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

ALKBH5 介导的 RNA 去甲基化在维持 HSPC 基因组稳定性中的作用

• 批准号: 10445661
• 负责人: Zhijian Qian
• 金额: $ 47.85万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445661
• 关键词: Aging; Apoptosis; Biological Process; CD34 gene; Cell Death; Cell Maintenance; Cells; Chromatin; DNA Damage; DNA Repair; DNA lesion; Development; Dysmyelopoietic Syndromes; Functional disorder; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genome Stability; Genomic Instability; Goals; Growth; Hematological Disease; Hematopoietic; Hematopoietic stem cells; Histone Deacetylase; Human; Human Cell Line; In Vitro; Life; Long-Term Effects; Maintenance; Malignant - descriptor; Malignant Neoplasms; Mediating; Mediator of activation protein; Messenger RNA; Methylation; Modeling; Modification; Molecular; Mus; Mutagenesis; Oncogene Activation; Oncogenes; Oxidative Stress; Pathway interactions; Patients; Phenocopy; Physiological; Play; Polyribosomes; Post-Transcriptional Regulation; Post-Translational Protein Processing; Process; RNA; RNA methylation; Reactive Oxygen Species; Role; Source; Speed; Stem Cell Development; Stress; System; Testing; Time; Transgenic Mice; Transgenic Organisms; Up-Regulation; Xenograft procedure; cell injury; crosslinking and immunoprecipitation sequencing; demethylation; epigenetic regulation; genome integrity; hematopoietic stem cell self-renewal; in vivo; insight; knock-down; leukemic transformation; novel; novel therapeutic intervention; overexpression; prevent; repaired; response; ribosome profiling; self-renewal; stem cell function; stem cells; transcriptome sequencing; tumorigenesis

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).

抽象的 骨髓增生异常综合征 (MDS) 是一组不同的恶性血液病， 源自造血干细胞（HSC）。活性氧 (ROS) 和 DNA 水平增加 MDS 患者的造血细胞经常出现损伤。 ROS水平升高， 由内源性或外源性来源（包括癌基因激活）产生，导致损失 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突变。 m6A RNA 甲基化起着 通过引入另一层转录后调控，在多种生物过程中发挥重要作用 细胞内的基因表达。该项目的目标是阐明 ALKBH5 介导的重要作用 表观遗传调控在维持造血干/祖细胞（HSPC）基因组稳定性中的作用 氧化应激期间的变化，以及 ALKBH5 的失调如何促进白血病转化 HSPC 在 MDS 的启动和发展中的作用。我们发现ROS显着增加了全局m6A 人类细胞系中的 RNA 甲基化，并且 m6A mRNA 甲基化的升高是快速 修复 ROS 诱导的 DNA 损伤并防止细胞死亡。有趣的是，我们发现 ALKBH5，即 m6A RNA 去甲基化酶负责 ROS 诱导的 m6A mRNA 甲基化升高。 ROS诱导后 ALKBH5 的翻译修饰，并抑制 ALKBH5 的去甲基酶活性。我们证明了 ALKBH5 的强制表达抑制 ROS 诱导的 m6A mRNA 甲基化并显着延迟修复 ROS 诱导的 DNA 损伤。因此，我们假设 ALKBH5 的异常表达会破坏 HSPC 通过对 HSPC 的基因组完整性和存活产生负面影响来发挥功能，从而导致白血病 MDS 的启动和发展过程中 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和潜在机制 氧化应激； 2）ALKBH5/Alkbh5过表达对小鼠和人类维持的影响 体内 ROS 应激期间的初级 HSPC； 3) 是否需要 ALKBH5/Alkbh5 来维持 MDS 中的前白血病干细胞 (pre-LSC)。我们的研究将为新机制提供新的见解 MDS 的发展和 HSPC 响应氧化的基因表达的表转录调控 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（前 LSC）。