Pathological reprogramming of the m6A epitranscriptome in uterine fibroids

项目摘要/摘要 子宫肌瘤(UF)是威胁全球妇女健康的最重要的良性肿瘤。不会再有了- 目前UF存在长期非侵入性治疗选择，对肿瘤病因的深入了解是开发的关键 更有效的治疗方法。在这方面，虽然UF的表观/遗传决定因素已经被表征 广泛地说，它们的潜在发病机制仍然不清楚，涉及到疾病中的其他因素。 开始了。在此，我们提出了一个新的基础来解释UF的发展通过病理性的重新编程 子宫肌层表位转录组学研究，为使用致癌抑制物进行治疗干预提供概念证据 由意外的m6A-染色质串扰驱动的增强子活性。作为体内最丰富的化学物质 MRNA的修饰，N6-甲基腺苷(M6A)是转录后mRNA命运的关键决定因素。 因此，细胞的身份和功能。因此，m6A动态平衡的破坏与多种不同的 慢性和急性人类疾病状况。然而，关于m6A在体内的作用尚不清楚。 尿崩症的发病机制。我们现在证明了M6A编写器METTL3和RBM15在UF中异常上调 与邻近子宫肌层(MM)比较。此外，我们还表明，METTL3缺失会触发UF细胞死亡和 转录抑制性组蛋白甲基化的全球上调，首次将m6A与UF肿瘤联系起来 生物学和揭示其与UF表观基因组的新颖串扰。整合的RNA甲基化和表达 在METTL3缺陷的UF细胞中的图谱显示了一种深刻改变的m6A修饰环境，并发现 高置信度m6A修饰的METTL3驱动UF细胞生长和存活的mRNA效应器。基于这些 发现，我们假设异常的依赖于METTL3的RNA甲基化重新编程MM 表观转录组，导致表观遗传失调和驱动UF启动和表达的基因表达改变 进步。因此，我们认为表观遗传抑制物通过抑制m6A驱动的PRO-2。 肿瘤形成途径，将为UF提供治疗益处。为了检验这些假设，我们将：(1)建立 UF中表位转录重编程的基础。我们将对比分析M6A改装的情况 来自成对的MM和UF肿瘤组织的mRNAs和染色体相关调控RNA(CarRNAs)以及 研究METTL3和RBM15在甲基组重编程中的功能协同作用；(2)描绘 依赖于METTL3的RNA甲基化在纤维化转化中的作用。我们会问METTL3，在某种程度上 依赖于其过表达和甲基转移酶的活性，可以在 体外和体内UF肿瘤形成；(3)阐明依赖METTL3的RNA甲基化对基因的影响 以UF表示。我们将评估m6A对mRNA稳定性和翻译以及carRNA的全球影响。 染色质状态和转录的依赖控制；(4)检查BRD抑制剂的治疗潜力 在人类尿失禁的临床前小鼠模型中。我们将评估选定的BRD抑制剂的治疗效果， 安全性和抗肿瘤活性的机制，包括对染色质状态和转录的影响。

PROJECT SUMMARY/ABSTRACT Uterine fibroids (UFs) are the most important benign neoplastic threat to women’s health worldwide. As no long- term non-invasive treatment option currently exists for UFs, deeper insight into tumor etiology is key to develop more effective therapies. In this regard, while the epi/genetic determinants of UFs have been characterized extensively, their underlying pathogenesis nonetheless remains obscure, implicating additional factors in disease onset. Herein, we propose a novel basis to explain UF development through pathological reprogramming of the myometrial epitranscriptome and offer proof of concept for therapeutic intervention using inhibitors of tumorigenic enhancer activity driven by unanticipated m6A-chromatin crosstalk. As the most abundant internal chemical modification in mRNA, N6-methyladenosine (m6A) is a key determinant of posttranscriptional mRNA fate and thus cell identity and function. Accordingly, disruption of m6A homeostasis is implicated in a diverse range of chronic and acute human disease conditions. However, nothing is known about the role of m6A in the pathogenesis of UFs. We now show that m6A writers METTL3 and RBM15 are aberrantly upregulated in UFs compared to adjacent myometrium (MM). Further, we show that METTL3 depletion triggers UF cell death and global upregulation of transcriptionally repressive histone methylation, linking m6A for the first time with UF tumor biology and revealing its novel crosstalk with the UF epigenome. Integrated RNA methylation and expression profiling in METTL3-deficient UF cells revealed a profoundly altered m6A modification landscape and identified high-confidence m6A-modified mRNA effectors of METTL3-driven UF cell growth and survival. Based on these findings, we hypothesize that aberrant METTL3-dependent RNA methylation reprograms the MM epitranscriptome, leading to epigenetic dysregulation and altered expression of genes that drive UF initiation and progression. Accordingly, we propose that epigenetic inhibitors, through suppression of m6A-driven pro- tumorigenic pathways, will provide therapeutic benefit in UFs. To test these hypotheses we will: (1) Establish the basis of epitranscriptomic reprogramming in UFs. We will comparatively profile the m6A modification landscape of mRNAs and chromosome-associated regulatory RNAs (carRNAs) from paired MM and UF tumor tissues and investigate functional cooperativity between METTL3 and RBM15 in methylomic reprogramming; (2) Delineate the role of METTL3-dependent RNA methylation in fibrotic transformation. We will ask if METTL3, in a manner dependent upon its overexpression and methyltransferase activity, can trigger MM stem cell transformation in vitro and UF tumor formation in vivo; (3) Elucidate the impact of METTL3-dependent RNA methylation on gene expression in UFs. We will assess the global impact of m6A on mRNA stability and translation as well as carRNA- dependent control of chromatin state and transcription; (4) Examine the therapeutic potential of BRD inhibitors in a preclinical mouse model of human UFs. We will evaluate select BRD inhibitors for therapeutic efficacy, safety, and mechanism of anti-tumor activity, including impact on chromatin status and transcription.