Research UK Centre at The Institute of Cancer Research and The Royal Marsden National Health Service (NHS) funding to the National Institute for Health Research (NIHR) Biomedical Research Centre at the same institutions. We thank A. Mirza, M. Richards and M. Liu for their assistance with NMR, mass spectrometry and HPLC. We thank S. Gaus (Merck Serono) for excellent technical assistance. We thank the team of Proteros Biostructures GmbH, Martinsried, Germany for the Reporter Displacement Assay and in particular E.V. Schneider and A. Lammens for the X-ray co-crystal structure of CCT251545 with CDK8–cyclin C. We thank G.J. Feng and B. Lloyd Lewis for assistance with organoid growth experiments and F. Rudge for development of the esiRNA protocol (BBSRC grant number BB/G016887/1). We thank N. Evans for editorial assistance.
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J.B., A.M., K. Schiemann, and D. Waalboer designed and synthesized all new compounds; P.C. and D.M. analyzed crystallographic data; D.S. performed SPR experiments; G.B., W.C., K.E., E.F., S.G., O.A.-P., O.P., R.T.P., M.-J.O.-R., M.S. and R.S.S. developed and performed cell-based assays; K.E., A.d.H.B. and M.V. performed in vivo studies. A.B., P.A.C., T.D., C.E., S.A.E., A.M., F. Rohdich, F. Raynaud, K. Schiemann, K. Schneider, R.S., P.W., D. Wienke and J.B. designed studies and analyzed results. J.B., P.A.C., T.D. and D. Wienke wrote the paper.
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T.D., P.A.C., W.C., D.W., O.A.P., M.O.R., A.M., R.S.S., M.S., E.F., R.T.P., A.H.B., M.V., G.B., S.G., F. Raynaud, P.W., S.A.E. and J.B. are current or former employees of The Institute of Cancer Research, which has a commercial interest in the development of WNT pathway inhibitors. C.E., P.C., F. Rohdich, D.M., K. Schiemann, K. Schneider, D.S., R.S., O.P., A.B. and D.W. are current or former employees of Merck Serono, which has a commercial interest in the development of WNT pathway inhibitors.
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Supplementary Text and Figures
Supplementary Results, Supplementary Tables 1–12 and Supplementary Figures 1–18. (PDF 22997 kb)
Supplementary Note
Compound synthetic methods. (PDF 635 kb)
Supplementary Data Set 1
Identification of significantly enriched transcription factor binding sites. (XLSX 30 kb)
Supplementary Data Set 2
Identification of significantly enriched pathways. (XLSX 25 kb)
Supplementary Data Set 3
Analysis of gene expression regulated by transcription factors with links to CDK8 and/or CDK19. (XLSX 110 kb)
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Dale, T., Clarke, P., Esdar, C. et al. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease. Nat Chem Biol 11, 973–980 (2015). https://doi.org/10.1038/nchembio.1952
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DOI: https://doi.org/10.1038/nchembio.1952
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