Abstract
Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.
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References
Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).
Shilatifard, A. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annu. Rev. Biochem. 75, 243–269 (2006).
Ernst, T. et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat. Genet. 42, 722–726 (2010).
Nikoloski, G. et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat. Genet. 42, 665–667 (2010).
De Raedt, T. et al. PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies. Nature 514, 247–251 (2014).
Lee, W. et al. PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors. Nat. Genet. 46, 1227–1232 (2014).
Ntziachristos, P. et al. Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nat. Med. 18, 298–301 (2012).
Bracken, A.P. et al. EZH2 is downstream of the pRB–E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 22, 5323–5335 (2003).
Yu, J. et al. A polycomb repression signature in metastatic prostate cancer predicts cancer outcome. Cancer Res. 67, 10657–10663 (2007).
Wang, C. et al. EZH2 Mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR. Cancer Res. 72, 315–324 (2012).
Morin, R.D. et al. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat. Genet. 42, 181–185 (2010).
Majer, C.R. et al. A687V EZH2 is a gain-of-function mutation found in lymphoma patients. FEBS Lett. 586, 3448–3451 (2012).
McCabe, M.T. et al. Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). Proc. Natl. Acad. Sci. USA 109, 2989–2994 (2012).
Yap, D.B. et al. Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood 117, 2451–2459 (2011).
Qi, W. et al. Selective inhibition of Ezh2 by a small-molecule inhibitor blocks tumor cells proliferation. Proc. Natl. Acad. Sci. USA 109, 21360–21365 (2012).
Campbell, J.E. et al. EPZ011989, A potent, orally-available EZH2 inhibitor with robust in vivo activity. ACS Med. Chem. Lett. 6, 491–495 (2015).
Garapaty-Rao, S. et al. Identification of EZH2 and EZH1 small-molecule inhibitors with selective impact on diffuse large B cell lymphoma cell growth. Chem. Biol. 20, 1329–1339 (2013).
Knutson, S.K. et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat. Chem. Biol. 8, 890–896 (2012).
Nasveschuk, C.G. et al. Discovery and optimization of tetramethylpiperidinyl benzamides as inhibitors of EZH2. ACS Med. Chem. Lett. 5, 378–383 (2014).
Konze, K.D. et al. An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem. Biol. 8, 1324–1334 (2013).
McCabe, M.T. et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492, 108–112 (2012).
Brooun, A. et al. Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance. Nat. Commun. 7, 11384 (2016).
Cao, R. & Zhang, Y. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Curr. Opin. Genet. Dev. 14, 155–164 (2004).
Cao, R. & Zhang, Y. SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol. Cell 15, 57–67 (2004).
Margueron, R. et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).
Jiao, L. & Liu, X. Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2. Science 350, aac4383 (2015).
Justin, N. et al. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2. Nat. Commun. 7, 11316 (2016).
Xu, C. et al. Binding of different histone marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2). Proc. Natl. Acad. Sci. USA 107, 19266–19271 (2010).
Han, Z. et al. Structural basis of EZH2 recognition by EED. Structure 15, 1306–1315 (2007).
Sneeringer, C.J. et al. Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc. Natl. Acad. Sci. USA 107, 20980–20985 (2010).
Yu, Y. et al. Quantitative profiling of combinational K27/K36 modifications on histone H3 variants in mouse organs. J. Proteome Res. 15, 1070–1079 (2016).
Yuan, W. et al. H3K36 methylation antagonizes PRC2-mediated H3K27 methylation. J. Biol. Chem. 286, 7983–7989 (2011).
Knutson, S.K. et al. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Mol. Cancer Ther. 13, 842–854 (2014).
Baker, T. et al. Acquisition of a single EZH2 D1 domain mutation confers acquired resistance to EZH2-targeted inhibitors. Oncotarget 6, 32646–32655 (2015).
Gibaja, V. et al. Development of secondary mutations in wild-type and mutant EZH2 alleles cooperates to confer resistance to EZH2 inhibitors. Oncogene 35, 558–566 (2016).
Baselga, J. et al. Lapatinib with trastuzumab for HER2-positive early breast cancer (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial. Lancet 379, 633–640 (2012).
Baselga, J. et al. Relationship between tumor biomarkers and efficacy in EMILIA, a phase III Study of trastuzumab emtansine in HER2-positive metastatic breast cancer. Clin. Cancer Res. 22, 3755–3763 (2016).
Mochizuki-Kashio, M. et al. Ezh2 loss in hematopoietic stem cells predisposes mice to develop heterogeneous malignancies in an Ezh1-dependent manner. Blood 126, 1172–1183 (2015).
Xu, C. & Min, J. Structure and function of WD40 domain proteins. Protein Cell 2, 202–214 (2011).
Zhang, C. & Zhang, F. The multifunctions of WD40 proteins in genome integrity and cell cycle progression. J Genomics 3, 40–50 (2015).
Stirnimann, C.U., Petsalaki, E., Russell, R.B. & Müller, C.W. WD40 proteins propel cellular networks. Trends Biochem. Sci. 35, 565–574 (2010).
Migliori, V., Mapelli, M. & Guccione, E. On WD40 proteins: propelling our knowledge of transcriptional control? Epigenetics 7, 815–822 (2012).
Cao, F. et al. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia. Mol. Cell 53, 247–261 (2014).
Li, Y. et al. Structural basis for activity regulation of MLL family methyltransferases. Nature 530, 447–452 (2016).
Antonysamy, S. et al. Crystal structure of the human PRMT5:MEP50 complex. Proc. Natl. Acad. Sci. USA 109, 17960–17965 (2012).
Ho, M.C. et al. Structure of the arginine methyltransferase PRMT5-MEP50 reveals a mechanism for substrate specificity. PLoS One 8, e57008 (2013).
Karatas, H., Townsend, E.C., Bernard, D., Dou, Y. & Wang, S. Analysis of the binding of mixed lineage leukemia 1 (MLL1) and histone 3 peptides to WD repeat domain 5 (WDR5) for the design of inhibitors of the MLL1–WDR5 interaction. J. Med. Chem. 53, 5179–5185 (2010).
Senisterra, G. et al. Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5. Biochem. J. 449, 151–159 (2013).
Sackton, K.L. et al. Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C. Nature 514, 646–649 (2014).
Li, S. et al. A liquid chromatography/mass spectrometry-based generic detection method for biochemical assay and hit discovery of histone methyltransferases. Anal. Biochem. 443, 214–221 (2013).
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).
Acknowledgements
We thank the following colleagues for their help with this manuscript: J.-H. Zhang for HTS screening; L. Zhong for cellular assay development; Y. Wei for LC–MS/MS sample analysis; M. Dillon for medicinal chemistry; L. Liu for protein expression, purification and characterization; Z. Chen for animal studies; Y. Fan for bioinformatics data handling; X. Luo and H. Liu for assistance in structural studies; Zhenting Gao for modeling analysis; Zhenhai Gao for PRC2 biology discussion; staff at SSRF for data collection.
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W.Q., K.Z., J.G., Y.H. and E.L. designed the study and interpreted data. K.Z., J.G., H.Z., M. Zhang, L.L., M. Zhao, Z.C., Leying F., Lijian F., Y.G., G.L., Y. Lin, M.S., Z.W., Y.Y., C. Zeng and S.Z. performed the biochemical, biophysical and structure-related experiments and data analysis. W.Q., Y.W., J.Z., L.T., S.C., C. Zhang, H.C., D.F., Q.F., H.G., X.G., Y. Liu, F.L., J.Z. and L.Z. are involved in the cellular and animal studies and data analysis. Y.H., Z.Y., A.L., L.W. and Q.Z. designed, synthesized and characterized the chemical compounds. P.A., C.O. and E.L. guided multiple aspects of this study and team collaboration. K.Z., J.G., W.Q. and E.L. wrote the manuscript with input from co-authors.
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All authors performed the work herein as employees of the Novartis Institutes for Biomedical Research, Inc.
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Qi, W., Zhao, K., Gu, J. et al. An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED. Nat Chem Biol 13, 381–388 (2017). https://doi.org/10.1038/nchembio.2304
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DOI: https://doi.org/10.1038/nchembio.2304
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