GPRASP family as novel regulators in hematopoietic stem cells

项目摘要 造血干细胞移植(HSCT)是治疗大多数血液病的唯一方法。 包括白血病在内的疾病。HSCT需要捐献者的HSCs参与适当的支持利基，生存下来， 增殖，并分化为成熟的血统。最近的研究表明，“应激造血”， 包括发生在HSCT后的，与稳态造血相比，受到明显的调控。 更好地理解调节移植的HSCs及其植入的分子机制是 有必要改进HSCT，特别是在移植受到细胞数量较少限制的情况下(例如脐带血 血液)或可能因体外操作(例如基因治疗)而受到损害。为此，主要目标是 本项目旨在识别和表征HSCT的新的关键内在调节因子。我的作品流露出高度的表现力 多个G蛋白偶联受体相关分类蛋白(如Gprasp1和Gprasp2)在 HSCs，相对于下游的祖细胞。我还发现GPRASP1和GPRASP2都是阴性的 HSCT的监管机构。Gprasp缺陷的HSCs表现出增强的归巢、存活和静止 移植。这种表型似乎依赖于CXCR4，它是HSCs功能的主要调节因子，其序列 包含一个假定的GAP结合基序。事实上，异位CXCR4增强了HSC的归巢、存活和静止 在移植过程中，Gprasp1或Gprasp2的表型复制丢失。此外，我的初步研究表明，在 Gprasp1或Gprasp2丢失时，CXCR4稳定并在HSC上积聚，使其对SDF-1敏感。CXCR4 也参与调节B细胞前体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止B细胞的产生而扰乱B细胞的生产 淋巴祖细胞的分化。最后，尽管作为一个群体的HSC在Gprasp1中高度丰富 和Gprasp2的表达，我最近对单细胞基因表达数据和单个HSCs的分析 免疫荧光显示，这些基因在单个HSC中的表达是不同的。 这种差异表达与不同的基因表达谱相对应(例如，干细胞、细胞周期、B细胞 差异化)。尽管克隆分析表明，即使是高纯度的造血干细胞也表现出多样性 分化潜能，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家族成员调控HSC移植和B细胞 分化，并与HSC功能异质性相关。此建议书的指导阶段将会发生 在圣犹大儿童研究医院，在香农·麦金尼-弗里曼博士的指导下。在独立阶段，我 我将证实并扩展我在HSCT和B细胞中GPRASPs和CXCR4之间的分子联系的研究 分化，以及利用新的分子报告程序来精炼HSC的功能异质性。这个 机构资源和学术环境以及我提案中概述的计划中的课程将确保 我成功地过渡到独立。

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.