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

DNMT3A 突变在克隆异质性和造血进化中的作用

• 批准号: 9977184
• 负责人: Olga A Guryanova
• 金额: $ 34.31万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977184
• 关键词: ATAC-seq; Acute; Address; Age; Age-Years; Aging; Animals; Architecture; Automobile Driving; Biological Assay; Blood; Blood Cells; Bone Marrow; Bone Marrow Cells; Cardiovascular Diseases; Cell Fate Control; Cell Separation; Cell Survival; Cells; Cellular Assay; Chromatin; Chromatin Structure; Clonal Evolution; Clone Cells; Color; Complement; Complex; DNA; DNA Methylation; DNA Modification Methylases; DNA Modification Process; DNA Sequence Alteration; DNA analysis; DNMT3a; Data; Disease; Dysmyelopoietic Syndromes; Epigenetic Process; Event; Evolution; Fluorescence-Activated Cell Sorting; Frequencies; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Fingerprintings; Genetic Transcription; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Heterogeneity; Human; Human Genetics; In Vitro; Individual; Knock-in Mouse; Knowledge; Lead; Leukemic Hematopoietic Stem Cell; Literature; Longevity; Maps; Marrow; Masks; Measures; Methods; Modeling; Molecular; Mutation; Myelogenous; Myeloproliferative disease; Nucleosomes; Pathway interactions; Periodicity; Phenotype; Population; Positioning Attribute; Precancerous Conditions; Process; Relaxation; Reporting; Resolution; Role; Sampling; Signal Pathway; Signal Transduction; Source; Stress; Stretching; System; Techniques; Technology; Time; Tobacco smoke; Work; age related; bisulfite sequencing; cancer therapy; cardiovascular risk factor; chemotherapy; chromatin remodeling; design; epigenome; epigenomics; fitness; genetic profiling; genetic variant; genotoxicity; hematopoietic stem cell fate; hematopoietic stem cell quiescence; in vivo; leukemia; mouse model; mutant; premalignant; pressure; prevent; prospective; self-renewal; single molecule; stem; stem cell fate; stem cells; temporal measurement; therapeutic target; transcription factor; transcriptome sequencing; transcriptomics

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. !

抽象的 克隆造血症，经常在70岁以上的人中发现，发生在一个造血 茎/祖细胞主导骨髓中的所有其他干细胞，并产生所有的大部分 分化的血细胞，并可能加重一致的心血管疾病或髓样的进展 恶性。遗传和表观遗传变异性的积累允许细胞采样一组不同的基因 表达状态允许选择具有最高适应性的造血克隆，尤其是在遗传毒素之后 暴露于烟草烟雾或先前的癌症治疗。表观遗传修饰剂（例如DNMT3A）的突变 是在与年龄有关的克隆造血作用中发生的祖先事件，并在释放前的肿瘤中富集 化学疗法后持续存在的白血病HSC。在DNMT3A突变的敲门小鼠中的初步研究 我们开发了DNMT3A突变的造血克隆的实质性变异性 控件。传统的合奏方法掩盖了由于人口平均而导致的细胞到细胞的异质性，而 单细胞技术不适合前瞻性分离用于功能测定的细胞。我们开发了一个 体内多色克隆跟踪系统，可以轻松地表征克隆成分 骨髓在动物的寿命上，可以在前所未有的临时捕获克隆进化 解决。重要的是，该系统适用于通过 荧光激活的细胞分选，实现功能和–omics分析。在此提案中，我们跟踪克隆 实时，稳态和选择性下的体内占主导地位克隆的结构和出现 压力，并评估特定遗传背景的贡献，例如存在DNMT3A突变 进入此过程。接下来，我们将占主导地位（“获奖者”）和非主导（“失败者”）克隆并进行表演 转录组，表观基因组和染色质分析分析，研究特定途径和表观遗传学 州驾驶克隆主导地位。这将使用FACS分类中的常规合奏方法完成 主要克隆（RNA-Seq，DNA Bisulfite测序，ATAC-SEQ），并通过Mapit Technology完成 允许简单地分析单个单个单一的DNA甲基化和核小体定位 DNA的分子，为表观基因组异质性研究增加粒度。这些结果是交叉验证的 通过功能性的长期自我更新测定在孤立的显性和非主要骨髓细胞中的体内， 并通过单细胞转录组分析。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，我们的研究结果将产生丰富的潜力来源 治疗靶标，以阻止或防止前进状态和弗兰克白血病的进展。 呢