GPRASP family as novel regulators in hematopoietic stem cells

项目摘要 造血干细胞（HSC）移植（HSCT）代表了大多数血液病患者的唯一治愈性疗法。 包括白血病在内的疾病。HSCT需要供体HSC与适当的支持生态位接合，存活， 增殖并分化成成熟的血液谱系。最近的研究表明，“应激造血”， 包括HSCT后发生的，与稳态造血相比，受到不同的调节。 更好地理解调控移植HSC及其植入的分子机制是 改善HSCT是必要的，特别是在移植受小细胞数量限制的情况下（如脐带血）。 血液）或可能受到离体操作（例如基因治疗）的损害。为此，主要目标 本项目旨在鉴定和表征HSCT的新型关键内在调节因子。我的作品展现了高表达 多种GPRASP（G-蛋白偶联受体相关分选蛋白）（例如GPrasp 1和GPrasp 2）在 HSC，相对于下游祖细胞。我还发现GPRASP 1和GPRASP 2都是负性的， HSCT的调节剂。Gprasp缺陷型HSC在移植过程中表现出增强的归巢、存活和静止。 移植这种表型似乎依赖于Cxcr 4，它是HSC功能的主要调节因子，其序列 含有推定的GASP结合基序。事实上，异位Cxcr 4增强HSC归巢、存活和静止 在移植期间，表型复制GPrasp 1或GPrasp 2的丢失。此外，我的初步研究表明， 当GPRasp 1或GPRasp 2缺失时，CXCR 4稳定并在HSC上积累，使它们对SDF-1敏感。CXCR4 还涉及调节B细胞祖细胞和基质细胞之间的细胞间接触。 有趣的是，我最近的数据表明，GPRASP的丢失也可能通过阻断B细胞的分泌来干扰B细胞的产生。 淋巴祖细胞的分化。最后，尽管HSC作为一个群体高度富集GPrasp 1， 和Gprasp 2表达，我最近通过对单个细胞基因表达数据和单个HSC的分析， 免疫荧光显示这些基因的表达在个体HSC中是异质的。 这种差异表达对应于不同的基因表达谱（例如干细胞性、细胞周期、B细胞增殖、细胞周期）。 差异化）。尽管克隆分析显示，即使是高度纯化的HSC， 尽管这种功能异质性与分化潜能有关，但目前对这种功能异质性的分子调控知之甚少。 因此，我假设多个GPRASP家族成员调节HSC移植和B细胞 分化，并与HSC功能异质性相关。本提案的辅导阶段将在 在圣裘德儿童研究医院，由香农·麦金尼-弗里曼医生指导在独立阶段，我 我的研究将进一步证实GPRASPs和CXCR 4在HSCT和B细胞中的分子联系 分化，以及开发新的分子报告基因来改善HSC功能异质性。的 机构资源和学术环境以及我建议中概述的计划课程将确保 我成功过渡到独立。

Project Summary Hematopoietic stem cell (HSC) transplantation (HSCT) represents the only curative therapy for most hematologic disease including leukemia. HSCT requires donor HSCs to engage with the proper supporting niche, survive, proliferate, and differentiate into mature blood lineages. Recent studies suggest that “stress hematopoiesis”, including that which occurs post-HSCT, is subject to distinct regulation compared steady-state hematopoiesis. A better understanding the molecular mechanisms that regulate transplanted HSCs and their engraftment is necessary to improve HSCT, especially in situations where transplant is limited by small cell numbers (e.g. cord blood) or potentially compromised by ex vivo manipulation (e.g. gene therapy). Towards this, the main goal of this project is to identify and characterize novel key intrinsic regulators of HSCT. My work reveals high expression of multiple GPRASPs (G-Protein Coupled Receptor Associated Sorting Proteins) (e.g. Gprasp1 and Gprasp2) in HSCs, relative to downstream progenitors. I have also found that both GPRASP1 and GPRASP2 act as negative regulators of HSCT. Gprasp-deficient HSCs present enhanced homing, survival and quiescence during transplant. This phenotype appears dependent on Cxcr4, a master regulator of HSCs function, whose sequence contains a putative GASP-binding motif. Indeed, ectopic Cxcr4 enhances HSC homing, survival and quiescence during transplant, phenocopying loss of Gprasp1 or Gprasp2. Further, my preliminary studies show that, upon Gprasp1 or Gprasp2 loss, CXCR4 is stabilized and accumulates on HSCs, sensitizing them to SDF-1. CXCR4 has also been implicated in regulating cell-to-cell contact between B-cell progenitors and stroma cells. Interesting, my recent data suggests that GPRASP loss may also perturb B-cell production by blocking the differentiation of lymphoid progenitors. Finally, although HSCs as a population are highly enriched for Gprasp1 and Gprasp2 expression, my recent analyses of single cell gene expression data and single HSCs by immunofluorescence reveals that the expression of these genes is heterogeneous amongst individual HSCs. This differential expression corresponds with distinct gene expression profiles (e.g. stemness, cell cycle, B-cell differentiation). Although clonal analyses have revealed that even highly purified HSCs display diverse differentiation potential, the molecular regulation of this functional heterogeneity is currently poorly understood. Thus, I hypothesize that multiple GPRASP family members regulate HSC transplantation and B-cell differentiation, and correlate with HSC functional heterogeneity. The mentored phase of this proposal will occur at St. Jude Children’s Research Hospital, under Dr. Shannon McKinney-Freeman. In the independent phase, I will confirm and extend my studies of the molecular link between GPRASPs and CXCR4 in HSCT and B-cell differentiation, as well as exploit novel molecular reporters to refine HSC functional heterogeneity. The institutional resources and academic environment and the planned courses outlined in my proposal will ensure my successful transition to independence.