Project 4 - Mechanisms of establishing clonal dominance

摘要 造血克隆失衡使个体易于发生造血功能障碍，有些进展为 脊髓发育不良在衰老过程中，突变出现，导致克隆优势。在移植或基因治疗中 克隆变异使治疗复杂化。体细胞基因突变可能赋予选择性生长 这是单个造血干细胞的优势，使得它最终将超过正常（野生型，WT）细胞 导致克隆失衡。早期检测这些细胞是治疗干预以阻止进展的关键 血液紊乱Zebrabow斑马鱼允许彩色条形码和跟踪个体造血 干细胞在体内分化成外周血细胞的过程中。最近开发的一项技术， 允许在斑马弓遗传背景中嵌合靶向基因。不需要移植， WT和单基因突变造血干细胞之间的稳健竞争测定可以使用 基于颜色的细胞条形码TWISTR系统。显性克隆可以通过流式细胞术分选， 与非显性克隆相比用于下游分析。ASXL 1的嵌合突变导致克隆 我们发现，使用CRISPR或化学物质抑制NR 4A 1抗炎通路， 抑制恢复克隆平衡。使用代谢组学，我们发现胆碱代谢在 显性asxl 1突变克隆。在目标1中，将进行药理学实验以评价NR 4A 1 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靶点。我们还发现巨噬细胞在干细胞中起作用， 通过与造血干细胞的物理相互作用实现细胞克隆性。巨噬细胞与“吃我”相互作用 在造血干细胞表面的钙网蛋白信号，以决定他们的命运。在目标2中，我们计划 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 在巨噬细胞相互作用后诱导的克隆扩增。我们还进行了化学遗传学研究 斑马鱼和人造血干/祖细胞表面钙网蛋白诱导剂的筛选 细胞，并将研究特定的化合物，可以诱导一个“吃我”的信号占主导地位的克隆。我们的研究将 对克隆平衡的基本理解产生影响，并将导致克隆平衡的潜在疗法。 支配地位

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.