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疾病， 来源于造血干细胞(HSCs)。活性氧(ROS)和DNA水平升高 MDS患者的造血细胞常可检测到损伤。ROS水平升高， 由内源或外源产生，包括癌基因激活，导致 肝星状细胞的静止和自我更新。ROS诱导的DNA损伤加速干细胞老化过程 并促进了与癌症发展相关的突变。M6A RNA甲基化在 通过引入另一层转录后调控，在多个生物学过程中发挥重要作用 细胞内的基因表达。这个项目的目标是阐明ALKBH5介导的重要作用 表观遗传调控在维持造血干/祖细胞基因组稳定性中的作用 在氧化应激过程中，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 人类细胞系中的RNA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ALKBH5的异常表达破坏了HSPC 通过对HSPC的基因组完整性和存活率产生负面影响而发挥作用，从而导致白血病 MDS发生发展过程中HSPC的转化。在本提案中，我们将确定1) ALKBH5在维持HSPC基因组稳定性中的作用及其机制 2)ALKBH5/Alkbh5过表达对小鼠和人的维持作用 体内ROS应激时的原代HSPC；3)ALKBH5/Alkbh5是否维持 MDS中的白血病前干细胞(pre-LSCs)。我们的研究将为新的机制提供新的见解 MDS的发生和HSPC氧化反应中基因表达的表型调控 压力。此外，我们的研究将提供第一组证据来支持ALKBH5的重要作用- 介导的m6A mRNA去甲基化在维持正常HSPC和白血病前干细胞(Pre-MSC)中的作用 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).