The role of H3.3K27M-induced gene repression in brainstem gliomagenesis

Abstract

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive pediatric brainstem tumor recently found to contain high incidence of H3.3K27M mutations. These mutations reprogram the H3K27me3 epigenetic landscape of DIPG by inhibiting the H3K27-specific histone methyltransferase EZH2. This leads to global reduction with focal gains of the repressive H3K27me3, a mark responsible for cell fate decisions. To date the tumor-driving effects of H3.3K27M remain largely unknown. We show H3.3K27M cannot form tumors alone, however it cooperates with PDGF signaling in vivo, enhancing gliomagenesis and reducing survival of p53 WT and knockout murine models of DIPG. We find H3.3K27M expression drives increased proliferation of tumor-derived murine neurospheres, suggesting cell cycle deregulation may contribute to increased malignancy in mutant tumors. RNA-Seq on tumor tissue from H3.3K27M expressing mice showed global upregulation of PRC2 target genes, and a subset of newly repressed genes enriched in regulators of development and cell proliferation. Strikingly, H3.3K27M induces targeted repression of the p16/ink4a locus, a critical regulator of the G0/G1 to S phase transition. We find increased levels of H3K27me3 at the p16 promoter, however pharmacological reduction of this promoter methylation does not rescue p16 expression. While DNA methylation is also present at this promoter, it is not K27M-dependent. Intriguingly, inhibition of DNA methylation restores p16 levels and is cytotoxic against murine tumor cells. Importantly, we show that H3.3K27M-mediated p16 repression is an important mechanism underlying the proliferation of H3.3K27M tumor cells as in vivo cdkn2a knockout eliminates the survival difference between H3.3K27M and H3.3WT tumor-bearing mice.