MOLECULAR MECHANISMS UNDERLYING CLONAL EXPANSION OF HEMATOPOIETIC STEM CELLS

项目总结 克隆性造血的不确定潜能(CHIP)与从头开始和 与治疗相关的血液肿瘤，表明芯片中发现的突变可能导致疾病 发展。肿瘤抑制基因TP53的获得性体细胞突变在 在芯片中发现了突变。临床研究表明，在造血干细胞(HSCs) TP53突变使老年人易患血液肿瘤和其他与衰老相关的疾病。 然而，关于TP53突变促进的机制仍有很大的认识空白。 HSC扩容。我们最近发现，在CHIP中发现的TP53突变促进了小鼠HSC的自我更新 在活体内。我们发现，在CHIP中发现的几种突变型P53蛋白，而不是野生型P53蛋白，与 表观遗传调控因子EZH2，增强其活性。此外，我们还发现突变型p53增加了 染色质对NLRP1炎症体基因的可及性以及NLRP1的表达上调 突变的HSC，导致促炎细胞因子IL-1β的分泌增加。我们假设那个变种人 P53通过两种不同的机制促进突变的HSC的扩增。首先，突变型p53与AND相互作用 增强表观遗传调节因子EZH2的活性，促进突变的HSC自我更新。第二，突变型p53 通过NLRP1炎症小体介导的促炎途径抑制野生型HSC的功能。在这 建议的研究，我们将利用生化、遗传学、分子、免疫学和药理学 研究细胞自主和非自主的方法和脊椎动物模型 突变型p53促进造血干细胞克隆性扩增的机制。检测突变型p53 HSC是否扩增 是通过增强EZH2活性和增加H3K27me3介导的，我们将确定 EZH2对小鼠突变型P53-HSC自我更新的遗传和药理抑制作用。到时候我们会的 利用逆转录病毒将芯片中发现的热点TP53突变导入人原代HSPC 检测突变型P53是否增加H3K27me3水平并促进HSPC的扩张。为了阐明细胞的非 突变型p53介导的NLRP1炎性小体激活对HSC扩增的自主机制 将确定突变型p53造血干细胞是否比野生型造血干细胞对IL-1β暴露更具抵抗力。我们还将 使用细胞因子阵列询问小鼠和人类造血细胞中细胞因子的分泌。最后，我们 将进行竞争性骨髓移植测试，以确定基因和 NLRP1炎性小体对小鼠和人克隆扩张的药理抑制作用 具有TP53突变的造血干细胞。我们的结果将为开发新的潜在目标提供新的见解 预防和治疗芯片和其他与衰老相关的疾病的治疗。

PROJECT SUMMARY Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risks of de novo and therapy-related hematological neoplasms, suggesting that mutations identified in CHIP likely drive disease development. Acquired somatic mutations in tumor suppressor gene TP53 rank in the top five among mutations identified in CHIP. Clinical studies suggest that expansion of hematopoietic stem cells (HSCs) with TP53 mutations predisposes the elderly to hematological neoplasms and other aging-related diseases. However, there is a significant gap in knowledge regarding the mechanisms by which TP53 mutations promote HSC expansion. We recently found that TP53 mutations identified in CHIP enhance HSC self-renewal in mice in vivo. We showed that several mutant p53 proteins found in CHIP, but not wild–type p53, interact with epigenetic regulator EZH2 and enhance its activity. Moreover, we discovered that mutant p53 increases the chromatin accessibility to the NLRP1 inflammasome gene and that NLRP1 expression was upregulated in mutant HSCs, leading to increased secretion of pro-inflammatory cytokine IL-1β. We hypothesize that mutant p53 promotes mutant HSC expansion through two distinct mechanisms. First, mutant p53 interacts with and enhances the activity of epigenetic regulator EZH2 to promote mutant HSC self-renewal. Second, mutant p53 suppresses wild type HSC function through NLRP1 inflammasome-mediated pro-inflammatory pathway. In this proposed research, we will utilize biochemical, genetic, molecular, immunologic, and pharmacological approaches as well as vertebrate models to investigate the cell autonomous and non-autonomous mechanisms by which mutant p53 promotes clonal expansion of HSCs. To test if mutant p53 HSC expansion is mediated through enhanced EZH2 activity and increased H3K27me3, we will determine the impact of genetic and pharmacological inhibition of EZH2 activity on mutant p53 HSC self-renewal in mice. We will then introduce hot-spot TP53 mutations identified in CHIP into human primary HSPCs using retroviruses and examine if mutant p53 increases H3K27me3 levels and promotes HSPC expansion. To elucidate the cell non- autonomous mechanisms of mutant p53-mediated NLRP1 inflammasome activation on HSC expansion, we will determine if mutant p53 HSCs are more resistant to IL-1β exposure than wild-type HSCs. We will also interrogate cytokine secretion in both mouse and human hematopoietic cells using cytokine arrays. Finally, we will perform competitive bone marrow transplantation assays to determine the impact of genetic and pharmacological inhibition of the NLRP1 inflammasome on the clonal expansion of both mouse and human HSCs with TP53 mutations. Our results will provide novel insights into developing new potential target-based therapeutics for prevention and treatment of CHIP and other aging-associated diseases.