DNA Methylation Potential Energy Landscape Analysis of Diffuse Midline Glioma/Diffuse intrinsic pontine glioma (DMG/DIPG RNA-Seq)

Diffuse intrinsic pontine glioma (DIPG) is a uniformly lethal brainstem tumor of childhood, driven by mutations in histone H3 at lysine 27 (K27M) leading to altered epigenetic regulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation. However, studies providing a static view of the epigenome do not adequately capture the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed whole-genome bisulfite sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls and applied a novel framework for analysis of DNA methylation variability, permitting the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increasingly stochastic DNA methylation localizing to key regulatory elements and genes. We demonstrate marked epigenetic instability at genes regulating pluripotency and developmental identity, potentially enabling cells to sample a diverse range of transcriptional programs and differentiation states. We then evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. Treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment elicited changes in gene expression, including upregulation of immune signaling, and sensitized DIPG cells to the effects of histone deacetylase inhibition. This study provides a resource for dissecting the epigenetic instability that underlies DIPG heterogeneity and plasticity, suggesting the application of epigenetic therapies to constrain the range of epigenetic states available to DIPG cells. RNA-seq of indicated patient-derived DIPG neurosphere cell lines after treatment with DMSO control of decitabine (DAC), in triplicate.

弥漫内生型桥脑胶质瘤（Diffuse intrinsic pontine glioma, DIPG）是一种致死性均一的儿童脑干肿瘤，由组蛋白H3赖氨酸27位点突变（K27M）驱动，导致表观遗传调控异常。DIPG的表观基因组分析显示，其存在全局抑制性染色质标记丢失，同时伴随DNA低甲基化。然而，仅提供表观基因组静态视图的研究，无法充分阐释DIPG肿瘤内异质性与表型可塑性的调控基础。为解决这一问题，本研究对23例原发性患者DIPG标本、4株患者来源的DIPG神经球细胞系以及5例正常对照样本开展了全基因组亚硫酸氢盐测序（Whole-genome bisulfite sequencing, WGBS），并应用全新的DNA甲基化变异分析框架，构建了可捕捉内在表观遗传变异的全基因组DNA甲基化势能景观。本研究证实，DIPG的表观基因组呈现显著紊乱状态，随机DNA甲基化富集于关键调控元件与功能基因。研究还发现，调控多能性与发育身份的基因存在显著表观遗传不稳定性，这可能使肿瘤细胞能够切换至多种不同的转录程序与分化状态。随后，本研究评估了DIPG表观基因组对药物调控的响应性：使用DNA去甲基化药物地西他滨（decitabine）处理后，细胞出现显著的全基因组去甲基化，且活性增强子、二价启动子及关键发育基因的启动子区域的DNA甲基化随机性显著降低。地西他滨处理可诱导基因表达改变，包括免疫信号通路的上调，同时使DIPG细胞对组蛋白去乙酰化酶抑制剂的作用更为敏感。本研究为解析DIPG异质性与可塑性背后的表观遗传不稳定性提供了研究资源，提示可通过表观遗传疗法限制DIPG细胞可获得的表观遗传状态范围。此外，本研究还对经二甲基亚砜（DMSO）对照或地西他滨（DAC）处理后的指定患者来源DIPG神经球细胞系进行了RNA测序，设置三次生物学重复。