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）是一组不同的恶性血液病， 来源于造血干细胞（HSC）。活性氧（ROS）和DNA水平增加 通常在MDS患者的造血细胞中检测到损伤。活性氧水平升高， 由内源性或外源性来源产生，包括癌基因激活，导致细胞凋亡的丧失。 HSC的静止和自我更新。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诱变。m6 A RNA甲基化在 通过引入另一层转录后调节， 细胞内的基因表达。该项目的目标是阐明ALKBH 5介导的 造血干/祖细胞基因组稳定性的表观遗传调控 以及ALKBH 5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，ROS显著增加了全球m6 A 在人类细胞系中，m6 A mRNA甲基化水平的升高是快速启动细胞凋亡所必需的。 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，m6 A RNA去甲基化酶负责ROS诱导的m6 A 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m6 A mRNA甲基化，并显著延迟修复 ROS引起的DNA损伤因此，我们假设ALKBH 5的异常表达破坏了HSPC， 通过负面影响HSPC的基因组完整性和存活发挥功能，从而促进白血病的发生。 在MDS的起始和发展期间HSPC的转化。在本提案中，我们将确定1） ALKBH 5在维持HSPC基因组稳定性中的作用和潜在机制 2）ALKBH 5/Alkbh 5过表达对维持小鼠和人类细胞凋亡的影响。 在体内ROS应激期间的原代HSPC;和3）ALKBH 5/Alkbh 5是否是维持 前白血病干细胞（前LSC）在MDS。我们的研究将提供新的见解，新的机制， 氧化应激对骨髓增生异常综合征的发生及HSPCs基因表达的表观调控 应力此外，我们的研究将提供第一组证据来支持ALKBH 5的重要作用。 介导的m6 A mRNA去甲基化在维持正常HSPC和白血病前干细胞（前 LSC）。

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 are commonly detected in hematopoietic cells from MDS patients. An elevated level of ROS, generated from either endogenous or exogenous sources including oncogene activation, leads to loss of quiescence and self-renewal of HSCs. ROS-induced DNA damage speeds up the aging process of stem cells and contributes to the mutagenesis associated with cancer development. m6A RNA methylation plays a significant role 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 significant role of ALKBH5-mediated epigenetic regulation 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 that aberrant expression of ALKBH5 disrupts HSPC functions by negatively influencing genome integrity and survival of HSPCs, thereby contributing to leukemic transformation of HSPCs during the initiation and development of MDS. 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 effects of ALKBH5/Alkbh5 overexpression on the maintenance of mouse and human primary HSPCs during ROS stress in vivo; and 3) whether ALKBH5/Alkbh5 is required for the maintenance of pre-leukemic stem cells (pre-LSCs) in MDS. Our study will provide new insights into novel mechanisms of MDS development and epitranscriptional regulation of gene expression in HSPCs in response to oxidative stress. Additionally, our study will provide the first set of evidence to support a significant role of ALKBH5- mediated m6A mRNA demethylation in the maintenance of normal HSPCs and pre-leukemic stem cell (pre- LSCs).