Targeting Notch2 in Hematopoietic Cell Therapy.

R01HL103827-06A1重新提交(周、兰)10.22.2018 摘要 造血干细胞(HSC)移植是治疗各种恶性肿瘤和肿瘤的唯一选择。 非恶性疾病很少。一个成功的结果取决于注入足够数量的 功能活跃的动员造血祖细胞(HPC)。病人行动不力 对目前的动员剂反应不佳仍然是一个临床问题。此外, 使用低于预期剂量的干细胞进行移植会增加干细胞植入的风险 失败了。因此，开发更有效和低风险的高性能红细胞动员方案和战略将 极大地改善了患者的预后。我们最近发现了Notch作为细胞表面的一种新作用 黏附以及信号分子将干细胞保留在骨髓微环境中。凹槽 是一种保守的细胞表面受体，调节茎的生长和分化。凹槽 转录激活是Notch受体与Notch配体功能结合的结果，这是 依赖于Notch受体对O-岩藻糖和条纹的翻译后修饰 Notch胞外区的表皮生长因子(EGF)模块。结构研究已经 证明O-岩藻糖结合在核心配体结合区的特定苏氨酸残基上 Notch EGF重复序列的功能是作为一种替代氨基酸与其进行特异性和功能性接触 缺口配体DLL4和JAG1。O-岩藻糖延伸为二糖(GlcNAcβ1- 3Fucose)进一步增加了与某些Notch配体的结合亲和力。在上一笔资金中 循环中，我们发现了保守的Notch-配体相互作用及其被O-修饰。 岩藻糖化作用促进肝星状细胞静止和维持生态位。我们提供了令人信服的证据表明 缺乏O-岩藻糖基化导致HSC静止和HSC与骨的粘附性降低 骨髓巢细胞增多，HSC从骨髓流出增多。我们还报告说，中和 DLL4或JAG1以及阻断Notch2显著增强了HSC向外周的出口。更多, 最近，我们发现一过性的Notch2阻断会导致更好的植入和造血。 与HSPC活性增强和内质网应激减弱相关的动员HSPC的恢复 激活。此外，我们还发现了带有核心配体的边缘修饰的重组Notch肽 结合EGF重复序列作为Notch配体诱骗诱导HSC及其祖细胞外逃的实验研究 成骨细胞球体。因此，我们假设Notch2与DLL4或JAG1相互作用调节 配体和小生境特定的HSPC保留。我们进一步假设Notch2-靶向动员 方案可能通过减轻内质网应激反应的激活来改善HCT的结局。我们将对此进行测试 三个目标中的假设。在目标1中，我们将研究JAG1和DLL4表达的不同角色 通过内皮细胞和未成熟的骨线细胞调节HSC生态位滞留， 位置和动员。在目标2中，我们将评估配体结合亲和力和效率。 边缘修饰的Notch多肽作为诱饵促进HSPC从HSC生态位外流。在《目标3》中，我们将 研究阻断Notch2增强HSPC活性的意义和机制 通过减弱内质网应激反应。在这项研究的最后，我们希望我们的发现将揭示出新的 Notch信号在HSC生态位生物学和内质网应激调节中的作用 了解Notch信号在应激性造血过程中控制HSC活性的资源 对确定改善HCT结果的方法的影响。

R01HL103827-06A1 Resubmission (Zhou, Lan) 10.22.2018 Abstract Hematopoietic stem cell (HSC) transplantation is the only curative option for various malignant and a few nonmalignant diseases. A successful outcome is dependent on infusing an adequate number of functionally active mobilized hematopoietic progenitor cells (HPCs). Inadequate mobilization in patients showing poor responses to current mobilizing agents remains a clinical problem. In addition, transplantation using lower-then-desired doses of stem cells increases risks of stem cell engraftment failure. Hence, developing more efficacious and low-risk HPC mobilization regimens and strategies will greatly improve patient outcomes. We recently identified a novel role of Notch as a cell surface adhesion as well as signaling molecule to retain stem cell in the bone marrow microenvironment. Notch is a conserved cell surface receptor that regulates stemness, growth, and differentiation. Notch transactivation is the result of functional engagement of Notch receptors with Notch ligands, which is dependent on the posttranslational modification of Notch receptors with O-fucose and Fringe on the epidermal growth factor (EGF) modules of Notch extracellular domain. Structural studies have demonstrated that O-fucose attached to the specific threonine residue of the core ligand binding region of Notch EGF repeats functions as a surrogate amino acid to make specific and functional contact to Notch ligand DLL4 and JAG1, respectively. Elongation of O-fucose to a disaccharide (GlcNAcβ1- 3Fucose) by Fringe further increases Notch binding affinity to some Notch ligands. In the last funding cycle, we identified conservative Notch-ligand adhesive interaction and its modification by O- fucosylation for HSC quiescence and niche maintenance. We provided compelling evidences showing that deficiency of O-fucosylation leads to decreased HSC quiescence and HSC adhesion to bone marrow niche cells, and increased HSC egress from the marrow. We also reported that neutralizing DLL4 or JAG1 as well as blocking Notch2 significantly enhances HSC egress to the periphery. More, recently, we found transient Notch2 blockade results in superior engraftment and hematopoietic recovery of mobilized HSPC associated with enhanced HSPC activity and attenuated ER stress activation. Further, we found that Fringe-modified recombinant Notch peptides bearing the core ligand binding EGF repeats function as decoys for Notch ligand to induce HSC and progenitor cell egress in osteoblastic spheroids. We thus hypothesize that Notch2 interaction with DLL4 or JAG1 regulates ligand- and niche-specific HSPC retention. We further hypothesize that Notch2-targeted mobilization regimen may improve HCT outcome by mitigating ER stress response activation. We will test this hypothesis in three aims. In Aim 1, we will examine the differential roles of JAG1 and DLL4 expressed by the endothelial and the immature osteolineage cells in the regulation of HSC niche retention, location and mobilization. In Aim 2, we will assess the ligand binding affinity and the efficiency of Fringe-modified Notch peptides as decoys to enhance HSPC egress from HSC niche. In Aim 3, we will examine the significance and the mechanism by which blocking Notch2 potentiates HSPC activity through attenuating ER stress response. At the end of this study, we hope our findings will reveal novel aspects of Notch signaling in HSC niche biology and in ER stress regulation which may provide a resource for understanding Notch signaling in the control of HSC activity during stress hematopoiesis, with implications for identifying ways to improve HCT outcomes.