Improving HSC transplantation by defining novel regulators of engraftment

通过定义新的植入调节因子来改善 HSC 移植

• 批准号: 10341128
• 负责人: SHANNON L MCKINNEY-FREEMAN
• 金额: $ 40.39万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10341128
• 关键词: Acute; Apoptosis; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Blood; Bone Marrow; CD34 gene; CXCR4 gene; Candidate Disease Gene; Cells; Cellular biology; Data; Disease; Engraftment; Environment; Family member; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Genes; Genetic; Goals; Growth; Hematological Disease; Hematopoiesis; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; Laboratories; Life; Lysosomes; Marrow; Molecular; Molecular Target; Mus; Pathway interactions; Patients; Phenotype; Physiological; Pilot Projects; Progenitor Cell Engraftment; Protein Binding Domain; Protein Family; Protein Sortings; Regulation; Reporting; Role; Stress; Testing; Transplantation; United States; Work; chemotherapy; conditioning; curative treatments; experience; hematopoietic transplantation; improved; in vivo; irradiation; knock-down; leukemia; member; novel; post-transplant; progenitor; protein degradation; small hairpin RNA; stem cell migration; stem cell survival; stem cells; transcriptome

Project Summary Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for many patients with hematologic disease or leukemia. In 2015, >22,000 HSCT were performed in the United States. Donor HSC experience many stresses during transplant, including myelo-toxic conditioning that alters the niche, ex vivo manipulation, and supra-physiological expansion. Although post-transplant stress hematopoiesis is likely subject to biological regulation distinct from steady-state hematopoiesis, the molecular regulation of HSCT is poorly understood. We seek to identify the key intrinsic regulators of HSCT. In a screen for genes whose depletion perturbs murine HSPC in vivo repopulation, we identified GASP (G-protein coupled receptor (GPCR) Associated Sorting Protein) family member, Gprasp2, as a novel negative regulator of HSCT. We discovered that loss of Gprasp2, or the highly related gene, Gprasp1, dramatically enhances HSPC competitive repopulating activity. Gprasp1 and Gprasp2 are both expressed by murine and human HSC. GPRASP1 and GPRASP2 bind a motif in the C-terminus of GPCRs to traffic them to lysosomes. Thus, loss of GPRASP1 and GPRASP2 likely stabilizes GPCRs that promote HSCT. CXCR4, a master regulator of HSC migration, survival and quiescence, contains a GASP-binding motif and is thus a putative GASP target. Indeed, Gprasp1 or Gprasp2 knockdown reduced apoptosis, increased quiescence and stabilized CXCR4 in HSPC ex vivo and acutely post-transplant. These phenotypes were abolished in Cxcr4-/- HSPC. Thus, these data implicate GASP family members as novel regulators of CXCR4. We hypothesize that GPRASP1 and GPRASP2 loss boosts HSCT by promoting HSPC survival and quiescence via CXCR4 stabilization. We will test this and also assess a role for GASPs in native hematopoiesis, according to the following Aims: 1) to illuminate cellular mechanisms that enhance HSC transplantation, 2) to identify molecular targets whose stabilization enhances HSC transplantation and 3) to identify novel molecular bottlenecks of human HSC transplantation. In Aim 1, we will use Gprasp1 and Gprasp2 shRNAs to examine Gprasp-deficient HSPC for apoptosis, quiescence, and niche retention acutely post- transplant. We will also use SclERT2-CreGprasp1fl/fl and SclERT2-CreGprasp2fl/fl mice to interrogate a role for Gprasp1 and Gprasp2 in steady-state hematopoiesis. In Aim 2, we will employ Cxcr4fl/flROSA26Cre-ERT2 mice to test if CXCR4 is a key functional target of GPRASP1 or GPRASP2 in HSPC. We will also examine CXCR4 stability and localization in Gprasp-deficient HSPCs and use standard biochemical assays to test for a physical interaction between GPRASP1, GPRASP2 and CXCR4. Finally, in Aim 3, we will treat human CD34+ HSPC with GPRASP1 or GPRASP2-shRNAs to test if GPRASP loss can enhance bone marrow engraftment of human cells when transplanted into NOD-scid IL2rγnull mice. This work will cement GASP family members as novel negative regulators of HSPC, reveal a new mechanism of CXCR4 regulation in HSC, and illuminate new molecular targets for improving HSCT.

项目摘要 造血干细胞移植(HSCT)是许多血液病患者的唯一治疗方法 疾病或白血病。2015年，美国进行了22,000例造血干细胞移植。捐献者HSC经历了许多 移植期间的应激，包括改变利基的骨髓毒性调节，体外操作，以及 超生理扩张。尽管移植后的应激性造血可能受到生物因素的影响 HSCT的分子调控与稳态造血不同，人们对其分子调控知之甚少。我们 寻求确定HSCT的关键内在调节因子。在筛选其耗尽扰乱小鼠的基因 HSPC体内再繁殖，我们鉴定了GAP(G蛋白偶联受体相关分类蛋白) 家族成员Gprasp2作为一种新的HSCT负性调节因子。我们发现Gprasp2的缺失，或者说 高度相关的基因Gprasp1显著增强HSPC竞争性再繁殖活性。Gprasp1和 Gprasp2在小鼠和人的HSC中均有表达。GPRASP1和GPRASP2在C末端结合一个基序 将它们运输到溶酶体。因此，GPRASP1和GPRASP2的缺失可能会稳定GPCRs，从而 推广HSCT。CXCR4是HSC迁移、存活和静止的主要调节因子，它含有一个GAP结合蛋白 Motif，因此可能是喘息目标。事实上，Gprasp1或Gprasp2基因敲除减少了细胞凋亡，增加了 CXCR4在HSPC体内的静止和稳定以及移植后的急性变化。这些表型被废除了 在CXCR4-/-HSPC中。因此，这些数据暗示GAP家族成员可能是CXCR4的新调节者。我们 假设GPRASP1和GPRASP2缺失通过促进HSPC存活和 通过CXCR4稳定化实现静止。我们将测试这一点，并评估喘息在本地的作用 造血，根据以下目的：1)阐明增强HSC的细胞机制 移植，2)确定其稳定性促进HSC移植的分子靶点，3) 发现人类造血干细胞移植的新的分子瓶颈。在目标1中，我们将使用Gprasp1和Gprasp2 ShRNAs用于检测Gprasp缺陷HSPC的细胞凋亡、静止和生态位保留 移植。我们还将使用SclERT2-CreGprasp1fl/fl和SclERT2-CreGprasp2fl/fl小鼠来询问Gprasp1的角色 和Gprasp2在稳态造血中的作用。在目标2中，我们将使用Cxcr4fl/flROSA26Cre-ERT2小鼠来测试是否 CXCR4是GPRASP1或GPRASP2在HSPC中的关键功能靶点。我们还将检查CXCR4的稳定性和 在Gprasp缺陷的HSPC中进行定位并使用标准生化分析来测试物理相互作用 GPRASP1、GPRASP2和CXCR4之间。最后，在目标3中，我们将用GPRASP1处理CD34+HSPC 或GPRASP2-shRNAs来测试当GPRASP缺失时是否可以增强人类细胞的骨髓植入 移植到NOD-SCID IL2rγ缺失小鼠体内。这项工作将巩固喘息的家庭成员作为新的负面影响 HSPC的调控，揭示了CXCR4在HSC中调控的新机制，并阐明了新的分子靶点 用于改善HSCT。