Defining the Niche-dependent Role of RNA Editing in Aged and MDS Hematopoietic Stem and Progenitor Cell Dysfunction

定义RNA编辑在老年和MDS造血干细胞中的小生境依赖性作用， 祖细胞功能障碍 项目摘要 在衰老过程中，造血干细胞和祖细胞（HSPC）的维持受损， 克隆DNA突变以及小生境驱动的RNA加工失调可以为 骨髓增生异常综合征（MDS）启动。最近，腺苷脱氨酶增加与 我们的研究小组和其他研究小组表明，RNA 1（ADAR 1）介导的A到I编辑 导致了一系列恶性肿瘤的治疗耐药性。同时，我们发现， HSPC中的慢病毒增强的ADAR 1表达增强了骨髓分化， PU.1的上调和休眠减少。全转录组RNA测序（RNA-seq） 分析表明，炎症细胞因子信号通路和RNA编辑增加， 在正常老化的HSPC进展为MDS期间。因此，我们假设， ADAR 1的RNA编辑为MDS提供了超过正常HSPC的竞争优势。大多数 ADAR 1介导的腺苷到肌苷（A-to-I）RNA编辑事件在人类中发生在双 由灵长类特异性Alu序列产生的单链RNA（dsRNA）环，其中包含10%的 人类基因组，从而强调了重要的ADAR 1功能差异之间存在 人HSPC与其鼠对应物的比较。然而，有限的研究工作旨在 已破译ADAR 1介导的RNA编辑在HSPC维持中的作用 主要是在小鼠模型中，而不是在高度纯化的人HSPC中。因为ADAR 1是由 炎症细胞因子加速老化和MDS的启动，我们的主要目标是确定利基- RNA编辑对人HSPC细胞命运和衰老过程中细胞周期调控的依赖性作用， MDS启动。我们将首先通过全转录组和单细胞来确定RNA编辑谱 RNA-seq、RESSqPCR和慢病毒RNA编辑报告基因。ADAR 1在HSPC中的功能作用 将在添加或不添加以下物质的基质共培养物中检查老化和MDS起始 炎性细胞因子、FUCCI 2BL细胞周期报告基因和人源化老化HSC和MDS 免疫受损小鼠模型。我们还将研究RNA编辑对APOBEC 3的影响。 家族的DNA脱氨酶功能在MDS的启动。拟议的研究是唯一的回应， PAS-13-033：刺激血液学研究：新的努力（SHINE），因为它将 确定ADAR 1介导的调控mRNA和miRNA编辑在HSPC髓系中的作用 炎症性骨中年龄依赖性MDS启动过程中的定型和细胞周期失调 骨髓龛本研究的最终目的是确定生物学，诊断和预后 与MDS起始相比，ADAR 1介导的RNA编辑在HSPC老化中的意义。

Title Defining the Niche-dependent Role of RNA Editing in Aged and MDS Hematopoietic Stem and Progenitor Cell Dysfunction Project Summary During aging, impaired hematopoietic stem and progenitor cell (HSPC) maintenance induced by clonal DNA mutations as well as niche-driven RNA processing deregulation can set the stage for myelodysplastic syndrome (MDS) initiation. Recently, increased adenosine deaminase associated with RNA1 (ADAR1)-mediated A-to-I editing was shown by our group and other research teams to contribute to therapeutic resistance in a broad array of malignancies. Also, we discovered that lentivirally enforced ADAR1 expression in HSPC enhanced myeloid differentiation commensurate with upregulation of PU.1 and reduced dormancy. Whole transcriptome RNA sequencing (RNA-seq) analysis demonstrated that inflammatory cytokine signaling pathways and RNA editing increased during normal aged HSPC evolution to MDS. Thus, we hypothesized that niche dependent activation of RNA editing by ADAR1 provides a competitive advantage for MDS over normal HSPCs. The majority of ADAR1 mediated adenosine-to-inosine (A-to-I) RNA editing events in humans occur within double- stranded RNA (dsRNA) loops created by primate-specific Alu sequences, which comprise 10 percent of the human genome, thereby underscoring that important ADAR1 functional differences exist between human HSPCs compared with their murine counterparts. However, the limited research effort aimed at deciphering the role of ADAR1-mediated RNA editing in HSPC maintenance has been performed primarily in mouse models rather than highly purified human HSPCs. Because ADAR1 is activated by inflammatory cytokines that accelerate aging and MDS initiation, our main goal is to define the niche- dependent role of RNA editing on human HSPC cell fate and cell cycle regulation during aging and MDS initiation. We will first determine the RNA editing profile by whole transcriptome and single cell RNA-seq, RESSqPCR and lentiviral RNA editing reporters. The functional role of ADAR1 in HSPC aging and MDS initiation will be examined in stromal co-cultures with or without addition of inflammatory cytokines, FUCCI2BL cell cycle reporters, and humanized aged HSC and MDS immunocompromised mouse models. We will also examine the effect of RNA editing on APOBEC3 family of DNA deaminase function during MDS initiation. The proposed study is uniquely responsive to PAS-13-033: Stimulating Hematology Investigation: New Endeavors (SHINE) because it will identify the role of ADAR1-mediated regulatory mRNA and miRNA editing in HSPC myeloid lineage commitment and cell cycle deregulation during age-dependent MDS initiation in the inflammatory bone marrow niche. The ultimate goal of this study is to determine the biological, diagnostic and prognostic significance of ADAR1-mediated RNA editing in HSPC aging compared with MDS initiation.