Project 4 - Mechanisms of establishing clonal dominance

摘要 造血克隆失衡使个人容易患上造血疾病，一些人进展到 骨髓发育不良。在衰老过程中，会出现导致克隆优势的突变。在移植或基因治疗中 试验，克隆改变使治疗复杂化。体细胞基因突变(S)可能会带来选择性生长 单个造血干细胞的优势，使其最终将超过正常(野生型)细胞 导致克隆不平衡。早期发现这类细胞是进行治疗干预以阻止进展的关键 一种血液病。斑马鱼允许对单个造血细胞进行彩色条形码和跟踪 干细胞在体内分化为外周血细胞。最近开发的一种名为TWISTR的技术 允许对斑马弓遗传背景中的基因进行马赛克靶向。无需移植， WT和单基因突变的造血干细胞之间的强大竞争分析可以用 基于颜色的蜂窝条形码TWISTR系统。优势克隆可以通过流式细胞术和 与用于下游分析的非优势克隆进行比较。ASXL1的嵌合诱变导致克隆 优势，我们发现，抑制NR4A1抗炎途径使用CRISPR或化学 抑制能恢复克隆平衡。利用代谢组学，我们发现胆碱代谢在 显性ASXL1突变克隆。在目标1中，将进行药理实验来评估NR4A1 促进克隆显性和针对胆碱相关代谢基因的遗传实验的途径将 确定抑制克隆显性的代谢靶点。我们还发现巨噬细胞在干细胞中发挥作用。 通过与造血干细胞的物理作用实现细胞克隆性。巨噬细胞与“吃我”相互作用 信号，钙网织蛋白，在造血干细胞表面决定它们的命运。在目标2中，我们计划 评估钙网蛋白同源基因的突变体，并检测其对克隆的影响。我们计划探索这一机制 巨噬细胞相互作用后诱导的克隆扩张。我们还进行了一种化学遗传 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，可以诱导出一个“吃我”的信号优势克隆。我们的研究将 对克隆平衡的基本理解产生了影响，并将导致克隆的潜在治疗方法 统治地位。

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.