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Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise1. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M)2,3. Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis4,5. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M6,7. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data5, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.

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Figure 1: H3K27M correlates with H3K27ac and is excluded from PRC2 targets.
Figure 2: PRC2 is required for the oncogenic potential of H3K27M DIPG cells.
Figure 3: Bromodomain-protein inhibition impairs proliferation and triggers differentiation of H3K27M-positive DIPG cells.
Figure 4: Bromodomain-protein inhibition significantly extends survival of DIPG xenograft model.

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References

  1. 1

    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 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2

    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  PubMed Central  Google Scholar 

  3. 3

    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 

  4. 4

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5

    Herz, H.M. et al. Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling. Science 345, 1065–1070 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6

    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  PubMed Central  Google Scholar 

  7. 7

    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 

  8. 8

    Hashizume, R. et al. Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nat. Med. 20, 1394–1396 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9

    Monje, M. et al. Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma. Proc. Natl. Acad. Sci. USA 108, 4453–4458 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10

    Justin, N. et al. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2. Nat. Commun. 7, 11316 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11

    Piunti, A. & Shilatifard, A. Epigenetic balance of gene expression by Polycomb and COMPASS families. Science 352, aad9780 (2016).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  12. 12

    Bracken, A.P. et al. The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev. 21, 525–530 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13

    Piunti, A. et al. Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication. Nat. Commun. 5, 3649 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14

    Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15

    Qi, J. Bromodomain and extraterminal domain inhibitors (BETi) for cancer therapy: chemical modulation of chromatin structure. Cold Spring Harb. Perspect. Biol. 6, a018663 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. 16

    Jonkers, I. & Lis, J.T. Getting up to speed with transcription elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 167–177 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17

    Galderisi, U., Jori, F.P. & Giordano, A. Cell cycle regulation and neural differentiation. Oncogene 22, 5208–5219 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18

    Gordon, J., Amini, S. & White, M.K. General overview of neuronal cell culture. Methods Mol. Biol. 1078, 1–8 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19

    An, W., Palhan, V.B., Karymov, M.A., Leuba, S.H. & Roeder, R.G. Selective requirements for histone H3 and H4 N termini in p300-dependent transcriptional activation from chromatin. Mol. Cell 9, 811–821 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20

    Filippakopoulos, P. & Knapp, S. Targeting bromodomains: epigenetic readers of lysine acetylation. Nat. Rev. Drug Discov. 13, 337–356 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21

    Gupta, S., Takebe, N. & Lorusso, P. Targeting the Hedgehog pathway in cancer. Ther. Adv. Med. Oncol. 2, 237–250 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22

    Whitfield, M.L., George, L.K., Grant, G.D. & Perou, C.M. Common markers of proliferation. Nat. Rev. Cancer 6, 99–106 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23

    Weiner, A. et al. Co-ChIP enables genome-wide mapping of histone mark co-occurrence at single-molecule resolution. Nat. Biotechnol. 34, 953–961 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24

    Pasini, D. et al. Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes. Nucleic Acids Res. 38, 4958–4969 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25

    Ferrari, K.J. et al. Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity. Mol. Cell 53, 49–62 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26

    Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27

    Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    CAS  Article  Google Scholar 

  28. 28

    Jones, C. & Baker, S.J. Unique genetic and epigenetic mechanisms driving paediatric diffuse high-grade glioma. Nat. Rev. Cancer 14, 651–661 (2014).

    CAS  Article  Google Scholar 

  29. 29

    Grasso, C.S. et al. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat. Med. 21, 827 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30

    Mazur, P.K. et al. Combined inhibition of BET family proteins and histone deacetylases as a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma. Nat. Med. 21, 1163–1171 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31

    Bhadury, J. et al. BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma. Proc. Natl. Acad. Sci. USA 111, E2721–E2730 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32

    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  PubMed Central  Google Scholar 

  33. 33

    Mueller, S. et al. Targeting Wee1 for the treatment of pediatric high-grade gliomas. Neuro-oncol. 16, 352–360 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34

    Chen, F.X. et al. PAF1, a molecular regulator of promoter-proximal pausing by RNA polymerase II. Cell 162, 1003–1015 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35

    Chen, F., Gao, X. & Shilatifard, A. Stably paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor triptolide. Genes Dev. 29, 39–47 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  36. 36

    Lee, T.I., Johnstone, S.E. & Young, R.A. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protoc. 1, 729–748 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37

    Bolger, A.M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  39. 39

    Zang, C. et al. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 25, 1952–1958 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40

    Saldanha, A.J. Java Treeview—extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. 42

    Anders, S., Pyl, P.T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43

    Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. 44

    Meerbrey, K.L. et al. The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc. Natl. Acad. Sci. USA 108, 3665–3670 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

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Acknowledgements

We thank all the members of the Shilatifard lab for their useful comments and suggestions. We thank M. Monje (Stanford University) for use of the SU-DIPG-IV cell line. We thank D. Pasini (European Institute of Oncology, IEO) for providing the SUZ12 and control vectors, and C. Rivetta for technical support. pInducer20 was a gift from S. Elledge (deposited in Addgene, plasmid #44012). A.P. is supported by a long-term EMBO fellowship (ALTF 372-2015), and his work in the Shilatifard lab is supported by AIRC and Marie Curie Actions—People—COFUND. R.H. is supported by US National Institutes of Health grant RO1NS093079, the Matthew Larson Foundation and the Bear Necessities Pediatric Cancer Foundation and Rally Foundation. Proteomics services were performed by the Northwestern Proteomics Core Facility, generously supported by NCI CCSG P30CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center, and the National Resource for Translational and Developmental Proteomics supported by P41GM108569. Studies in regards to the development of targeted therapeutics for DIPG within our groups are partially supported by the generous support of J. McNicholas Pediatric Brain Tumor Foundation. Studies in the Shilatifard laboratory are supported by NCI grant R35CA197569.

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Contributions

A.P. and A.S. designed the study. A.P. performed the majority of the experiments, part of the analyses and wrote the manuscript. R.H., C.D.J., A.P. and A.S. designed the in vivo studies. R.H. and Q.M. performed and analyzed the in vivo experiments. M.A.M. performed the MNase-IP experiment. A.R.W. performed the initial bioinformatics analyses on the studies related to the role of PRC2 in DIPG. E.T.B. performed all other bioinformatics analyses. C.M.H. performed and analyzed the immunohistochemistry studies. S.A.M. and E.J.R. generated and sequenced the next-generation sequencing (NGS) libraries. Y.-h.T. provided technical help. A.V.M.performed the FACS studies. N.A.A. and N.L.K. designed the mass spectrometry studies. N.A.A. performed the mass spectrometry experiments. R.R.L. and A.M.S. provided clinical supervision in the interpretation of data. A.P., M.A.M., C.D.J. and A.S. revised the manuscript. All authors commented on the manuscript and approved data included in it.

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Correspondence to C David James or Ali Shilatifard.

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The authors declare no competing financial interests.

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Supplementary Methods & Supplementary Figures 1–18 (PDF 6282 kb)

Supplementary Table 1

Mass spectrometry data (XLSX 41 kb)

Supplementary Table 2

Antibody's list (XLSX 10 kb)

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Piunti, A., Hashizume, R., Morgan, M. et al. Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas. Nat Med 23, 493–500 (2017). https://doi.org/10.1038/nm.4296

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