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）是一组起源于骨髓增生异常综合征的多种恶性血液病。 造血干细胞（HSCs）活性氧（ROS）和DNA损伤水平增加 在MDS患者的造血细胞中检测到。高水平的活性氧可以产生自 内源性和外源性来源以及癌基因激活，导致静止期的丧失， HSC的自我更新。ROS诱导的DNA损伤加速干细胞的衰老过程，并有助于 与癌症发展相关的突变。m6A RNA甲基化在多种肿瘤中具有重要作用。 通过引入另一层基因表达的转录后调节， 细胞这个项目的目标是阐明RNA表观遗传调控和DNA之间的串扰的作用 造血干/祖细胞（HSPCs）基因组稳定性的损伤修复 氧化应激，以及ALKBH5的失调如何促进白血病转化， HSPCs在MDS的发生和发展中的作用我们发现活性氧显着增加了全球m6A RNA 在人类细胞系中，m6A mRNA甲基化的升高是快速表达所必需的。 修复ROS诱导的DNA损伤和防止细胞死亡。有趣的是，我们发现ALKBH 5，m6A RNA去甲基化酶负责ROS诱导的m6A mRNA甲基化升高。ROS诱导的后 ALKBH 5的翻译修饰，并抑制ALKBH 5的脱甲基酶活性。我们发现 ALKBH 5的强制表达抑制ROS诱导的m6A mRNA甲基化，并显著延迟修复 ROS引起的DNA损伤因此，我们假设：1）ALKBH 5介导的m6A修饰具有 在维持基因组的完整性和细胞对氧化应激的反应中的存活中起重要作用， HSPC;和2）ALKBH 5的失调可能破坏HSPC功能，从而促进白血病 HSPCs的转化。在这个提议中，我们将确定1）Alkbh 5的作用和潜在机制 在维持HSPCs的基因组稳定性中对氧化应激的反应; 2） ALKBH 5/Alkbh 5在体内ROS应激期间维持小鼠和人原代HSPC中的作用;以及3） ALKBH 5/Alkbh 5如何促进del（5q）MDS的发展。我们将使用基因鼠 模型以及患者来源的异种移植（PDX）模型，以研究ALKBH 5在 维持HSPC在体内，并将结合联合收割机转录组和表位转录组分析，以确定 介导ALKBH 5在HSPC中作用的关键下游靶点和相关下游途径 有或没有ROS引起的应力。我们的研究将为潜在的新机制提供新的见解。 基因表达的表观调控，以及发现新的机制，调节活性 ALKBH5在氧化应激中的作用这项研究将提供第一组证据来支持一个 ALKBH 5介导的m6A mRNA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.