The role of PTEN in epigenetic and metabolic regulation of IDH-mutant gliomas

摘要 中枢神经系统(CNS)的星形细胞肿瘤是一种难治性肿瘤，预后不佳。 缺乏有针对性的治疗选择。超过70%的星形细胞肿瘤在TCA循环中携带突变- 相关基因异柠檬酸脱氢酶1(IDH1)。野生型IDH1催化氧化脱羧基 异柠檬酸生成α-酮戊二酸(αKG)，而突变的IDH1m将αKG代谢成 代谢物D-2-羟基戊二酸(D2HG)。包括组蛋白和DNA在内的几种表观遗传双加氧酶 去甲基酶需要αKG去甲基化组蛋白残基和DNACpG岛。具有竞争力的D2HG 由于其结构与αKG相似，因此可抑制这些酶。这会导致组蛋白和DNA的增加 甲基化称为胶质瘤-CpG岛甲基化表型(G-CIMP)。然而，DNA甲基化 在IDH1M星形细胞瘤中，G-CIMP水平并不均匀。IDH1M星形细胞瘤低G-高水平与高G- CIMP的前景尤其严峻。我的初步数据表明，PTEN杂合性缺失(LOH)是 G-CIMP低与G-CIMP高的肿瘤顶端基因改变。此外，PTEN LOH独立存在 与预后不良有关。然而，目前尚不清楚PTEN杂合性缺失是如何促进IDH1m的致瘤性的 星形细胞瘤。我们的前提是观察到PTEN是一种关键的肿瘤抑制因子。在其他 肿瘤，PTEN杂合性缺失激活PI3K/AKT-mTOR信号通路，通过多种途径促进肿瘤生长 包括代谢重新编程在内的机制。我的初步数据显示细胞增殖增加 在IDH1M星形细胞系中，部分PTEN基因敲除(KD)伴随着D2HG水平的降低。 根据我的假设和初步数据，我假设PTEN LOH激活了PI3K/AKT-mTOR信号 对IDH1M星形细胞瘤进行代谢重编程以降低D2HG水平，从而调节DNA的降低 甲基化和G-CIMP水平。因此，靶向PI3K/AKT-mTOR信号转导系统是一个很有前途的研究方向 治疗开发的候选人。为了检验这一假设，我提出了两个具体目标。目标1将映射 人和鼠源性IDH1M星形细胞瘤中产生D2HG的代谢途径的改变 或使用新的宫内电穿孔(IUE)Pten+/-IDH1m模型不降低PTEN。同时，我会 使用下一代评估相应的全基因组表观遗传改变，包括DNA甲基化 基于测序的表观遗传学分析与基因表达变化的关系。目标2将决定是否 靶向PTEN LOH驱动的PI3K/AKT-mTOR激活在体外具有治疗作用，在动物中也是一种原则证明 模特们。总之，我的工作将通过定义PTEN Loh如何推动 通过代谢重编程获得侵袭性IDH1M星形细胞瘤亚群并为开发奠定基础 恶性IDH1m星形细胞瘤的靶向有效治疗。

ABSTRACT Astrocytic tumors of the central nervous system (CNS) are intractable tumors and harbor dismal outcomes due to a lack of targeted treatment options. More than 70% of astrocytic tumors bear mutations in the TCA cycle- related gene, isocitrate dehydrogenase 1 (IDH1). Wildtype IDH1 catalyzes the oxidative decarboxylation of isocitrate to generate α-Ketoglutarate (αKG), while mutated IDH1 (IDH1m) metabolizes αKG into the oncometabolite D-2-hydroxyglutarate (D2HG). Several epigenetic dioxygenases including histone and DNA demethylases require αKG to demethylate histone residues and DNA CpG islands. D2HG competitively inhibits these enzymes due to its structural similarity to αKG. This results in an increase in histone and DNA methylation referred to as the glioma-CpG island methylation phenotype (G-CIMP). However, DNA methylation G-CIMP levels are not uniform in IDH1m astrocytomas. IDH1m astrocytomas with low versus high levels of G- CIMP bear a particularly grim prognosis. My preliminary data identifies PTEN loss of heterozygosity (LOH) as the top genetic alteration in G-CIMP low versus G-CIMP high tumors. Moreover, PTEN LOH independently related with a poor prognosis. However, it remains unknown how PTEN LOH drives tumorigenicity in IDH1m astrocytomas. Our premise is based on the observation that PTEN is a critical tumor suppressor. In other cancers, PTEN LOH activates PI3-kinase (PI3K)/AKT-mTOR signaling to drive tumor growth through various mechanisms including metabolic reprogramming. My preliminary data demonstrates increased cell proliferation in IDH1m astrocytic cell lines with partial PTEN knockdown (KD) accompanied by a reduction in D2HG levels. Based on my premise and preliminary data, I hypothesize that PTEN LOH activates PI3K/AKT-mTOR signaling to metabolically reprogram IDH1m astrocytomas to reduce D2HG levels and thereby mediate lowered DNA methylation and G-CIMP levels. Subsequently, targeting PI3K/AKT-mTOR signaling represents a promising candidate for therapeutic development. To test this hypothesis, I propose two specific aims. Aim 1 will map alterations in metabolic pathways that generate D2HG in human and murine-derived IDH1m astrocytomas with or without PTEN reduction using a novel in-utero electroporation (IUE) Pten +/- IDH1m model. In parallel, I will assess corresponding genome wide epigenetic alterations including DNA methylation using next generation sequencing-based epigenetic assays in relation to changes in gene expression. Aim 2 will determine if targeting PTEN LOH-driven PI3K/AKT-mTOR activation is therapeutic in vitro and proof-of-principle in animal models. Together, my work will address a critical gap in our knowledge by defining how PTEN LOH drives a subset of aggressive IDH1m astrocytomas via metabolic reprograming and lay the groundwork for developing targeted and effective therapies for malignant IDH1m astrocytomas.