Abstract Background Diffuse midline glioma (DMG) is a fatal pediatric brainstem tumor driven by the histone H3 K27M mutation. The known downstream consequences of this mutation include loss of repressive H3K27me3 marks, global DNA hypomethylation, and altered gene expression. However, since static views of the epigenome do not adequately capture the regulatory underpinnings of DIPG heterogeneity and plasticity, we sought to comprehensively map the dynamic DNA methylation landscape of DMG using methods that capture epigenetic variation, and to assess the responsiveness of the DMG epigenetic landscape to drugs aimed at the epigenome. Methods We carried out whole-genome bisulfite sequencing on 23 primary DMG patient samples, 4 neurosphere cell lines, and normal controls, and applied a novel framework for analysis of DNA methylation variability, permitting derivation of comprehensive genome-wide methylation potential energy landscapes that capture intrinsic epigenetic variation. We investigated the responsiveness of the epigenetic landscape of DMG neurosphere cell lines to the DNA methyltransferase inhibitor decitabine, alone or in combination with HDAC inhibitors or G9a/GLP inhibitors. Results We found that DMG has a markedly disordered epigenome with increased methylation stochasticity (quantified as methylation entropy), enriched at genes regulating pluripotency and developmental identity, potentially enabling DMG cells to sample diverse transcriptional programs and differentiation states. Despite global hypomethylation, DMG cells exhibit focal hypermethylation at specific regulatory regions including bivalent promoters. Decitabine treatment led to profound genome-wide demethylation, accompanied by a reduction in methylation stochasticity at active enhancers and bivalent promoters. Decitabine treatment elicited induction of STING/interferon signaling, expression of cancer-testis antigens such as PRAME, and de-repression of targets such as CXCR4 and CDKN2A/B. We found significant overlap of differentially methylated regions in DMG with targets of gene-repressive H3K9me2/3 modification, and showed that G9a/GLP inhibition further upregulates STING/interferon signaling. Conclusions The DMG methylome is highly disordered but responsive to pharmacologic modulation. Epigenetic therapies can constrain the range of epigenetic states available to DMG cells, potentially reducing heterogeneity/plasticity. These results also suggesst that targeting the aberrant epigenetic landscape in DMG can upregulate immune signaling, induce expression of novel antigens, and reactivate silenced tumor suppressor genes.
Tetens et al. (Tue,) studied this question.