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EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas

Abstract

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16INK4A. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.

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Figure 1: A mouse model of DIPG.
Figure 2: EZH2 inhibition affects the growth of H3K27M-positive mouse DIPG-like tumor cells in vitro and in vivo.
Figure 3: EZH2 inhibition affects the growth of primary H3K27M-positive human DIPG cells.
Figure 4: Effect of EZH2 inhibition on cell proliferation is dependent on functional p16INK4A.
Figure 5: Genome-wide effects of H3K27M at CGIs are linked to H3K27me3, SUZ12 and RING1B enrichment in H3WT.
Figure 6: H3K27M and EZH2 inhibition leads to significant gene-expression changes.

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References

  1. Varambally, S. et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419, 624–629 (2002).

    CAS  PubMed  Article  Google Scholar 

  2. Helin, K. & Dhanak, D. Chromatin proteins and modifications as drug targets. Nature 502, 480–488 (2013).

    CAS  PubMed  Article  Google Scholar 

  3. Morin, R.D. et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 476, 298–303 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Pasqualucci, L. et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat. Genet. 43, 830–837 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 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).

    CAS  Article  PubMed  Google Scholar 

  7. 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).

    CAS  PubMed  Article  Google Scholar 

  8. McCabe, M.T. et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492, 108–112 (2012).

    CAS  Article  PubMed  Google Scholar 

  9. Konze, K.D. et al. An orally bioavailable chemical probe of the Lysine Methyltransferases EZH2 and EZH1. ACS Chem. Biol. 8, 1324–1334 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Ernst, T. et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat. Genet. 42, 722–726 (2010).

    CAS  PubMed  Article  Google Scholar 

  11. Ntziachristos, P. et al. Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nat. Med. 18, 298–301 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Hargrave, D., Bartels, U. & Bouffet, E. Diffuse brainstem glioma in children: critical review of clinical trials. Lancet Oncol. 7, 241–248 (2006).

    PubMed  Article  Google Scholar 

  13. Laigle-Donadey, F., Doz, F. & Delattre, J.-Y. Brainstem gliomas in children and adults. Curr. Opin. Oncol. 20, 662–667 (2008).

    PubMed  Article  Google Scholar 

  14. Schwartzentruber, J. et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482, 226–231 (2012).

    CAS  PubMed  Article  Google Scholar 

  15. Wu, G. et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat. Genet. 44, 251–253 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Buczkowicz, P. et al. Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations. Nat. Genet. 46, 451–456 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Taylor, K.R. et al. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nat. Genet. 46, 457–461 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Fontebasso, A.M. et al. Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nat. Genet. 46, 462–466 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Lewis, P.W. et al. Inhibition of PRC2 activity by a gain-of-function H3 H3 mutation found in pediatric glioblastoma. Science 340, 857–861 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Bender, S. et al. Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas. Cancer Cell 24, 660–672 (2013).

    CAS  PubMed  Article  Google Scholar 

  21. Chan, K.-M. et al. The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression. Genes Dev. 27, 985–990 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Sturm, D. et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell 22, 425–437 (2012).

    CAS  PubMed  Article  Google Scholar 

  23. Verma, S.K. et al. Identification of potent, selective, cell-active inhibitors of the histone lysine methyltransferase EZH2. ACS Med. Chem. Lett. 3, 1091–1096 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Knutson, S.K. et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc. Natl. Acad. Sci. USA 110, 7922–7927 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Gil, J. & Peters, G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat. Rev. Mol. Cell Biol. 7, 667–677 (2006).

    CAS  PubMed  Article  Google Scholar 

  26. Petitjean, A. et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum. Mutat. 28, 622–629 (2007).

    CAS  PubMed  Article  Google Scholar 

  27. Schroeder, K.M., Hoeman, C.M. & Becher, O.J. Children are not just little adults: recent advances in understanding of diffuse intrinsic pontine glioma biology. Pediatr. Res. 75, 205–209 (2013).

    PubMed  Article  CAS  Google Scholar 

  28. Verhaak, R.G.W. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98–110 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Pollard, S.M. et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4, 568–580 (2009).

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  31. Deaton, A.M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Hambardzumyan, D., Amankulor, N.M., Helmy, K.Y., Becher, O.J. & Holland, E.C. Modeling adult gliomas using RCAS/t-va technology. Transl. Oncol. 2, 89–95 (2009).

    PubMed  PubMed Central  Article  Google Scholar 

  33. Funato, K., Major, T., Lewis, P.W., Allis, C.D. & Tabar, V. Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation. Science 346, 1529–1533 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Zhang, P. et al. ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-Inhibitors. Int. J. Cancer 137, 2007–2018 (2015).

    CAS  PubMed  Article  Google Scholar 

  35. Jiang, Y., Boije, M., Westermark, B. & Uhrbom, L. PDGF-B Can sustain self-renewal and tumorigenicity of experimental glioma-derived cancer-initiating cells by preventing oligodendrocyte differentiation. Neoplasia 13, 492–503 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Hashizume, R. et al. Characterization of a diffuse intrinsic pontine glioma cell line: implications for future investigations and treatment. J. Neurooncol. 110, 305–313 (2012).

    PubMed  Article  Google Scholar 

  37. Caretti, V. et al. Monitoring of tumor growth and post-irradiation recurrence in a diffuse intrinsic pontine glioma mouse model. Brain Pathol. 21, 441–451 (2011).

    PubMed  Article  Google Scholar 

  38. Debacq-Chainiaux, F., Erusalimsky, J.D., Campisi, J. & Toussaint, O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat. Protoc. 4, 1798–1806 (2009).

    CAS  PubMed  Article  Google Scholar 

  39. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Shechter, D., Dormann, H.L., Allis, C.D. & Hake, S.B. Extraction, purification and analysis of histones. Nat. Protoc. 2, 1445–1457 (2007).

    CAS  PubMed  Article  Google Scholar 

  41. Sidoli, S. et al. Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones. Proteomics 14, 2200–2211 (2014).

    CAS  PubMed  Article  Google Scholar 

  42. Lin, S. & Garcia, B.A. Examining histone posttranslational modification patterns by high-resolution mass spectrometry. Methods Enzymol. 512, 3–28 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

We thank members of the Helin laboratory for discussions. F.M. was supported by a postdoctoral fellowship from EMBO (874-2011). D.P. was supported by a postdoctoral fellowship from EMBO (1411-2011) and the Danish Medical Research Council. I.C. was supported by a fellowship from the Lundbeck Foundation. The work in the Helin laboratory was supported by the Danish Medical Research Council (DFF – 4004-00081), the Danish National Research Foundation (DNRF 82), and through a center grant from the Novo Nordisk Foundation (The Novo Nordisk Foundation Section for Stem Cell Biology in Human Disease). A.M.C. acknowledges support from the Xarxa de Bancs de Tumors de Catalunya (XBTC) sponsored by Pla Director d'Oncologia de Catalunya, and funding from the Fondo Alicia Pueyo, AECC Scientific Foundation, European Union Seventh Framework Programme (FP7/2007-2013) under Marie Curie International Reintegration Grant (PIRG-08-GA-2010-276998) and ISCIII-FEDER (CP13/00189). We thank S. Pollard for the gift of GNS cell lines, N. Gupta for providing the SF7761 and SF8628 cell lines and L. Uhrbom and P. Lewis for plasmids.

Author information

Authors and Affiliations

Authors

Contributions

F.M. performed the majority of the experiments; S.W., D.P.P., J.W.H., C.Z. and I.C. performed the remaining experiments. The bioinformatics analyses were performed by B.L. and J.V.J.; N.R. and B.T.P. contributed to gene-expression analysis. A.T. and O.N.J. performed MS analysis. N.G.O., C.L., M.S., C.d.T., J.M. and A.M.C. analyzed the primary tumor samples. A.M.C provided DIPG cell lines. F.M. and K.H. prepared the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Kristian Helin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–13 and Supplementary Tables 1 and 4 (PDF 11060 kb)

Supplementary Table 2

List of genes corresponding to the four categories and gene expression changes in H3K27M cells (XLSX 294 kb)

Supplementary Table 3

GO term enrichment analysis on "unchanged" genes using annotation clustering tool available on the DAVID bioinformatics resources (XLSX 73 kb)

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Mohammad, F., Weissmann, S., Leblanc, B. et al. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat Med 23, 483–492 (2017). https://doi.org/10.1038/nm.4293

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