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

摘要 中枢神经系统（CNS）的星形细胞肿瘤是难治性肿瘤， 缺乏针对性的治疗方案。超过70%的星形细胞肿瘤在TCA循环中携带突变- 异柠檬酸脱氢酶1（IDH 1）。野生型IDH 1催化 异柠檬酸生成α-酮戊二酸（αKG），而突变的IDH 1（IDH 1 m）将αKG代谢为 癌代谢产物D-2-羟基戊二酸（D2 HG）。几种表观遗传双加氧酶，包括组蛋白和DNA 脱甲基酶需要αKG使组蛋白残基和DNA CpG岛脱甲基。D2 HG竞争力 由于其与αKG的结构相似性而抑制这些酶。这导致组蛋白和DNA的增加 这种甲基化被称为胶质瘤-CpG岛甲基化表型（G-CIMP）。然而，DNA甲基化 IDH 1 m星形细胞瘤中G-CIMP水平不均匀。G-水平低与高的IDH 1 m星形细胞瘤 CIMP的前景尤其严峻。我的初步数据确定了PTEN杂合性缺失（洛）， 在G-CIMP低相对于G-CIMP高的肿瘤中最高的遗传改变。此外，PTEN洛独立地 与预后不良有关。然而，PTEN洛缺失如何驱动IDH 1 m的致瘤性仍不清楚 星形细胞瘤我们的前提是基于观察到PTEN是一个重要的肿瘤抑制因子。换句 在癌症中，PTEN洛激活PI 3-激酶（PI 3 K）/AKT-mTOR信号传导以通过各种途径驱动肿瘤生长。 包括代谢重编程的机制。我的初步数据显示细胞增殖 在IDH 1 m星形胶质细胞系中，部分PTEN敲除（KD）伴随着D2 HG水平的降低。 基于我的前提和初步数据，我假设PTEN洛激活PI 3 K/AKT-mTOR信号传导 代谢重编程IDH 1 m星形细胞瘤以降低D2 HG水平，从而介导DNA降低 甲基化和G-CIMP水平。随后，靶向PI 3 K/AKT-mTOR信号转导代表了一种有希望的 治疗开发的候选人。为了验证这一假设，我提出了两个具体目标。目标1将映射 在人和鼠源性IDH 1 m星形细胞瘤中产生D2 HG的代谢途径的改变， 或不使用新的子宫内电穿孔（IUE）Pten +/-IDH 1 m模型进行PTEN减少。与此同时，我将 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分析与基因表达的变化有关。目标2将确定是否 靶向PTENLOH驱动PI 3 K/AKT-mTOR活化在体外是治疗性的且在动物中是原理验证的 模型总之，我的工作将通过定义PTEN洛如何驱动一个基因， 通过代谢重编程治疗侵袭性IDH 1 m星形细胞瘤亚群，并为开发 恶性IDH 1 m星形细胞瘤的靶向和有效治疗。

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.