The role of DNMT3A mutations in clonal heterogeneity and evolution of hematopoiesis

摘要 克隆性造血，常见于70岁以上的人，当一种单一的造血细胞 干细胞/祖细胞支配着骨髓中的所有其他干细胞，并产生ALL的很大一部分 分化的血细胞，并可能加重同时发生的心血管疾病或进展为髓系 恶毒。遗传和表观遗传变异性的积累允许细胞对一组不同的基因进行采样 表达状态允许选择具有最高适合度的造血细胞克隆，特别是在基因毒性之后 暴露于烟草烟雾或以前的癌症治疗。DNMT3A等表观遗传修饰因子的突变 是发生在与年龄相关的克隆性造血中的祖先事件，并在白血病前期和 化疗后持续存在的白血病HSCs。DNMT3A基因敲除突变小鼠的初步研究 我们发现在DNMT3A突变的造血克隆中，与 控制。传统的集成方法由于总体平均而掩盖了细胞到细胞的异质性，而 单细胞技术不适合用于功能分析的预期细胞分离。我们开发了一种 体内多色克隆跟踪系统，允许轻松表征克隆成分 在动物的整个生命周期中的骨髓，使得能够在前所未有的时间捕捉克隆进化 决议。重要的是，该系统适用于通过以下方式分离单个造血克隆 荧光激活的细胞分类，实现功能和组学分析。在这个提案中，我们跟踪克隆 在活体中实时、稳定和选择性地构建和出现优势克隆 压力，并评估特定遗传背景的贡献，如DNMT3A突变的存在 进入这一过程。接下来，我们分离优势克隆(“赢家”)和非优势克隆(“输家”)并执行 转录、表观基因组和染色质图谱分析，以研究特定的途径和表观遗传学 状态(S)驱动克隆显性。这将在FACS中使用传统的集成方法来完成-分类 优势克隆(RNA-SEQ、DNA亚硫酸氢盐测序、ATAC-SEQ)，并由MapIt技术补充 允许同时分析DNA甲基化和核小体定位 DNA分子，以增加表观基因组异质性研究的粒度。这些结果是交叉验证的 通过在分离的优势和非优势骨髓细胞中进行体外功能长期自我更新试验， 并进行单细胞转录分析。这些发现将加深我们对克隆进化的理解 造血系统。更重要的是，我们的研究成果将产生丰富的潜力来源 治疗靶点，以阻止或防止进展到癌前状态和坦率的白血病。 好了！

ABSTRACT Clonal hematopoiesis, frequently found in people over 70 years of age, occurs when a single hematopoietic stem/progenitor cell dominates all other stem cells in the bone marrow and yields a large fraction of all differentiated blood cells, and may aggravate coincident cardiovascular disease or progress to myeloid malignancy. Accumulation of genetic and epigenetic variability permits the cells to sample a diverse set of gene expression states allowing selection of hematopoietic clones with the highest fitness, especially after genotoxic exposures such as tobacco smoke or prior cancer therapy. Mutations in epigenetic modifiers such as DNMT3A are ancestral events occurring in age-related clonal hematopoiesis, and are enriched in pre-leukemic and leukemic HSCs that persist following chemotherapy. Preliminary studies in Dnmt3a-mutant knock-in mice that we developed show substantial functional variability among Dnmt3a-mutant hematopoietic clones compared to controls. Conventional ensemble methods mask cell-to-cell heterogeneity due to population averaging, while single-cell techniques are not amenable to prospective isolation of cells for functional assays. We developed an in vivo multicolor clonal tracking system, allowing easy characterization of clonal composition in the bone marrow over the lifespan of an animal, enabling capture of clonal evolution at unprecedented temporal resolution. Importantly, this system is suitable for isolation of individual hematopoietic clones by fluorescence-activated cell sorting, enabling functional and –omics analyses. In this proposal, we track clonal architecture and emergence of dominant clones in vivo in real time, in steady-state and under selective pressures, and evaluate the contribution of specific genetic backgrounds such as presence of Dnmt3a mutations into this process. Next, we isolate dominant (“winners”) and non-dominant (“losers”) clones and perform transcriptomic, epigenomic, and chromatin profiling analyses, to investigate specific pathways and epigenetic state(s) driving clonal dominance. This will be done using conventional ensemble methods in FACS-sorted dominant clones (RNA-seq, DNA bisulfite sequencing, ATAC-seq), and complemented by MAPit technology allowing simultaneous analysis of DNA methylation and nucleosome positioning over long stretches of single molecules of DNA, to add granularity to the epigenome heterogeneity studies. These results are cross-validated by functional long-term self-renewal assays ex vivo in isolated dominant and non-dominant bone marrow cells, and by single-cell transcriptomic analysis. These findings will deepen our understanding of clonal evolution in the hematopoietic system. More importantly, the results of our studies will generate a rich source of potential therapeutic targets to hinder or prevent the progression to pre-malignant states and to frank leukemia. !