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

基本信息

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
项目类别：
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

项目摘要

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）是许多血液学患者的唯一治疗疗法疾病或白血病。 2015年，在美国进行了> 22,000 HSCT。捐助者HSC经验很多移植过程中的应力，包括改变利基市场的髓毒条件，离体操纵和跨生理学扩张。尽管移植后应力造血可能会受到生物学的影响与稳态造血的调节，HSCT的分子调节知之甚少。我们寻求确定HSCT的关键内在调节因子。在耗尽鼠的基因的屏幕中 HSPC在体内重生中，我们确定了GASP（G蛋白偶联受体（GPCR）相关排序蛋白）家庭成员GPRASP2是HSCT的新型负调节剂。我们发现GPRASP2或高度相关的基因GPRASP1极大地增强了HSPC竞争重生活性。 gprasp1和 GPRASP2均由Murine和Human HSC表示。 GPRASP1和GPRASP2在C末端结合了一个基序 GPCR的渗透到溶酶体。这是GPRASP1和GPRASP2的丢失可能稳定了GPCR 促进HSCT。 CXCR4是HSC迁移，生存和静止的主要调节剂，含有喘着粗气的结合主题，因此是推定的喘气目标。实际上，GPRASP1或GPRASP2敲低降低了凋亡，增加了 HSPC离体和移植后急性的静止和稳定的CXCR4。这些表型被废除了在CXCR4 - / - HSPC中。这些数据暗示GASP家族成员是CXCR4的新调节剂。我们假设GPRASP1和GPRASP2损失通过促进HSPC存活和通过CXCR4稳定静止。我们将测试这一点，并评估喘气的作用造血，根据以下目的：1）阐明增强HSC的细胞机制移植，2）确定稳定增强HSC移植的分子靶标，3）确定人类HSC移植的新型分子瓶颈。在AIM 1中，我们将使用GPRASP1和GPRASP2 shRNA检查GPRASP缺陷HSPC的凋亡，静止和利基保留急性急性移植。我们还将使用sclert2-cregprasp1fl/fl和sclert2-cregprasp2fl/fl小鼠来询问GPRASP1的角色稳态造血中的GPRASP2。在AIM 2中，我们将使用CXCR4FL/FLROSA26CRE-ERT2小鼠来测试是否 CXCR4是HSPC中GPRASP1或GPRASP2的关键功能目标。我们还将检查CXCR4稳定性和在GPRASP缺乏HSPC中进行定位，并使用标准的生化测定测试物理相互作用在GPRASP1，GPRASP2和CXCR4之间。最后，在AIM 3中，我们将使用GPRASP1处理人类CD34+ HSPC 或GPRASP2-SHRNA测试GPRASP损失是否可以增强人类细胞的骨髓植入移植到nod-ScIDIL2RγNull小鼠中。这项工作将以新的负面为基础，巩固喘息的家庭成员 HSPC的调节剂，揭示了HSC中CXCR4调控的新机制，并阐明了新的分子靶标用于改善HSCT。