Glioblastoma Stem Cells Reprogram Chromatin In Vivo To Generate Selective Therapeutic Dependencies [ChIP-Seq]

Glioblastomas are universally fatal cancers that contain self-renewing glioblastoma stem cells (GSCs) that initiate tumors. Traditional anti-cancer drug discovery based on in vitro cultures tends to identify targets with poor therapeutic index and fails to accurately model the effects of the tumor microenvironment. Here, leveraging in vivo genetic screening, we identified the H3K4me3 regulator DPY30 as an in vivo-specific glioblastoma dependency. Based on the hypothesis that in vivo epigenetic regulation may define critical GSC dependencies, we interrogated active chromatin landscapes of GSCs derived from intracranial xenografts and cell culture through Histone H3 lysine 4 trimethylation (H3K4me3) chromatin immunoprecipitation and transcriptome analysis. Intracranial-specific genes marked by H3K4me3 included FOS, NFkB, and phosphodiesterase family members. In intracranial tumors, DPY30 regulated angiogenesis and hypoxia pathways in an H3K4me3-dependent manner, but was dispensable in cultured GSCs. PDE4B was a key downstream effector of DPY30; the PDE4 inhibitor, rolipram, preferentially targeted DPY30-expressing cells and impaired in vivo glioblastoma growth without affecting cultured tumor cells. Collectively, DPY30 selectively regulates H3K4me3 modification on genes critical to support angiogenesis and tumor growth in vivo, laying the foundation for the DPY30-PDE4B axis as a specific and novel therapeutic target in glioblastoma with a broad therapeutic index. Overall design: H3K4me3 ChIP was performed in 5 glioblastoma stem cell (GSC) lines grown either in cell culture or in immunocompromised mice for 3 weeks

胶质母细胞瘤（Glioblastoma）是一类普遍致命的恶性肿瘤，其体内含有可启动肿瘤发生的自我更新型胶质母细胞瘤干细胞（GSCs）。传统基于体外培养的抗癌药物研发模式，往往会筛选出治疗指数不佳的靶点，且无法准确模拟肿瘤微环境的生物学效应。
本研究借助体内遗传筛选技术，将组蛋白H3赖氨酸4三甲基化（Histone H3 lysine 4 trimethylation，H3K4me3）调控因子DPY30鉴定为胶质母细胞瘤的体内特异性必需依赖基因。基于"体内表观遗传调控可决定关键GSC必需依赖通路"这一假说，我们通过H3K4me3染色质免疫共沉淀（ChIP）与转录组分析，解析了源自颅内异种移植瘤与体外细胞培养的GSCs的活性染色质景观。经H3K4me3标记的颅内特异性基因包括FOS、NF-κB以及磷酸二酯酶家族成员。
在颅内肿瘤中，DPY30以H3K4me3依赖的方式调控血管生成与缺氧通路，但在体外培养的GSCs中却非必需。PDE4B是DPY30的关键下游效应因子；PDE4抑制剂咯利普兰（rolipram）可优先靶向表达DPY30的细胞，并在不影响体外培养肿瘤细胞的前提下，抑制体内胶质母细胞瘤的生长。
综上，DPY30可选择性调控体内支撑血管生成与肿瘤生长的关键基因的H3K4me3修饰，为将DPY30-PDE4B轴作为具备宽泛治疗指数的胶质母细胞瘤特异性新型治疗靶点奠定了理论基础。
实验整体设计：对5株分别在体外培养与免疫缺陷小鼠体内培养3周的胶质母细胞瘤干细胞（GSCs）系进行H3K4me3 ChIP检测。