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患有造血疾病，有些进展到 骨髓增生。在衰老期间，会导致克隆主导地位的突变。在移植或基因治疗中 试验，克隆改变使治疗复杂。体细胞基因突变可能会赋予选择性生长 单个造血干细胞的优势，使其最终会超过正常（野生型，WT）细胞 引起克隆失衡。早期检测到这种细胞是热干预以阻止进展的关键 血液疾病。斑马鱼斑马鱼允许颜色条形码和跟踪个体造血 干细胞在体内分化为外周血细胞。最近开发的名为Twistr的技术 允许在斑马遗传背景中基因的镶嵌靶向。无需移植， 可以使用WT和单基因突变体造血干细胞之间的强大竞争分析。 基于颜色的蜂窝条形码互块系统。可以通过流式细胞仪和 与非优势克隆进行下游分析相比。 ASXL1的镶嵌诱变导致克隆 优势，我们发现使用CRISPR或Chemical抑制NR4A1抗炎途径 抑制恢复克隆平衡。使用代谢组学，我们发现胆碱代谢发生了变化 主要的ASXL1突变克隆。在AIM 1中，将进行药物实验以评估NR4A1 促进克隆优势和靶向胆碱代谢基因的遗传实验的途径将 确定代谢靶标抑制克隆优势。我们还发现巨噬细胞在STEM中起作用 细胞克隆性通过与造血干细胞的物理相互作用。巨噬细胞与“吃我”互动 信号，钙网蛋白，在造血干细胞表面确定其命运。在AIM 2中，我们计划 评估钙网蛋白直系同源物的突变体并检查对克隆性的影响。我们计划探究机制 巨噬细胞相互作用后诱导的克隆膨胀。我们也进行了化学通用 屏幕在斑马鱼和人造血茎和祖细胞表面找到钙网蛋白的诱导剂 细胞并将研究可能诱导主要克隆信号的特定化合物。我们的研究将 对克隆平衡的基本理解有影响，并将导致克隆的潜在疗法 主导地位。