Role of inflammation in TET2 mediated clonal hematopoiesis in the context of bone marrow niche

克隆性造血（CH）是由于造血干细胞中体细胞突变的积累而发生的， 祖细胞（HSPC）。CH的存在增加了血液系统疾病发生的风险。 恶性肿瘤增加了12倍，心脏病增加了2倍多。TET 2，一个参与胞嘧啶调节的基因 甲基化是CH最常见的突变驱动因素之一。 显示具有Tet 2功能丧失（Tet 2-KO）的HSPC的扩增被炎症强烈促进。 不幸的是，驱动Tet 2-KO HSPC扩增的潜在机制，以及相对表达量， 小生境的贡献，没有定义。我的长期目标是了解哪些目标路径是 通过炎症应激调节，以促进CH。确定的因素可以作为早期干预和疾病 监测战略。我们已经在体内发现白细胞介素1 β（IL 1 β）促进白血病前期细胞增殖， Tet 2-KO HSPC相对于健康细胞的骨髓增殖和扩增。此外，体外评估显示， Tet 2-KO HSPC的自我更新能力在IL 1 β暴露后显著增加。有趣的是，IL 1 β- Tet 2-KO HSPC介导的骨髓扩增也得到非造血骨髓小生境的支持 细胞进一步地，当Tet 2-KO造血细胞被激活时，IL 1 β改变了骨髓基质生态位的组成。 都在场鉴定影响Tet 2-KO自我更新和骨髓扩增的转录变化 HSPC，骨髓来源的Tet 2-KO和健康HSPC的单细胞RNA测序，用或不用 进行IL 1 β检测。这些数据揭示了与自我更新、细胞因子和细胞因子相关的途径和基因的上调。 在IL 1 β处理的Tet 2-KO HSPC中，相对于健康HSPC，IL 1 β处理的Tet 2-KO HSPC中的信号传导和髓样分化。 累积这些数据使我得出了我的总体假设，即IL 1 β驱动Tet 2-KO HSPC的扩增 通过改变转录和表观遗传信号，同时影响与 HSPC。我将通过以下两个目标来阐述我的假设。目标1将确定推动扩张的机制 Tet 2-KO HSPC在IL 1 β诱导的慢性炎症背景下的表达。我会利用遗传学和药理学 抑制方法，以确定影响Tet 2-KO HSPC适应性的候选基因相对于 健康的细胞此外，我将使用快速免疫沉淀质谱内源性蛋白质（RIME） 和染色质免疫沉淀（ChIP-seq），以确定IL 1 β和TET 2如何改变甲基化， Tet 2-KO HSPC的扩增。在目标2中，我将确定IL-1 β是如何重新编程骨髓小生境的， 影响Tet 2-KO HSPC相对于健康细胞的适应性。具体来说，我将使用飞行时间流式细胞术 （CyTOF）和分化测定，以表征IL 1 β如何改变骨髓基质成分 利基最后，我将研究响应IL 1 β的小生境细胞如何影响Tet 2-KO HSPCs的扩增， 体外共培养测定。累积起来，这些研究将确定炎症介导的机制， TET 2 CH，通过降低TET 2 HSPC的适应性为早期干预和检测提供靶标。

Clonal hematopoiesis (CH) occurs due to the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). The presence of CH increases the risk for the development of hematological malignancies by 12-fold and heart disease by more than 2-fold. TET2, a gene involved in regulation of cytosine methylation, is one of the most frequently mutated drivers of CH. Mounting evidence and our preliminary data shows that expansion of HSPCs with Tet2 loss-of-function (Tet2-KO) is strongly promoted by inflammation. Unfortunately, the underlying mechanisms driving expansion of Tet2-KO HSPCs, as well as the relative contribution of the niche, is not defined. My long-term goal is to understand which targetable pathways are regulated by inflammatory stress to promote CH. Identified factors could serve as early intervention and disease monitoring strategies. We have found in vivo that interleukin 1 beta (IL1β) promotes preleukemic myeloproliferation and expansion of Tet2-KO HSPCs over healthy cells. Furthermore, in vitro assessment shows that self-renewal ability of Tet2-KO HSPCs is significantly increased upon IL1β exposure. Interestingly, IL1β- mediated myeloid expansion of Tet2-KO HSPCs is also supported by non-hematopoietic bone marrow niche cells. Further IL1β alters the composition of the bone marrow stromal niche when Tet2-KO hematopoietic cells are present. To identify transcriptional changes impacting self-renewal and myeloid expansion of Tet2-KO HSPCs, single cell RNA sequencing of bone marrow derived Tet2-KO and healthy HSPCs treated with or without IL1β was performed. These data revealed upregulation of pathways and genes related to self-renewal, cytokine signaling, and myeloid differentiation in the IL1β treated Tet2-KO HSPCs relative to healthy HSPCs. Cumulatively these data led me to my overarching hypothesis that IL1β drives expansion of Tet2-KO HSPCs through the alteration of transcriptional and epigenetic signaling while impacting niche interactions with HSPCs. I will address my hypothesis by following two aims. Aim 1 will identify the mechanism driving expansion of Tet2-KO HSPCs in the context of IL1β induced chronic inflammation. I will utilize genetic and pharmacological inhibition approaches to identify the role of candidate genes impacting fitness of Tet2-KO HSPCs relative to healthy cells. Further, I will use rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) and chromatin immunoprecipitation (ChIP-seq) to identify how IL1β and TET2 alter methylation to promote expansion of Tet2-KO HSPCs. In Aim 2, I will determine how reprogramming of the bone marrow niche by IL1β impacts fitness of Tet2-KO HSPCs relative to healthy cells. Specifically, I will use cytometry by time of flight (CyTOF) and differentiation assays to characterize how IL1β alters the stromal component of the bone marrow niche. Finally, I will examine how niche cells in response to IL1β impacts Tet2-KO HSPCs expansion using in vitro coculture assays. Cumulatively, these studies will define the inflammation-mediated mechanisms driving TET2 CH, providing targets for early intervention and detection by reducing the fitness of TET2 HSPCs.