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

克隆造血（CH）是通过获得造血干细胞（hsc）中复发性白血病相关基因的体细胞突变而发生的。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具有适应性和生长优势，这些机制进一步受到内在调节和外在环境因素的协调。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系统恶性肿瘤的风险。因此，了解由最相关的CH驱动基因突变引起的克隆显性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。在HERZBLUT研究单位的密切合作下，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，使用创新的细胞条形码和单细胞测序技术在小鼠和人类ch突变的HSC发育中。我们将研究并比较剪接因子基因SRSF2和SF3B1以及表观遗传修饰基因DNMT3A和TET2两种高度流行的ch突变基因引起的机制。特别是，SRSF2和SF3B1中的CH突变与高干细胞适应度评分和加速CH相关疾病的风险相关。我们的目的是首先研究1)在同基因小鼠移植模型和单细胞分辨率的体外分化实验中，CH突变的造血干细胞的克隆竞争、局部分布和分化行为。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突变克隆的生长和行为。2)其次，我们将通过连接细胞状态和细胞命运来研究ch突变造血干细胞的分子和代谢谱的变化。为此，我们开发了基于条形码的配对子分析，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。我们将研究造血干细胞中CH突变影响下的线粒体电位、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，将我们的发现转化为患者的人类CH。并希望找到CH突变的干细胞/祖细胞的异常表面标记谱，以进行CH的高级检测。该项目将揭示由重要的CH驱动基因引起的克隆优势的分子和功能机制，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影响克隆发育。

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.