Quantitative assessment of clonal architecture, stem cell competition and lineage fate in CH (Project B2)

CH 克隆结构、干细胞竞争和谱系命运的定量评估（项目 B2）

• 批准号: 533779356
• 负责人: Professor Dr. Michael Rieger
• 金额: --
• 依托单位: Klinik 2 - Hämatologie/Onkologie, Rheumatologie und Infektiologie/HIV
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/533779356?language=en
• 关键词: Quantitative; assessment; clonal; architecture; stem

Clonal hematopoiesis (CH) occurs by the acquisition of somatic mutations in recurrent leukemia-associated genes in hematopoietic stem cells (HSCs). These CH driver mutations cause a fitness and growth advantage of CH-mutated over non-mutated HSCs by various mechanisms, which are further orchestrated by intrinsic regulation and extrinsic environmental factors. Many studies revealed a clinical association of the presence of CH mutations and the affected mutant clone size in blood cells, and their risk to aggravate cardiovascular diseases and to progress into hematologic malignancies. Therefore, understanding the pathomechanisms of clonal dominance and growth caused by mutations in the most relevant CH driver genes is of utmost importance for early prevention and clinical management strategies. In close collaboration within the Research Unit HERZBLUT we want to study the clonal architecture, dominance and interference at single clone and cell level, using innovative cellular barcoding and single cell sequencing technologies in mouse and human CH-mutated HSC development. We will investigate and compare the mechanisms that are caused by two highly prevalent classes of CH-mutated genes, in the splicing factor genes SRSF2 and SF3B1 and in the epigenetic modifier genes DNMT3A and TET2. In particular, CH mutations in SRSF2 and SF3B1 are associated with a high stem cell fitness score and accelerated risk to aggravate CH-related diseases. We aim to study first 1) the clonal competition, local distribution, and differentiation behavior of CH-mutated HSCs in syngeneic CH mouse transplantation models and in in vitro differentiation assays at single cell resolution. Therefore, we will apply lentiviral barcoding, single cell sequencing and time-lapse microscopy-based single cell tracking to quantitatively assess CH-mutated clonal outgrowth and behavior. 2) Second, we will investigate changes in the molecular and metabolic profile of CH-mutated HSCs by linking cell state with cell fate. To this end, we have developed barcode-based paired daughter assays to understand molecular correlations between the cell of clonal origin (state) and its future differentiation behavior (fate). We will investigate the link between mitochondrial potential, energy metabolism and dormancy under the influence of CH mutations in HSCs 3) Last, we aim to translate our findings to human CH in patients, by studying differentiation trajectories in patients carrying CH mutations in splicing factor and epigenetic regulator genes, and want to find aberrant surface marker profiles of CH-mutated stem/progenitor cells for advanced detection of CH. This project will enlighten the molecular and functional mechanisms of clonal dominance caused by important CH driver genes and how the interplay between intrinsic and extrinsic factors shapes clonal development over time.

克隆性造血（CH）是通过造血干细胞（HSC）中复发性白血病相关基因的体细胞突变发生的。这些CH驱动突变通过各种机制导致CH突变的HSC相对于非突变的HSC的适应性和生长优势，这些机制进一步由内在调节和外在环境因素协调。许多研究揭示了CH突变的存在与血细胞中受影响的突变克隆大小的临床关联，以及它们加重心血管疾病和进展为血液恶性肿瘤的风险。因此，了解最相关的CH驱动基因突变引起的克隆优势和生长的病理机制对于早期预防和临床管理策略至关重要。通过与HERZBLUT研究单位的密切合作，我们希望在单克隆和细胞水平上研究克隆结构，优势和干扰，在小鼠和人类CH突变HSC的发展中使用创新的细胞条形码和单细胞测序技术。我们将调查和比较的机制是由两个高度流行的CH突变基因，在剪接因子基因SRSF 2和SF 3B 1和表观遗传修饰基因DNMT 3A和TET 2。特别是，SRSF 2和SF 3B 1中的CH突变与高干细胞适应性评分和加速CH相关疾病加重的风险相关。我们的目的是首先研究1）在同基因CH小鼠移植模型中CH突变的HSC的克隆竞争、局部分布和分化行为，以及在单细胞分辨率下的体外分化测定。因此，我们将应用慢病毒条形码、单细胞测序和基于延时显微镜的单细胞跟踪来定量评估CH突变的克隆生长和行为。2)其次，我们将通过将细胞状态与细胞命运联系起来来研究CH突变的HSC的分子和代谢特征的变化。为此，我们开发了基于条形码的配对子代测定，以了解克隆起源细胞（状态）与其未来分化行为（命运）之间的分子相关性。我们将研究HSC中CH突变影响下线粒体电位、能量代谢和休眠之间的联系3）最后，我们的目标是将我们的发现转化为患者中的人类CH，通过研究剪接因子和表观遗传调节基因中携带CH突变的患者的分化轨迹，并希望找到CH突变的干细胞/本项目的研究将有助于阐明慢性乙型肝炎的重要病原体引起克隆优势的分子和功能机制驱动基因以及内在和外在因素之间的相互作用如何随着时间的推移塑造克隆发展。