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

项目摘要/摘要 这项提案的目的是了解Tetraspanin家族成员CD53是如何保护造血干细胞的 炎症应激所致的细胞(HSCs)。虽然对正常免疫系统功能很重要，但炎症性 信号转导可损害HSC功能，促进血液系统恶性肿瘤的发生发展。在预赛中 数据，我们发现CD53是炎症应激背景下HSC功能的关键调节因子。CD53是一种 Tetraspanin跨膜蛋白家族的成员，它组织多个蛋白质网络来调节 各种各样的细胞过程，如增殖、迁移和存活。虽然通常在非常 在低水平的HSC中，CD53在包括炎症在内的多种应激源的反应中显著上调 细胞因子、Toll样受体激动剂和动员剂。使用我们新生成的CD53-/-鼠标，我们发现 CD53的丢失导致HSC再生能力显著降低，并在面临 炎性应激。RNA测序和邻近标记研究表明CD53促进HSC 炎症反应中的静止通过激活“梦”，一种转录抑制复合体，涉及 抑制细胞周期基因表达的Rb样家族成员p107/Rbl1和p130/Rbl2 与p53和p21的激活有关。基于这些数据，我们假设CD53促进了梦的复合体介导 炎症应激反应抑制HSC细胞周期相关基因，从而促进HSC 静息和保护HSC功能。值得注意的是，在缺乏CD53的HSC中，CD53的表达显著增加 TET2或DNMT3A。这些表观基因组调节因子的突变通常与年龄相关的克隆有关 造血(CH)，包括炎症驱动的突变HSCs的扩张和增加的风险 白血病转化。我们预测CD53可能会促进突变型HSCs在CH中的克隆优势。 结合蛋白质组学、转录学和体内HSC功能工具，我们将：1)确定 CD53在促进炎症刺激下的HSC功能中的作用；2)确定CD53如何调节HSC 3)确定CD53升高是否促进了克隆优势 突变的造血干细胞。我们将进行过度表达研究，以阐明持续的CD53表达的影响 在HSC功能上，将使用Dream基因敲除小鼠来确定该复合体在介导 CD53对造血干细胞的影响我们将确定CD53调节HSC循环和梦想的机制 使用邻近连接试验激活以表征CD53相互作用的伙伴。最后，我们将表演 利用CD53和DNMT3A基因敲除小鼠进行功能研究和嵌合建模实验 CD53是否促进突变型HSCs的克隆扩增。总之，我们提议的研究将描述一种 使HSCs抵抗炎性应激的新机制。归根结底，理解这些机制 正常和突变的HSC都能抵抗炎症应激，这对治疗的发展至关重要 促进健康的HSC功能和防止突变的HSC在CHS中扩张的策略。

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.