The role of ALKBH5-mediated RNA demethylation in the maintenance of genomic stability in HSPCs

摘要 骨髓增生异常综合征(MDS)是一组起源于 来自造血干细胞(HSCs)。活性氧(ROS)水平升高和DNA损伤 在MDS患者的造血细胞中检测到。ROS水平提升，可通过以下方式生成 内源和外源以及癌基因的激活，都会导致静止和 造血干细胞的自我更新。ROS诱导的DNA损伤加速了干细胞的老化过程，并有助于 与癌症发展相关的突变。M6A RNA甲基化在多个 通过在基因表达中引入另一层转录后调控来实现生物过程 细胞。这个项目的目标是阐明rna表观遗传调控和dna之间的串扰作用。 损伤修复在维持造血干/祖细胞基因组稳定性中的作用 氧化应激，以及ALKBH5的解除调控如何有助于促进白血病转化 HSPC在MDS的发生发展中的作用。我们发现，ROS显著增加了全球m6A RNA 人类细胞系中的甲基化，而m6A mRNA甲基化的快速上调是 修复ROS诱导的DNA损伤，防止细胞死亡。有趣的是，我们发现ALKBH5，M6A RNA去甲基酶负责ROS诱导的m6A基因甲基化的上调。ROS诱导的后遗症 对ALKBH5进行翻译修饰，并抑制ALKBH5的去甲基酶活性。我们向大家展示了 强制表达ALKBH5抑制ROS诱导的m6A基因甲基化并显著延迟修复 ROS诱导的DNA损伤。因此，我们假设1)ALKBH5介导的m6A修饰具有 在维持基因组完整性和细胞对氧化应激的反应中的重要作用 HSPC；2)ALKBH5的解除管制可能扰乱HSPC功能，从而促进白血病 HSPC的改造。在这项提案中，我们将确定1)Alkbh5的作用和潜在机制 氧化应激在维持HSPC基因组稳定性中的作用；2) ALKBH5/Alkbh5在小鼠和人原代HSPC体内ROS应激维持中的作用 AlkBH5/Alkbh5如何促进del(5q)MDS的发展。我们将利用两种基因小鼠 模型以及患者来源的异种移植(PDX)模型，以研究ALKBH5在 体内维持HSPC，并将结合转录组和表位转录组学分析来鉴定 调节ALKBH5在HSPC中作用的关键下游靶点和相关下游通路 有或没有ROS诱导的应激反应。我们的研究将为新的机制提供新的见解 基因表达的表位转录调控以及发现调节活性的新机制 ALKBH5的氧化应激反应。这项研究将提供第一组证据来支持 ALKBH5介导的m6A基因去甲基化在HSPC维持中的重要作用

Abstract Myelodysplastic syndromes (MDS) are a group of diverse malignant hematological disorders that originate from hematopoietic stem cells (HSCs). Increased levels of reactive oxygen species (ROS) and DNA damage were detected in hematopoietic cells from MDS patients. An elevated level of ROS that can be generated from both endogenous and exogenous sources as well as oncogene activation, leads to loss of quiescence and self-renewal of HSCs. ROS-induced DNA damage speeds up aging process of stem cells and contributes to the mutagenesis associated with cancer development. m6A RNA methylation has significant roles in multiple biological processes by introducing another layer of post-transcriptional regulation of gene expression within cells. The goal of this project is to elucidate the role of crosstalk between RNA epigenetic regulation and DNA damage repair in the maintenance of genomic stability in hematopoietic stem/progenitor cell (HSPCs) during oxidative stress, and how deregulation of ALKBH5 contributes to promotion of leukemic transformation of HSPCs in the initiation and development of MDS. We found that ROS significantly increased global m6A RNA methylation in human cell lines, and that the elevation of m6A mRNA methylation is required for rapidly repairing ROS-induced DNA lesions and preventing cell death. Interestingly, we found that ALKBH5, the m6A RNA demethylase, is responsible for ROS-induced elevation of m6A mRNA methylation. ROS induced post- translational modification of ALKBH5, and inhibited the demethylase activity of ALKBH5. We showed that forced expression of ALKBH5 inhibited ROS-induced m6A mRNA methylation and significantly delayed repair of ROS-induced DNA damage. Thus, we hypothesize 1) that ALKBH5-mediated m6A modification has a significant role in the maintenance of genome integrity and survival of cells in response to oxidative stress in HSPCs; and 2) that deregulation of ALKBH5 may disrupt HSPC functions, thereby promoting leukemic transformation of HSPCs. In this proposal, we will determine 1) the role and underlying mechanism of Alkbh5 in the maintenance of genomic stability in HSPCs in response to oxidative stress; 2) the role of ALKBH5/Alkbh5 in the maintenance of mouse and human primary HSPCs during ROS stress in vivo; and 3) how ALKBH5/Alkbh5 contributes to the development of del(5q) MDS. We will employ both genetic murine models as well as patient-derived xeno-transplantation (PDX) models to investigate the role of ALKBH5 in the maintenance of HSPCs in vivo, and will combine transcriptome and epitranscriptome analysis to identify the key downstream targets and associated downstream pathways that mediate the role of ALKBH5 in HSPCs with or without ROS-induced stress. Our study will provide new insights into novel mechanisms underlying epitranscriptional regulation of gene expression as well as uncover novel mechanisms that regulate the activity of ALKBH5 in response to oxidative stress. This study will provide the first set of evidence to support a significant role of ALKBH5-mediated m6A mRNA demethylation in the maintenance of HSPCs.