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

克隆性造血(CH)是由于体细胞突变在造血干细胞中的积累而发生的 祖细胞(HSPC)。CH的存在增加了发生血液病的风险 恶性肿瘤增加12倍，心脏病增加2倍以上。参与胞嘧啶调控的基因TET2 甲基化是CH最常见的突变驱动因素之一。越来越多的证据和我们的初步数据 显示TET2功能丧失的HSPC(TET2-KO)的扩张受到炎症的强烈促进。 不幸的是，驱动TET2-KO HSPC扩张的潜在机制以及相对的 利基的贡献，没有被定义。我的长期目标是了解哪些是有针对性的途径 受炎症应激调节，促进CH。确定的因素可以作为早期干预和疾病 监控策略。我们在体内发现白介素1β(IL1β)促进白血病前期 TET2-KO HSPC在健康细胞上的骨髓增殖和扩增。此外，体外评估显示 IL-1β暴露后，TET2-KO HSPC的自我更新能力显著增强。有趣的是，IL1β- TET2-KO-HSPC介导的髓系扩增也受到非造血系骨髓龛的支持 细胞。IL-1β进一步改变TET2-KO造血细胞骨髓基质龛的组成 都在现场。鉴定影响TET2-KO自我更新和髓系扩增的转录变化 骨髓来源TET2-KO和健康HSPC处理前后HSPC的单细胞RNA测序 行IL-1β。这些数据揭示了与自我更新相关的途径和基因的上调，细胞因子 与健康的HSPC相比，IL1TET2-KOβ处理的HSPC中的信号转导和髓系分化。 累积起来，这些数据让我得出了我的总体假设，即IL1β推动了TET2-KO HSPC的扩张 通过改变转录和表观遗传信号，同时影响生态位相互作用 HSPC。我将通过以下两个目标来解释我的假设。目标1将确定推动扩张的机制 在IL-1β诱导慢性炎症的背景下，TET2-KO HSPC的表达。我将利用遗传学和药理学 识别影响TET2-KO HSPC相对适合度的候选基因作用的抑制方法 健康的细胞。此外，我将使用内源蛋白的快速免疫沉淀质谱仪(RIME) 和染色质免疫沉淀(ChIP-SEQ)，以确定IL1β和TET2如何改变甲基化以促进 TET2-KO HSPC的扩展。在目标2中，我将确定IL1β如何对骨髓利基重新编程 影响TET2-KO HSPC相对于健康细胞的适合性。具体地说，我将使用飞行时间的细胞术 (细胞周期分析)和分化试验研究白细胞介素1β如何改变骨髓基质成分 利基市场。最后，我将使用in检查针对IL1β的利基细胞如何影响TET2-KO HSPC的扩展 体外共培养实验。总而言之，这些研究将确定炎症介导的驱动机制 TET2-CH，通过降低TET2-HSPC的适合度为早期干预和检测提供靶点。

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.