Project description:Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive but genetically simple pediatric central nervous system tumor, defined by biallelic inactivation of the chromatin regulator SMARCB1 with remarkably few other cooperating mutations. Despite its genetic homogeneity, ATRT exhibits profound clinical and epigenetic heterogeneity, with three major subgroups (ATRT-TYR, ATRT-MYC, and ATRT-SHH) defined by DNA methylation and transcriptional signatures. Beyond these subgroup-defining features, we aimed to investigate epigenetic variability within tumors by applying whole-genome bisulfite sequencing and probabilistic modeling to quantify stochastic DNA methylation in primary ATRT samples encompassing all three subgroups. We show that ATRT exhibits a destabilized and increasingly stochastic methylome. While ATRT global methylation patterns diverge according to subgroup, some methylation perturbations, such as hypermethylation and increased methylation entropy over bivalent promoters, are consistent across subgroups. We find that methylation stochasticity alterations map onto potential drivers of ATRT, such as LIN28a, the HOXD cluster for ATRT-MYC, and OTX2 for ATRT-TYR, and identify actionable targets, such as hypermethylation of the tumor suppressor CDKN2a across all subgroups. We investigate the sensitivity of the aberrant DNA methylation landscape of ATRT to pharmacologic DNA methyltransferase inhibition and histone deacetylase inhibition (HDACi). We show that decitabine leads to profound demethylation of patient-derived ATRT cell lines, including reversal of hypermethylation at bivalent promoters and the CDKN2a locus. The addition of HDACi leads to dramatic gene expression changes, including upregulation of innate immune signaling pathways, such as STING/interferon signaling, genes under the regulation of bivalent promoters, and reactivation of the tumor suppressor CDKN2A. The combination of DNMTi and HDACi synergistically reduces cell viability. Taken together, we show that ATRT has a highly stochastic methylome sensitive to epigenetic manipulation.

Project description:Background Thousands of mammalian promoters are defined by co-enrichment of the histone tail modifications H3K27me3 (repressive) and H3K4me3 (activating) and are thus termed bivalent. It was previously observed that bivalent genes in human ES cells (hESC) are frequent targets for hypermethylation in human cancers, and depletion of DNA methylation in mouse embryonic stem cells has a marked impact on H3K27me3 distribution at bivalent promoters. However, only a fraction of bivalent genes in stem cells are targets of hypermethylation in cancer, and it is currently unclear whether all bivalent promoters are equally sensitive to DNA hypomethylation and whether H3K4me3 levels play a role in the interplay between DNA methylation and H3K27me3. Results We report the sub-classification of bivalent promoters into two groups—promoters with a high H3K27me3:H3K4me3 (hiBiv) ratio or promoters with a low H3K27me3:H3K4me3 ratio (loBiv). HiBiv are enriched in canonical Polycomb components, show a higher degree of local intrachromosomal contacts and are highly sensitive to DNA hypomethylation in terms of H3K27me3 depletion from broad Polycomb domains. In contrast, loBiv promoters are enriched in non-canonical Polycomb components, show lower intrachromosomal contacts and are less sensitive to DNA hypomethylation at the same genomic resolution. Multiple systems reveal that hiBiv promoters are more depleted of Polycomb complexes than loBiv promoters following a reduction in DNA methylation, and we demonstrate that H3K27me3 re-accumulates at promoters when DNA methylation is restored. In human cancer, we show that hiBiv promoters lose H3K27me3 and are more susceptible to DNA hypermethylation than loBiv promoters. Conclusion We conclude that bivalency as a general term to describe mammalian promoters is an over-simplification and our sub-classification has revealed novel insights into the interplay between the largely antagonistic presence of DNA methylation and Polycomb systems at bivalent promoters. This approach redefines molecular pathologies underlying disease in which global DNA methylation is aberrant or where Polycomb mutations are present.

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Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT [WGBS]

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