Protection of stressed hematopoietic stem cells by the tetraspanin family member CD53

项目总结/摘要 本提案的目的是了解四跨膜蛋白家族成员CD 53如何保护造血干细胞 细胞（HSC）的炎症应激。虽然对正常免疫系统功能很重要，但炎症 信号传导可损害HSC功能并促进造血系统恶性肿瘤的发展。初步 数据，我们确定了CD 53作为炎症应激背景下HSC功能的关键调节因子。CD 53是一种 跨膜蛋白四跨膜蛋白家族的成员，组织多蛋白网络以调节 多种细胞过程，例如增殖、迁移和生存。虽然通常表达在非常 在HSC中低水平，CD 53响应于多种应激源（包括炎性细胞因子）而显著上调。 细胞因子、Toll样受体激动剂和动员剂。使用我们新生成的Cd 53-/-小鼠，我们发现 CD 53的缺失导致HSC再生能力的显著降低和在面对疾病时循环的增加。 炎症应激RNA测序和邻近标记研究表明，CD 53促进HSC 通过激活“DREAM”（一种转录抑制因子复合物， Rb样家族成员p107/Rb 11和p130/Rb 12抑制细胞周期基因的表达， p53和p21的激活。基于这些数据，我们假设CD 53促进DREAM复合物介导的 抑制HSC中响应炎症应激的细胞周期相关基因，从而促进HSC 静止和保护HSC功能。值得注意的是，CD 53表达在缺乏造血干细胞中显著增加。 Tet 2或Dnmt 3a。这些表观基因组调控因子的突变通常与年龄相关的克隆性 造血（CH），其涉及突变HSC的炎症驱动的扩增和造血干细胞增殖的风险增加。 白血病转化我们预测，CD 53可能因此促进突变型HSC在CH中的克隆优势。 使用蛋白质组学、转录组学和体内HSC功能工具的组合，我们将：1）确定HSC的作用。 CD 53在促进HSC对炎症刺激的反应中的作用; 2）确定CD 53如何调节HSC 循环和DREAM复合物活性;和3）确定升高的CD 53是否促进克隆优势 突变的HSC。我们将进行过表达研究，以阐明持续的CD 53表达的影响， DREAM基因敲除小鼠将用于确定这种复合物在介导HSC功能中的作用， CD 53对HSC的影响我们将确定CD 53调节HSC循环和DREAM的机制。 使用邻近连接测定来表征CD 53相互作用配偶体。最后，我们将表演 使用Cd 53和Dnmt 3a敲除小鼠的功能研究和嵌合建模实验来确定 CD 53是否促进突变型HSC的克隆扩增。总之，我们提出的研究将描述一个 新的机制，使HSC抵抗炎症应激。最终，了解这些机制 正常和突变型HSC抵抗炎症应激的机制对于开发治疗性HSC至关重要。 促进健康HSC功能和防止CH中突变HSC扩增的策略。

Project Summary/Abstract The goal of this proposal is to understand how the tetraspanin family member CD53 protects hematopoietic stem cells (HSCs) from inflammatory stress. While important for normal immune system function, inflammatory signaling can impair HSC function and promote the development of hematopoietic malignancies. In preliminary data, we identified CD53 as a critical regulator of HSC function in the context of inflammatory stress. CD53 is a member of the tetraspanin family of transmembrane proteins that organize multi-protein networks to regulate a wide variety of cellular processes such as proliferation, migration, and survival. While normally expressed at very low levels in HSCs, CD53 is markedly upregulated in response to multiple stressors including inflammatory cytokines, toll like receptor agonists and mobilizing agents. Using our newly-generated Cd53-/- mouse, we found that loss of CD53 causes a significant reduction in HSC repopulating ability and increased cycling in the face of inflammatory stress. RNA sequencing and proximity labeling studies suggest that CD53 promotes HSC quiescence in response to inflammation via activation of “DREAM,” a transcriptional repressor complex involving the Rb-like family members p107/Rbl1 and p130/Rbl2 that inhibits the expression of cell cycle genes in response to p53 and p21 activation. Based on this data, we hypothesize that CD53 promotes DREAM complex-mediated repression of cell cycle-related genes in HSCs in response to inflammatory stress, thereby promoting HSC quiescence and protecting HSC function. Notably, CD53 expression is markedly increased in HSCs deficient for Tet2 or Dnmt3a. Mutations in these epigenome regulators are commonly associated with age-related clonal hematopoiesis (CH), which involves inflammation-driven expansion of mutant HSCs and increased risk of leukemic transformation. We predict that CD53 may thus promote the clonal advantage of mutant HSCs in CH. Using a combination of proteomic, transcriptomic and in vivo HSC functional tools, we will: 1) Determine the role of CD53 in promoting HSC function in response to inflammatory stimuli; 2) Determine how CD53 regulates HSC cycling and DREAM complex activity; and 3) Determine whether elevated CD53 promotes the clonal advantage of mutant HSCs. We will perform overexpression studies to elucidate the effects of sustained CD53 expression on HSC function, and DREAM knockout mice will be used to determine the role of this complex in mediating the effects of CD53 on HSCs. We will determine the mechanisms by which CD53 regulates HSC cycling and DREAM activation using proximity ligation assays to characterize CD53- interacting partners. Finally, we will perform functional studies and chimeric modeling experiments using Cd53 and Dnmt3a knockout mice to determine whether CD53 promotes the clonal expansion of mutant HSCs. Together, our proposed studies will describe a novel mechanism that enables HSCs to resist inflammatory stress. Ultimately, understanding the mechanisms by which both normal and mutant HSCs resist inflammatory stress is essential to the development of therapeutic strategies to promote healthy HSC function and to prevent the expansion of mutant HSCs in CH.