Project 4 - Mechanisms of establishing clonal dominance

项目 4 - 建立克隆优势的机制

• 批准号: 10641543
• 负责人: LEONARD Ira ZON
• 金额: $ 51.79万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-07 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10641543
• 关键词: Adult; Aftercare; Aging; Anti-Inflammatory Agents; Automobile Driving; Bar Codes; Biochemistry; Biological Assay; Blood Cells; Cell Communication; Cells; Chemicals; Choline; Clonal Hematopoietic Stem Cell; Clonality; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Coupled; DNA Sequence Alteration; DNMT3a; DNMT3a mutation; Dependence; Development; Disease; Dysmyelopoietic Syndromes; Early Diagnosis; Eating; Embryo; Epigenetic Process; Equilibrium; Experimental Genetics; Fishes; Flow Cytometry; Gene therapy trial; Genes; Genetic; Genetic Screening; Goals; Growth; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneity; Human; Individual; Lead; Macrophage; Metabolic; Metabolic Pathway; Metabolism; Methods; Modeling; Monitor; Mutagenesis; Mutate; Mutation; NR4A1 gene; Orthologous Gene; Outcome; Pathway interactions; Persons; Role; Signal Transduction; Sorting; Surface; Syndrome; System; Techniques; Technology; Testing; Therapeutic; Transplantation; Work; Zebrafish; adult stem cell; aged; antagonist; calreticulin; cell type; chemical genetics; experimental study; genetic variant; in vivo; inhibitor; mature animal; metabolomics; mosaic; mutant; pharmacologic; small molecule; stem; stem cell niche; stem cells; targeted treatment; transplantation therapy

ABSTRACT Hematopoietic clonal imbalance predisposes individuals to hematopoietic disorders, some progressing to myelodysplasia. During aging, mutations arise that lead to clonal dominance. In transplantation or gene therapy trials, clonal alterations complicate therapy. Somatic genetic mutation(s) may bestow a selective growth advantage to a single hematopoietic stem cell so that it will eventually outgrow normal (wild type, WT) cells causing clonal imbalance. Early detection of such cells is key to therapeutically intervene to block progression to a blood disorder. The Zebrabow zebrafish allows color barcoding and tracking of individual hematopoietic stem cells during differentiation into peripheral blood cells in vivo. A recently developed technique called TWISTR allows mosaic targeting of genes in the Zebrabow genetic background. Without the need for transplantation, a robust competition assay between WT and single gene mutant hematopoietic stem cells can be monitored using the color-based cellular barcoding TWISTR system. The dominant clone can be sorted via flow cytometry and compared to non-dominant clones for downstream analysis. Mosaic mutagenesis of ASXL1 leads to clonal dominance, and we found that inhibition of the NR4A1 anti-inflammatory pathway using CRISPR or chemical inhibition restores clonal balance. Using metabolomics, we have found that choline metabolism is altered in dominant asxl1 mutant clones. In Aim 1, pharmacologic experiments will be performed to evaluate the NR4A1 pathway in facilitating clonal dominance and genetic experiments targeting choline-related metabolic genes will identify metabolic targets for suppressing clonal dominance. We also found that macrophages play a role in stem cell clonality through physical interactions with hematopoietic stem cells. Macrophages interact with the “eat me” signal, Calreticulin, on the surface of hematopoietic stem cells to determine their fate. In Aim 2, we plan to evaluate mutants of Calreticulin orthologs and examine the effects on clonality. We plan to probe the mechanism of the induced clone expansion after macrophage interactions. We also have undertaken a chemical genetic screen to find inducers of Calreticulin on the surface of zebrafish and human hematopoietic stem and progenitor cells and will study specific compounds that can induce an “eat-me” signal for dominant clones. Our studies will have an impact on the basic understanding of clonal balance and will lead to potential therapies for clonal dominance.

抽象的 造血克隆失衡使个体容易出现造血障碍，有些会发展为 骨髓增生异常在衰老过程中会出现突变，导致移植或基因治疗中的克隆优势。 试验表明，克隆改变复合疗法可能会带来选择性生长。 单个造血干细胞的优势，使其最终会生长超过正常（野生型，WT）细胞 早期检测此类细胞是治疗干预以阻止进展的关键。 Zebrabow 斑马鱼可以对个体造血系统进行彩色条形码和跟踪。 干细胞在体内分化为外周血细胞的过程中，最近开发了一种称为 TWISTR 的技术。 允许斑马弓遗传背景中的基因嵌合靶向，无需移植。 WT 和单基因突变造血干细胞之间的强大竞争测定可以使用以下方法进行监测 基于颜色的细胞条形码 TWISTR 系统可以通过流式细胞术进行分选和 与下游分析的非显性克隆相比，ASXL1 的镶嵌突变导致克隆。 优势，我们发现使用 CRISPR 或化学物质抑制 NR4A1 抗炎途径 使用代谢组学，我们发现胆碱代谢处于正常状态。 在目标 1 中，将进行药理学实验来评估 NR4A1。 促进克隆优势的途径和针对胆碱相关代谢基因的遗传实验将 确定抑制克隆优势的代谢目标我们还发现巨噬细胞在干细胞中发挥作用。 细胞克隆通过与造血干细胞的物理相互作用与“吃我”相互作用。 在目标 2 中，我们计划通过造血干细胞表面的钙网蛋白信号来决定它们的命运。 评估钙网蛋白直向同源物的突变体并检查对克隆性的影响我们计划探索其机制。 我们还进行了化学遗传学研究。 筛选斑马鱼和人类造血干细胞和祖细胞表面的钙网蛋白诱导物 细胞，并将研究可以诱导优势克隆“吃我”信号的特定化合物。 对克隆平衡的基本理解产生影响，并将带来克隆平衡的潜在治疗方法 统治地位。