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，发生在单一造血干细胞缺乏时。 干/祖细胞在骨髓中占所有其他干细胞的主导地位，并产生所有干细胞中的大部分。 分化的血细胞，并可能加重并发的心血管疾病或进展到骨髓 恶性肿瘤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应性的造血克隆，特别是在遗传毒性后， 暴露于烟草烟雾或既往癌症治疗。表观遗传修饰因子（如DNMT 3A）的突变 是发生在年龄相关的克隆造血中的祖先事件，并且在白血病前和白血病后的造血中富集。 化疗后持续存在的白血病HSC。在Dnmt 3a突变基因敲入小鼠中的初步研究， 我们开发的Dnmt 3a突变造血克隆与 对照传统的集成方法掩盖了由于群体平均而导致的细胞间异质性，而 单细胞技术不适合用于功能测定的细胞的预期分离。我们开发了一个 在体内克隆跟踪系统，允许容易表征克隆组成中， 骨髓在动物的一生中，使捕捉克隆进化在前所未有的时间 分辨率重要的是，该系统适用于通过以下方法分离单个造血克隆： 荧光激活细胞分选，使功能和组学分析。在这个建议中，我们跟踪克隆 在真实的时间、稳态和选择性条件下， 压力，并评估特定遗传背景的贡献，如Dnmt 3a突变的存在 进入这个过程。接下来，我们分离出显性（“赢家”）和非显性（“输家”）克隆， 转录组学、表观基因组学和染色质谱分析，以研究特定途径和表观遗传学 国家（S）驱动克隆优势。这将在FACS分类中使用传统的集合方法来完成。 优势克隆（RNA-seq、DNA亚硫酸氢盐测序、ATAC-seq），并通过MAPit技术进行补充 允许同时分析DNA甲基化和核小体定位在长时间的单一的 DNA分子，以增加表观基因组异质性研究的粒度。这些结果经过交叉验证 通过在分离的显性和非显性骨髓细胞中的离体功能性长期自我更新测定， 和单细胞转录组学分析。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。 !

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. !