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 ...