Targeting m6A RNA methylation for glioma therapy

项目摘要/摘要 胶质瘤是最常见的成人原发脑癌，每年影响超过2万名美国人。尽管 过去的分子特征定义了表现出不同疾病轨迹的胶质瘤亚型和 在治疗反应方面，仍然缺乏个性化的治疗方法。这项研究的目标是 描述和开发一种由RNA高甲基化控制的降低恶性程度的可能机制。 表现出异柠檬酸脱氢酶1(IDHMUT)特征突变的胶质瘤生长明显减慢 而不是IDHWT肿瘤。这赋予了明显的临床优势。IDHMUT干扰正常的IDH1酶活性， 导致一种名为D-2-羟基戊二酸(D2HG)的肿瘤代谢物的产生。D2HG直接抑制α- 酮戊二酸依赖的双加氧酶参与一系列细胞功能，包括 DNA和RNA。D2HG介导的Tet家族DNA去甲基酶抑制导致DNA高甲基化 IDHMUT胶质瘤的甲基化程度与生存率的改善呈正相关。 FTO和AlkBH5是已知的仅有的两种RNA去甲基酶，都对D2HG介导的RNA去甲基酶敏感 抑制力。然而，RNA高甲基化在胶质瘤中还没有被研究过。这项提议旨在 对IDHMUT和IDHWT胶质瘤进行第一次‘表位转录’分析，评估 患者肿瘤样本和患者来源的胶质瘤干细胞。RNA甲基化最常发生在 N6-甲基腺苷(M6A)的形式，与许多转录后过程有关，包括 信使核糖核酸的稳定性、细胞内定位和翻译。我将采用最近开发的一种方法 用于研究m6A丰度和定位的M6A RNA免疫沉淀测序(MERIP-Seq) 具有文字记录级别的分辨率。MERIP-Seq是RNA-Seq的修改版本，我将使用这两种技术 鉴定富含m6A的转录本，并使用这些各自的 接近了。这将使我能够研究IDHMUT胶质瘤是否表现出一组可复制的m6A- 丰富的转录本，并评价m6A富集物对转录稳定性的影响。我还将评估M6A 作为改善预后的生物标记物的患者肿瘤的概况。为了阐明m6A超甲基化是否 作为临床上看好的IDHMUT亚型中抑制细胞增殖的关键因素，我将抑制FTO IDHWT胶质瘤细胞并确定抑制是否重现IDHMUT m6A高甲基化表型， 减少肿瘤增殖，提高总体存活率。综上所述，这项工作将提供对 M6A基因调控与脑胶质瘤恶性程度的机制及临床评价 抑制FTO作为一种新的治疗方法的疗效。

PROJECT SUMMARY/ABSTRACT Glioma is the most common primary adult brain cancer, affecting over 20,000 Americans each year. Though past molecular characterizations defined glioma subtypes exhibiting different disease trajectories and treatment responses, there remains a paucity of personalized therapeutic approaches. The goal of this study is to delineate and exploit a putative mechanism of decreased malignancy governed by RNA hypermethylation. Gliomas exhibiting characteristic mutations in isocitrate dehydrogenase 1 (IDHMUT) grow significantly slower than IDHWT tumors. This confers a clear clinical advantage. IDHMUT disrupts normal IDH1 enzymatic activity, leading to the production of an oncometabolite called D-2-hydroxyglutarate (D2HG). D2HG directly inhibits α- ketoglutarate-dependent dioxygenases involved in a range of cellular functions, including demethylation of DNA and RNA. D2HG-mediated inhibition of TET-family DNA demethylases leads to hypermethylated DNA profiles in IDHMUT gliomas, with the degree of methylation being positively associated with improved survival. FTO and AlkBH5 are the only two known RNA demethylases, and both are sensitive to D2HG-mediated inhibition. However, RNA hypermethylation has not been previously investigated in glioma. This proposal aims to perform the first `epitranscriptomic' profiling of IDHMUT and IDHWT gliomas, evaluating mRNA methylation in patient tumor samples and patient-derived glioma stem cells. RNA methylation occurs most frequently in the form of N6-methyladenosine (m6A), and is linked to a number of post-transcriptional processes including mRNA stability, intracellular localization, and translation. I will employ a recently developed methodology known as m6A RNA immunoprecipitation sequencing (MeRIP-Seq) to study m6A abundance and localization with transcript-level resolution. MeRIP-Seq is a modified version of RNA-Seq, and I will utilize both techniques to identify m6A-enriched transcripts and quantify overall transcript abundances using these respective approaches. This will allow me to investigate whether IDHMUT gliomas exhibit a reproducible set of m6A- enriched transcripts, and evaluate the effect of m6A enrichment on transcript stability. I will also assess m6A profiles in patient tumors as a biomarker of improved outcomes. To elucidate whether m6A hypermethylation is a key factor in reducing cell proliferation as seen in the clinically favored IDHMUT subtype, I will inhibit FTO in IDHWT glioma cells and determine if inhibition recapitulates the IDHMUT m6A hypermethylation phenotype, decreases tumor proliferation, and increases overall survival. Taken together, this work will provide insight into the mechanism of m6A-mediated gene regulation and malignancy in glioma, while evaluating the clinical efficacy of FTO inhibition as a novel therapeutic approach.