Determining the role of DNMT-interacting RNAs in myeloid disorders

描述（由申请人提供）：DNA甲基化是在基因表达中起介导作用的关键表观遗传特征。许多研究已经建立了异常基因组甲基化和癌症之间的联系。在骨髓增生异常综合征（MDS）中，一组异质性的恶性造血系统疾病，DNA甲基化异常在30%的病例中发生的急性髓系白血病（AML）进展过程中起着关键作用。迄今为止，有害的基因组甲基化的原因仍然难以捉摸。最近，我们发现了一类新的RNA，能够与DNA甲基转移酶1（DNMT 1），DNMT 1相互作用RNA（DiR），并抑制DNMT 1酶活性，从而调节基因组甲基化模式和相应基因的表达。我们已经证明，DiR起源于甲基化敏感基因CEBPA的基因座-编码外CEBPA（ecCEBPA）-通过与DNMT 1相互作用阻止CEBPA基因座甲基化来调节CEBPA表达。此外，通过对与DNMT 1相关的转录物进行深度测序，结合基因组规模的甲基化和表达谱分析，我们将这一发现的普遍性扩展到了许多基因位点。在这里，我们假设转录谱的失调触发DNA甲基化的变化，促进继发性白血病的发生。该项目的两个目的是：1）鉴定在MDS演变为AML中参与建立异常DNA甲基化模式的DiR; 2）验证其作为重置异常基因组甲基化的工具的潜力。在丹尼尔G Tenen教授（哈佛医学院）和John L. Rinn（哈佛大学），分别是白血病发生和RNA生物学领域的世界领先专家。通过将原代配对的MDS（诊断）/AML（进展）骨髓单个核细胞的DiR和DiR调节的基因的转录谱与相应基因组区域的DNA甲基化状态相关联，DNA甲基化和基因表达谱将与RNA-DNMT关联的存在或不存在相关联。这将导致鉴别与MDS-AML转化中的异常DNA甲基化模式相关的DiR（目的1）。将在R 00阶段、体外和体内系统（目标2）中对选定的候选物进行功能验证。这种方法将允许使用RNA分子校正基因特异性异常DNA甲基化。目前，唯一批准用于治疗应用的去甲基化剂是阿扎胞苷和地西他滨。然而，使用这些药物的主要缺点包括细胞毒性作用和全局非特异性去甲基化。这项研究有望实现真正的靶向治疗，而不是名义上的靶向治疗。虽然在短期内，这项研究将导致识别有助于白血病发生的新型ky调节剂;从长远来看，它将为开发期待已久的基因特异性去甲基化工具铺平道路，用于治疗癌症和其他由DNA甲基化异常引发的疾病。

DESCRIPTION (provided by applicant): DNA methylation is a key epigenetic signature playing mediating role in gene expression. Numerous studies have established a link between aberrant genomic methylation and cancers. In Myelodysplastic Syndromes (MDS), a heterogeneous group of malignant hematopoietic disorders, DNA methylation abnormalities play a pivotal role during the progression to Acute Myeloid Leukemia (AML) occurring in 30 percent of the cases. To date the cause of a deleterious genomic methylation remains elusive. Recently we identified a novel class of RNAs able to interact with DNA methyltransferase 1 (DNMT1), DNMT1-interacting RNAs (DiRs) and inhibit DNMT1 enzymatic activity thus regulating genomic methylation patterns and expression of the corresponding genes. We have shown that DiR originating within the locus of the methylation sensitive gene CEBPA - the extra-coding CEBPA (ecCEBPA) - regulates CEBPA expression by preventing CEBPA gene locus methylation through its interaction with DNMT1. Furthermore, by deep sequencing of the transcripts associated with DNMT1, combined with genome-scale methylation and expression profiling we have extended the generality of this finding to numerous gene loci. Here, we hypothesize that dysregulation of transcriptional profile triggers DNA methylation changes promoting secondary leukemogenesis. The two aims of this project are: 1) to identify DiRs involved in the establishment of aberrant DNA methylation patterns in MDS evolution to AML: and 2) to validate their potential as a tool to reset aberrant genomic methylation. Aim 1 will be accomplished during the K99 mentored phase, under the guidance of Prof. Daniel G Tenen (Harvard Medical School) and Prof. John L. Rinn (Harvard University), world-leading experts in the field of leukemogenesis and RNA biology, respectively. By correlating DiRs and DiRs-regulated genes' transcription profiles of primary paired MDS (diagnosis)/AML (progression) bone marrow mononuclear cells with DNA methylation status of the correspondent genomic regions, DNA methylation and gene expression profiles will be linked to the presence or absence of RNA-DNMT association. This will lead to the identification of DiRs associated with aberrant DNA methylation patterns in MDS-AML transformation (Aim 1). Chosen candidates will be functionally validated during the R00 phase, in vitro and in vivo systems (Aim 2). This approach will allow correcting gene-specific aberrant DNA methylation using RNA molecules. Currently, the only demethylating agents approved for therapeutic applications are Azacitidine and Decitabine. However major downsides for using these drugs include cytotoxic effects and global non-specific demethylation. This study holds promise for a genuine, rather than nominal, targeted therapy. While in the short terms this research will lead to the identification of novel ky regulators contributing to leukemogenesis; in the long terms it will pave the way for the development of a long-awaited gene-specific demethylating tool for the treatment of cancer and other diseases triggered by DNA methylation abnormalities.