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

Nature Medicine volume 23, pages 483–492 (2017)Cite this article

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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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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.

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Author notes

  1. Simon Weissmann, Benjamin Leblanc and Deo P Pandey: These authors contributed equally to this work.

Authors and Affiliations

  1. Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark

    Faizaan Mohammad, Simon Weissmann, Benjamin Leblanc, Deo P Pandey, Jonas W Højfeldt, Itys Comet, Chunqin Zheng, Jens Vilstrup Johansen, Nicolas Rapin, Bo T Porse & Kristian Helin

  2. Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark

    Faizaan Mohammad, Simon Weissmann, Benjamin Leblanc, Deo P Pandey, Jonas W Højfeldt, Itys Comet, Chunqin Zheng, Andrey Tvardovskiy, Ole N Jensen & Kristian Helin

  3. The Danish Stem Cell Center (Danstem), University of Copenhagen, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Benjamin Leblanc, Nicolas Rapin, Bo T Porse & Kristian Helin

  4. The Finsen Laboratory, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Nicolas Rapin & Bo T Porse

  5. Department of Biology, The Bioinformatics Centre, University of Copenhagen, Copenhagen, Denmark

    Nicolas Rapin

  6. Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark

    Andrey Tvardovskiy & Ole N Jensen

  7. Department of Pediatric Hematology and Oncology, Hospital Sant Joan de Déu Barcelona, Barcelona, Spain

    Nagore G Olaciregui, Cinzia Lavarino, Carmen de Torres, Jaume Mora & Angel M Carcaboso

  8. Pathology, Hospital Sant Joan de Déu Barcelona, Barcelona, Spain

    Mariona Suñol

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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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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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