Improving HSC transplantation by defining novel regulators of engraftment

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，在美国进行了超过22，000例HSCT。捐助者HSC经历了许多 移植过程中的应激，包括改变生态位的骨髓毒性调节，离体操作， 超生理扩张尽管移植后应激造血可能受到生物学的影响， 调控不同于稳态造血，HSCT的分子调控知之甚少。我们 寻求确定HSCT的关键内在调节因子。在筛选基因的过程中， HSPC体内再增殖，我们鉴定了GASP（G蛋白偶联受体（GPCR）相关分选蛋白） 家族成员GPrasp 2作为HSCT的一种新的负调节因子。我们发现GPRASP 2的缺失， 高度相关的基因Gprasp 1显著增强HSPC竞争性再增殖活性。GPRasp 1和 GPrasp 2均由鼠和人HSC表达。GPRASP 1和GPRASP 2结合C末端的基序 将它们运送到溶酶体。因此，GPRASP 1和GPRASP 2的缺失可能使GPCR稳定， 推广HSCT。CXCR 4是HSC迁移、存活和静止的主要调节因子，含有GASP结合蛋白。 基序，因此是推定的GASP靶标。事实上，GPrasp 1或GPrasp 2敲低减少了细胞凋亡，增加了细胞凋亡。 在HSPC中，CXCR 4在离体和移植后急性静止和稳定。这些表型被消除 在Cxcr 4-/- HSPC中。因此，这些数据暗示GASP家族成员是CXCR 4的新型调节剂。我们 假设GPRASP 1和GPRASP 2缺失通过促进HSPC存活来促进HSCT， 通过CXCR 4稳定化的静止。我们将对此进行测试，并评估GASP在本地 造血，根据以下目的：1）阐明增强HSC的细胞机制 移植，2）鉴定其稳定化增强HSC移植的分子靶标，和3） 鉴定人HSC移植新的分子瓶颈。在目标1中，我们将使用GPRasp 1和GPRasp 2 shRNA检测Gprasp缺陷型HSPC的细胞凋亡、静止和急性后的生态位保留。 移植我们还将使用SclERT 2-CreGprasp 1fl/fl和SclERT 2-CreGprasp 2fl/fl小鼠来研究Gprasp 1的作用。 和GPrasp 2在稳态造血中的作用。在目标2中，我们将使用Cxcr 4fl/flROSA 26 Cre-ERT 2小鼠来测试是否存在以下情况： CXCR 4是HSPC中GPRASP 1或GPRASP 2的关键功能靶标。我们还将检查CXCR 4的稳定性， 在Gprasp缺陷型HSPC中定位，并使用标准生化测定来测试物理相互作用 GPRASP 1、GPRASP 2和CXCR 4之间的差异。最后，在目标3中，我们将用GPRASP 1治疗人CD 34 + HSPC。 或GPRASP 2-shRNA来测试GPRASP缺失是否可以增强人细胞的骨髓植入， 移植到NOD-scid IL 2 r γnull小鼠中。这项工作将水泥GASP家庭成员作为新的负面 HSPC的调节因子，揭示了HSC中CXCR 4调节的新机制，并阐明了新的分子靶点 来改善HSCT。

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.