Abstract 3084: Epigenetic deregulation in H3.3-K27M mutant pediatric high-grade gliomas

Abstract

Abstract A recurrent mutation substituting lysine 27 with a methionine (K27M) on the amino-terminal tail of histone variant H3.3 was recently identified in about 25% of pediatric high-grade gliomas (pHGGs), the most common malignant brain tumor in childhood. K27M mutant tumors represent a clinically and biologically distinct subgroup of pHGGs, characterized by a unique genome-wide DNA methylation pattern as well as transcriptomic profile. Interestingly, tumors harboring the K27M mutation display a global reduction of the repressive histone modification H3K27me3, which is caused by a dominant-negative effect of K27M mutant H3.3 protein. The purpose of this study was to further elucidate the dominant-negative effect of K27M mutant H3.3 and investigate the connection between K27M-induced reduction of H3K27me3, DNA methylation and gene expression. Furthermore, we aimed to clarify if immunohistochemical (IHC) testing of H3K27me3 represents a reliable tool for clinical application to identify patients suffering from K27M mutant pHGGs, which might benefit from more intense treatment options. Co-immunoprecipitation (CoIP) experiments and histone methyltransferase (HMT) assays were conducted in three different cell lines to elucidate the dominant-negative function of K27M mutant H3.3. The potential impact on different levels of epigenetic gene regulation was analyzed by performing H3K27me3 chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq), whole genome bisulfite sequencing (WGBS) and gene expression profiling on fresh-frozen primary tumors with and without K27M mutations. As a result, we demonstrate that IHC reliably identifies K27M mutant pHGGs with high specificity and sensitivity, and confirm that H3K27me3 levels are globally reduced due to the dominant-negative effect of K27M mutant H3.3. As indicated by our CoIPs, this is a result of an increased interaction of K27M mutant H3.3 with several components of the Polycomb repressive complex 2 (PRC2), including enhancer of zeste homolog 2 (EZH2), which is responsible for di- and trimethylation of H3K27. EZH2 catalytic activity measured by HMT assay is significantly reduced in the presence of K27M mutant H3.3 protein. Genome-wide H3K27me3 ChIP-Seq data of fresh-frozen pHGG samples confirms globally reduced levels of H3K27me3, but also identifies a subset of loci with focal enrichment of this histone modification. By integrating with WGBS data, we show that K27M mutant pHGGs are characterized by reduced H3K27me3 occupancy and concomitant global DNA hypomethylation. Strikingly, 74% of genes found to be specifically upregulated in K27M mutant pHGGs show changes in one or both of these levels of epigenetic regulation. In conclusion, epigenetic deregulation in K27M mutant pHGGs reflected by reduced H3K27me3 occupancy and/or global DNA hypomethylation is a key modulator of gene activity in these highly aggressive brain tumors. Citation Format: Sebastian Bender, Yujie Tang, Anders M. Lindroth, Volker Hovestadt, Marc Zapatka, David T.W. Jones, Marcel Kool, Paul A. Northcott, Dominik Sturm, Peter Lichter, Christoph Plass, Yoon-Jae Cho. Epigenetic deregulation in H3.3-K27M mutant pediatric high-grade gliomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2014-3084