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

骨髓生态位背景下炎症在 TET2 介导的克隆造血中的作用

基本信息

批准号：
10802101
负责人：
John H McClatchy
金额：
$ 4.77万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10802101
关键词：
Aging Apoptosis Automobile Driving Biological Assay Bone Marrow Candidate Disease Gene Cardiovascular Diseases Cell Cycle Cells ChIP-seq Chronic Coculture Techniques Cytokine Signaling Cytometry Cytosine DNMT3a Data Development Disease Early Diagnosis Early Intervention Epigenetic Process Exhibits Exposure to Frequencies Gene Expression Regulation Genes Genetic Genetic Transcription Goals Guanine + Cytosine Composition Heart Diseases Hematologic Neoplasms Hematopoiesis Hematopoietic Hematopoietic stem cells Hypermethylation Immunoprecipitation In Vitro Inflammation Inflammatory Interleukin-1 Receptors Interleukin-1 beta Intervention Knock-out Knockout Mice Knowledge Mass Spectrum Analysis Mediating Mesenchymal Stem Cells Methylation Mitogen-Activated Protein Kinases Mitosis Modeling Monitor Mus Mutate Myelogenous Myeloproliferation Myocardial Ischemia NF-kappa B Pathway interactions Patient-Focused Outcomes Phase Transition Population Dynamics Preleukemia Process Production Proliferating Proteins Regulation Risk Risk Factors Role Signal Transduction Somatic Mutation Splenomegaly Stress Testing Time Transcription Alteration Transplantation Up-Regulation bisulfite sequencing bone marrow mesenchymal stem cell chromatin immunoprecipitation cytokine demethylation fitness gene repression hematopoietic differentiation improved in vivo inflammatory milieu leukemia loss of function overexpression pharmacologic programs response self-renewal single-cell RNA sequencing transcription factor transcriptome

项目摘要

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.

克隆性造血（CH）是由于造血干和细胞中体细胞突变的积累而发生的。 CH 的存在会增加发生血液病的风险。恶性肿瘤的发病率增加了 12 倍，心脏病的发病率增加了 2 倍以上。TET2 是一种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。越来越多的证据和我们的初步数据。显示炎症强烈促进 Tet2 功能丧失 (Tet2-KO) 的 HSPC 的扩增。不幸的是，驱动 Tet2-KO HSPC 扩展的潜在机制以及相关的利基市场的贡献尚未定义，我的长期目标是了解哪些是可定位的途径。受炎症应激调节促进 CH 的因素可作为早期干预和疾病治疗。我们在体内发现白细胞介素 1 β (IL1β) 会促进白血病前期。此外，体外评估显示 Tet2-KO HSPC 相对于健康细胞的骨髓增殖和扩增。暴露 IL1β 后，Tet2-KO HSPC 的自我更新能力显着增强。 Tet2-KO HSPC 介导的骨髓扩张也得到非造血骨髓生态位的支持当Tet2-KO造血细胞时，IL1β进一步改变骨髓基质生态位的组成。确定影响 Tet2-KO 自我更新和骨髓扩张的转录变化。 HSPC、骨髓来源的 Tet2-KO 和经过或未经处理的健康 HSPC 的单细胞 RNA 测序这些数据揭示了与自我更新、细胞因子相关的途径和基因的上调。相对于健康 HSPC，IL1β 处理的 Tet2-KO HSPC 中的信号传导和骨髓分化。累积起来，这些数据让我得出了一个总体假设：IL1β 驱动 Tet2-KO HSPC 的扩增通过改变转录和表观遗传信号，同时影响生态位相互作用我将通过以下两个目标来阐述我的假设：确定驱动扩张的机制。在 IL1β 诱导的慢性炎症的背景下，我将利用遗传和药理学研究 Tet2-KO HSPC。抑制方法来确定候选基因影响 Tet2-KO HSPC 相对于此外，我将使用内源性蛋白质的快速免疫沉淀质谱法（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 的扩增在目标 2 中，我将确定 IL1β 如何对骨髓生态位进行重编程。影响 Tet2-KO HSPC 相对于健康细胞的适应性具体来说，我将通过飞行时间使用细胞计数。 (CyTOF) 和分化测定来表征 IL1β 如何改变骨髓基质成分最后，我将研究利基细胞对 IL1β 的反应如何影响 Tet2-KO HSPC 的扩增。总的来说，这些研究将确定炎症介导的驱动机制。 TET2 CH，通过降低 TET2 HSPC 的适应度为早期干预和检测提供目标。