Determining the role of DNMT-interacting RNAs in myeloid disorders

描述(申请人提供)：DNA甲基化是一个关键的表观遗传特征，在基因表达中起着中介作用。许多研究已经确定了基因组甲基化异常与癌症之间的联系。在骨髓增生异常综合征(MDS)中，DNA甲基化异常在30%的急性髓系白血病(AML)进展中起着关键作用。到目前为止，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(DNMT1)相互作用，并抑制DNMT1的酶活性，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-额外编码的CEBPA(EcCEBPA)-通过与DNMT1的相互作用阻止CEBPA基因位点的甲基化来调节CEBPA的表达。此外，通过对与DNMT1相关的转录本进行深度测序，结合基因组规模的甲基化和表达谱，我们将这一发现的一般性扩展到了许多基因位点。这里，我们假设转录模式的失调触发DNA甲基化改变，促进继发性白血病的发生。本项目的两个目标是：1)鉴定在MDS向AML演变过程中参与建立异常DNA甲基化模式的DIR；2)验证它们作为重置基因组异常甲基化的工具的潜力。目标1将在K99指导阶段完成，分别在白血病发生和RNA生物学领域的世界领先专家Daniel G Tenen教授(哈佛医学院)和John L.Rinn教授(哈佛大学)的指导下完成。通过将原配对MDS(诊断)/AML(进展)骨髓单个核细胞DIRS和DIRS调控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关联的存在与否联系在一起。这将导致识别与MDS-AML转化中异常DNA甲基化模式相关的DIRS(目标1)。选定的候选者将在R00阶段、体外和体内系统中进行功能验证(目标2)。这种方法将允许使用RNA分子纠正特定基因的异常DNA甲基化。目前，唯一被批准用于治疗应用的去甲基化药物是氮杂替丁和地西他滨。然而，使用这些药物的主要缺点包括细胞毒性作用和全球非特异性去甲基化。这项研究为真正的、而不是名义上的靶向治疗带来了希望。虽然在短期内，这项研究将导致确定有助于白血病发生的新的Ky调控因子；但从长期来看，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.