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.

摘要