Abstract A15: Variant histone H3 mutations associate with histone modification, DNA methylation, and gene expression changes in pediatric high-grade gliomas

High-grade gliomas (HGG) account for twenty percent of primary brain tumors in children and have a devastating outcome with a 2-year survival rate of less than 30%. While adult HGG arise frequently in the cerebral cortex, pediatric HGG can form in cerebral cortex as well as midline regions including thalamus and brainstem, and are genetically distinct from adult tumors. Brainstem HGGs, referred to as diffuse intrinsic pontine glioma (DIPG), are mostly exclusive to children and confer a dismal less than 10% 2-year survival rate. Recent genome-wide sequence analysis of pediatric DIPG and non-brainstem HGG revealed a predominance of mutually exclusive somatic heterozygous H3F3A or HIST1H3B mutations encoding a K27M variant of histone H3.3 or H3.1, respectively. These mutations were found in 78% of DIPG patients and 22% of non-brainstem pediatric HGG. An additional mutually exclusive somatic heterozygous H3F3A mutation encoding histone H3.3 G34R was identified in 14% of non-brainstem HGG. We established xenografts from human non-brainstem HGGs carrying wild-type H3 or the H3.3 G34R mutant, and DIPGs with H3.3 or H3.1 K27M mutations. H3 K27M mutant xenografts showed a marked reduction in H3K27 trimethylation (H3K27me3), while tumors with wild-type or H3.3 G34R mutations retain H3K27me3 as demonstrated by immunohistochemistry and Western blot analyses. To determine if this was a dominant effect of K27M mutation or a developmental signature of DIPG we transduced primary astrocyte cultures derived from p53-deficient neonatal mouse cortex or brainstem with retrovirus expressing wild-type, K27M, K27A or G34R H3 mutant variants. Low-level expression of exogenous H3.3 K27M compared to endogenous H3 was sufficient to recapitulate prominent H3K27me3 reduction observed in the xenografts. Furthermore, we evaluated correlation between H3 mutation and H3K27me3 status in a large cohort of primary pediatric HGG including DIPG. From this we identified a significant relationship between H3 K27M mutation and H3K27me3 loss in DIPG. However, we also observed rare wild-type H3 DIPG cases exhibiting reduced H3K27me3 indicating a potential alternative mechanism for this methylation state. In addition, we evaluated the association of H3K27me3 and K27M mutations with genome-wide DNA methylation patterns, and gene expression signatures. Together, these results implicate histone H3 K27M and G34R mutations as unique, dominant genetic drivers in distinct subtypes of pediatric HGG. Current efforts focus on delineating the divergent pathological roles of H3 K27M and G34R mutations during DIPG and non-brainstem HGG tumorigenesis. Citation Format: Jon D. Larson, Troy A. McEachron, Chunxu Qu, Xiaoyan Zhu, Alexander K. Diaz, David W. Ellison, Brent A. Orr, Suzanne J. Baker. Variant histone H3 mutations associate with histone modification, DNA methylation, and gene expression changes in pediatric high-grade gliomas. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A15.