MOLECULAR MECHANISMS UNDERLYING CLONAL EXPANSION OF HEMATOPOIETIC STEM CELLS

项目摘要 不确定潜能的克隆性造血（CHIP）与新生儿和 治疗相关的血液肿瘤，表明CHIP中发现的突变可能导致疾病 发展肿瘤抑制基因TP 53获得性体细胞突变排在前五位 在CHIP中识别的突变。临床研究表明，扩增造血干细胞（HSC）， TP 53突变使老年人易患血液肿瘤和其他衰老相关疾病。 然而，关于TP 53突变促进细胞凋亡的机制， HSC扩展。我们最近发现，在CHIP中鉴定的TP 53突变增强了小鼠HSC的自我更新， in vivo.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， 表观遗传调节因子EZH 2并增强其活性。此外，我们发现突变型p53增加了 染色质对NLRP 1炎性体基因的可及性，并且NLRP 1表达在 突变HSC，导致促炎细胞因子IL-1β分泌增加。我们假设变异人 p53通过两种不同的机制促进突变型HSC扩增。首先，突变型p53与 增强表观遗传调节因子EZH 2的活性以促进突变型HSC自我更新。第二，突变型p53 通过NLRP 1炎性体介导的促炎途径抑制野生型HSC功能。在这 建议的研究，我们将利用生物化学，遗传学，分子，免疫学和药理学 方法以及脊椎动物模型来研究细胞自主和非自主 突变型p53促进HSC克隆扩增的机制。为了测试突变型p53 HSC扩增是否 是通过增强EZH 2活性和增加H3 K27 me 3介导的，我们将确定 EZH 2活性对小鼠中突变型p53 HSC自我更新的遗传和药理学抑制。然后我们将 使用逆转录病毒将CHIP中鉴定的热点TP 53突变引入人原代HSPC中， 检查突变型p53是否增加H3 K27 me 3水平并促进HSPC扩增。为了阐明细胞非- 突变型p53介导的NLRP 1炎性小体激活对HSC扩增的自主机制，我们 将确定突变型p53 HSC是否比野生型HSC对IL-1β暴露更具抗性。我们还将 使用细胞因子阵列研究小鼠和人造血细胞中细胞因子分泌。最后我们 将进行竞争性骨髓移植试验，以确定遗传和 NLRP 1炎性小体对小鼠和人的克隆扩增的药理学抑制 具有TP 53突变的HSC。我们的研究结果将为开发新的潜在靶向药物提供新的见解。 用于预防和治疗CHIP和其他衰老相关疾病的治疗剂。

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.