Increased 5-hydroxymethylcytosine and decreased 5-methylcytosine are indicators of global epigenetic dysregulation in diffuse intrinsic pontine glioma

Sama Ahsan, Eric H Raabe, Michael C Haffner, Ajay Vaghasia, Katherine E Warren, Martha Quezado, Leomar Y Ballester, Javad Nazarian, Charles G Eberhart, Fausto J Rodriguez, Sama Ahsan, Eric H Raabe, Michael C Haffner, Ajay Vaghasia, Katherine E Warren, Martha Quezado, Leomar Y Ballester, Javad Nazarian, Charles G Eberhart, Fausto J Rodriguez

Abstract

Introduction: Diffuse intrinsic pontine glioma (DIPG) is a malignant pediatric brain tumor associated with dismal outcome. Recent high-throughput molecular studies have shown a high frequency of mutations in histone-encoding genes (H3F3A and HIST1B) and distinctive epigenetic alterations in these tumors. Epigenetic alterations described in DIPG include global DNA hypomethylation. In addition to the generally repressive methylcytosine DNA alteration, 5-hydroxymethylation of cytosine (5hmC) is recognized as an epigenetic mark associated with active chromatin. We hypothesized that in addition to alterations in DNA methylation, that there would be changes in 5hmC. To test this hypothesis, we performed immunohistochemical studies to compare epigenetic alterations in DIPG to extrapontine adult and pediatric glioblastoma (GBM) and normal brain. A total of 124 tumors were scored for histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 9 trimethylation (H3K9me3) and 104 for 5hmC and 5-methylcytosine (5mC). An H-score was derived by multiplying intensity (0-2) by percentage of positive tumor nuclei (0-100%).

Results: We identified decreased H3K27me3 in the DIPG cohort compared to pediatric GBM (p < 0.01), adult GBM (p < 0.0001) and normal brain (p < 0.0001). H3K9me3 was not significantly different between tumor types. Global DNA methylation as measured by 5mC levels were significantly lower in DIPG compared to pediatric GBM (p < 0.001), adult GBM (p < 0.01), and normal brain (p < 0.01). Conversely, 5hmC levels were significantly higher in DIPG compared to pediatric GBM (p < 0.0001) and adult GBM (p < 0.0001). Additionally, in an independent set of DIPG tumor samples, TET1 and TET3 mRNAs were found to be overexpressed relative to matched normal brain.

Conclusions: Our findings extend the immunohistochemical study of epigenetic alterations in archival tissue to DIPG specimens. Low H3K27me3, decreased 5mC and increased 5hmC are characteristic of DIPG in comparison with extrapontine GBM. In DIPG, the relative imbalance of 5mC compared to 5hmC may represent an opportunity for therapeutic intervention.

Figures

Figure 1
Figure 1
Differential H3K27me3, 5mC and 5hmC immunoreactivity in DIPG and extrapontine GBM. Representative micrographs (400×) of immunoreactivity for Histone 3 Lysine 27 trimethylation (H3K27me3), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) in control brain, pediatric glioblastoma (pGBM), adult glioblastoma (aGBM), and diffuse intrinsic pontine glioma (DIPG).
Figure 2
Figure 2
H3K27me3 is decreased in DIPG. H-scores for Histone 3 Lysine 27 trimethylation (H3K27me3) immunoreactivity in normal brain (Control), adult glioblastoma (aGBM), pediatric glioblastoma (pGBM), and diffuse intrinsic pontine glioma (DIPG) tissue (p < 0.0001, Kruskal-Wallis ANOVA Test) (a). H3K27me3 median H-score was significantly lower for DIPGs compared to pediatric GBM (p < 0.01, Mann–Whitney Test), adult GBM (p < 0.0001), and normal brain (p < 0.0001) tissue. A significant difference was also noted in immunoreactivity between control and adult GBM (p < 0.0001) and also control and pediatric GBM (p < 0.0001). No difference was noted between adult and pediatric GBM immunoreactivity for H3K27me3 (p > 0.05). Number of samples per group and median H-scores are specified in b.
Figure 3
Figure 3
H3K9me3 immunoreactivity is no different in DIPG, adult GBM, and pediatric GBM. H-scores for Histone 3 Lysine 9 trimethylation (H3K9me3) immunoreactivity in normal brain (Control), adult glioblastoma (aGBM), pediatric glioblastoma (pGBM), and diffuse intrinsic pontine glioma (DIPG) tissue (p < 0.0001, Kruskal-Wallis ANOVA Test) (a). H3K9me3 immunoreactivity was relatively preserved in DIPG and not significantly different from extrapontine GBM (adult GBM p > 0.05, pediatric GBM p > 0.05, Kruskal-Wallis test of multiple comparisons). A significant difference was noted between tumor groups and normal brain (p < 0.05, Kruskal-Wallis test of multiple comparisons). Number of samples per group and median H-scores are specified in b.
Figure 4
Figure 4
5hmC is increased in DIPG. H-scores for 5-hydroxymethylcytosine (5hmC) immunoreactivity in normal brain (control), adult glioblastoma (aGBM), pediatric glioblastoma (pGBM), and diffuse intrinsic pontine glioma (DIPG) tissue (p < 0.0001, Kruskal-Wallis ANOVA Test) (a). Number of samples per group and median H-scores are specified in b.
Figure 5
Figure 5
5mC is decreased in DIPG. H-scores for 5-methylcytosine (5mC) immunoreactivity in normal brain (Control), adult glioblastoma (aGBM), pediatric glioblastoma (pGBM), and diffuse intrinsic pontine glioma (DIPG) tissue (p < 0.0024, Kruskal-Wallis ANOVA Test). (a). Number of samples per group and median H-scores are specified in b.
Figure 6
Figure 6
TET1andTET3expression are increased in DIPG tumors relative to normal brain. mRNA expression profiling of H3F3A mutant DIPG tumor tissue shows relative increased expression of TET1 and TET3, but not TET2, in tumor relative to adjacent non-neoplastic brain control.
Figure 7
Figure 7
Overproduction of 5hmC and disruption of histone methylation may contribute to DIPG tumorigenesis. Normal neural development is controlled by histone and DNA methylation executed by enhancer of zeste homologue 2 (EZH2) methyltransferase, DNA methyltransferase (DNMT), Ten Eleven Translocation (TET), Thymine-DNA glycosylase (TDG), and base excision repair (BER). Dysregulation of the epigenome by H3F3A mutation/EZH2 inhibition and corresponding overproduction of 5hmC by TET in DIPG may promote DIPG tumorigenicity.

References

    1. Ballester L, Wang Z, Shandilya S, Miettinen M, Burger P, Eberhart C, Rodriguez F, Raabe E, Nazarian J, Warren K, Quezado M. Morphologic characteristics and immunohistochemical profile of diffuse intrinsic pontine gliomas. Am J Surg Pathol. 2013;37(9):1357–1364. doi: 10.1097/PAS.0b013e318294e817.
    1. Paugh BS, Broniscer A, Qu C, Miller CP, Zhang J, Tatevossian RG, Olson JM, Geyer JR, Chi SN, Da Silva NS, Onar-Thomas A, Baker JN, Gajjar A, Ellison DW, Baker SJ. Genome-wide analyses identify recurrent amplifications of receptor tyrosine kinases and cell-cycle regulatory genes in diffuse intrinsic pontine glioma. J Clin Oncol. 2011;29(30):3999–4006. doi: 10.1200/JCO.2011.35.5677.
    1. Paugh BS, Zhu X, Qu C, Endersby R, Diaz AK, Zhang J, Bax DA, Carvalho D, Reis RM, Onar-Thomas A, Broniscer A, Wetmore C, Zhang J, Jones C, Ellison DW, Baker SJ. Novel oncogenic PDGFRA mutations in pediatric high-grade gliomas. Cancer Res. 2013;73(20):6219–6229. doi: 10.1158/0008-5472.CAN-13-1491.
    1. Saratsis AM, Kambhampati M, Snyder K, Yadavilli S, Devaney JM, Harmon B, Hall J, Raabe EH, An P, Weingart M, Rood BR, Magge SN, Macdonald TJ, Packer RJ, Nazarian J. Acta Neuropathol. 2013. Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes.
    1. Buczkowicz P, Hoeman C, Rakopoulos P, Pajovic S, Letourneau L, Dzamba M, Morrison A, Lewis P, Bouffet E, Bartels U, Zuccaro J, Agnihotri S, Ryall S, Barszczyk M, Chornenkyy Y, Bourgey M, Bourque G, Montpetit A, Cordero F, Castelo-Branco P, Mangerel J, Tabori U, Ho KC, Huang A, Taylor KR, Mackay A, Bendel AE, Nazarian J, Fangusaro JR, Karajannis MA, et al. Nat Genet. 2014. Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations.
    1. Taylor KR, Mackay A, Truffaux N, Butterfield YS, Morozova O, Philippe C, Castel D, Grasso CS, Vinci M, Carvalho D, Carcaboso AM, De Torres C, Cruz O, Mora J, Entz-Werle N, Ingram WJ, Monje M, Hargrave D, Bullock AN, Puget S, Yip S, Jones C, Grill J. Nat Genet. 2014. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma.
    1. Dewoskin V, Million R. The epigenetics pipeline. Nat Rev Drug Discov. 2013;12(9):661–662. doi: 10.1038/nrd4091.
    1. Schwartzentruber J, Korshunov A, Liu X-Y, Jones D, Pfaff E, Jacob K, Sturm D, Fontebasso A, Quang D-AK, Tönjes M, Hovestadt V, Albrecht S, Kool M, Nantel A, Konermann C, Lindroth A, Jäger N, Rausch T, Ryzhova M, Korbel J, Hielscher T, Hauser P, Garami M, Klekner A, Bognar L, Ebinger M, Schuhmann M, Scheurlen W, Pekrun A, Frühwald M, et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature. 2012;482(7384):226–231. doi: 10.1038/nature10833.
    1. Sturm D, Witt H, Hovestadt V, Khuong-Quang D-A, Jones D, Konermann C, Pfaff E, Tönjes M, Sill M, Bender S, Kool M, Zapatka M, Becker N, Zucknick M, Hielscher T, Liu X-Y, Fontebasso A, Ryzhova M, Albrecht S, Jacob K, Wolter M, Ebinger M, Schuhmann M, van Meter T, Frühwald M, Hauch H, Pekrun A, Radlwimmer B, Niehues T, von Komorowski G, et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell. 2012;22(4):425–437. doi: 10.1016/j.ccr.2012.08.024.
    1. Bender S, Tang Y, Lindroth A, Hovestadt V, Jones D, Kool M, Zapatka M, Northcott P, Sturm D, Wang W, Radlwimmer B, Højfeldt J, Truffaux N, Castel D, Schubert S, Ryzhova M, Seker-Cin H, Gronych J, Johann P, Stark S, Meyer J, Milde T, Schuhmann M, Ebinger M, Monoranu C-M, Ponnuswami A, Chen S, Jones C, Witt O, Collins V, et al. Reduced H3K27me3 and DNA Hypomethylation Are Major Drivers of Gene Expression in K27M Mutant Pediatric High-Grade Gliomas. Cancer Cell. 2013;24(5):660–672. doi: 10.1016/j.ccr.2013.10.006.
    1. Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J, Qu C, Ding L, Huether R, Parker M, Zhang J, Gajjar A, Dyer MA, Mullighan CG, Gilbertson RJ, Mardis ER, Wilson RK, Downing JR, Ellison DW, Zhang J, Baker SJ, St. Jude Children’s Research Hospital-Washington University Pediatric Cancer Genome P Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet. 2012;44(3):251–253. doi: 10.1038/ng.1102.
    1. Khuong-Quang DA, Buczkowicz P, Rakopoulos P, Liu XY, Fontebasso AM, Bouffet E, Bartels U, Albrecht S, Schwartzentruber J, Letourneau L, Bourgey M, Bourque G, Montpetit A, Bourret G, Lepage P, Fleming A, Lichter P, Kool M, von Deimling A, Sturm D, Korshunov A, Faury D, Jones DT, Majewski J, Pfister SM, Jabado N, Hawkins C. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta Neuropathol. 2012;124(3):439–447. doi: 10.1007/s00401-012-0998-0.
    1. Lewis P, Müller M, Koletsky M, Cordero F, Lin S, Banaszynski L, Garcia B, Muir T, Becher O, Allis C. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science (New York, NY) 2013;340(6134):857–861. doi: 10.1126/science.1232245.
    1. Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer. 2006;6(11):846–856. doi: 10.1038/nrc1991.
    1. Reddington J, Perricone S, Nestor C, Reichmann J, Youngson N, Suzuki M, Reinhardt D, Dunican D, Prendergast J, Mjoseng H, Ramsahoye B, Whitelaw E, Greally J, Adams I, Bickmore W, Meehan R: Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes.Genome Biol 2013.,14(3): doi:10.1186/gb-2013–14–3-r25
    1. Pfeifer G, Kadam S, Jin S-G. 5-hydroxymethylcytosine and its potential roles in development and cancer. Epigenetics Chromatin. 2013;6(1):10. doi: 10.1186/1756-8935-6-10.
    1. Sun W, Guan M, Li X. Stem Cells Dev. 2014. 5-hydroxymethylcytosine-mediated DNA demethylation in stem cells and development.
    1. Hahn M, Qiu R, Wu X, Li A, Zhang H, Wang J, Jui J, Jin S-G, Jiang Y, Pfeifer G, Lu Q. Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis. Cell Rep. 2013;3(2):291–300. doi: 10.1016/j.celrep.2013.01.011.
    1. Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, He C, Zhang Y. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;333(6047):1300–1303. doi: 10.1126/science.1210597.
    1. Haffner MC, Chaux A, Meeker AK, Esopi DM, Gerber J, Pellakuru LG, Toubaji A, Argani P, Iacobuzio-Donahue C, Nelson WG, Netto GJ, De Marzo AM, Yegnasubramanian S. Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers. Oncotarget. 2011;2(8):627–637.
    1. Orr B, Haffner M, Nelson W, Yegnasubramanian S, Eberhart C: Decreased 5-hydroxymethylcytosine is associated with neural progenitor phenotype in normal brain and shorter survival in malignant glioma.PLoS One 2012.,7(7): doi:10.1371/journal.pone.0041036
    1. Venneti S, Garimella MT, Sullivan LM, Martinez D, Huse JT, Heguy A, Santi M, Thompson CB, Judkins AR. Evaluation of histone 3 lysine 27 trimethylation (H3K27me3) and enhancer of Zest 2 (EZH2) in pediatric glial and glioneuronal tumors shows decreased H3K27me3 in H3F3A K27M mutant glioblastomas. Brain Pathol. 2013;23(5):558–564. doi: 10.1111/bpa.12042.
    1. Odia Y, Orr BA, Bell WR, Eberhart CG, Rodriguez FJ. cMYC expression in infiltrating gliomas: associations with IDH1 mutations, clinicopathologic features and outcome. J Neurooncol. 2013;115(2):249–259. doi: 10.1007/s11060-013-1221-4.
    1. Haffner M, Pellakuru L, Ghosh S, Lotan T, Nelson W, De Marzo A, Yegnasubramanian S. Tight correlation of 5-hydroxymethylcytosine and Polycomb marks in health and disease. Cell Cycle. 2013;12(12):1835–1841. doi: 10.4161/cc.25010.

Source: PubMed

Sponsor e collaboratori

Condizioni mediche

Interventi farmacologici

3
Sottoscrivi