Targeting Notch2 in Hematopoietic Cell Therapy.

R 01 HL 103827 - 06 A1重新提交（Zhou，Lan）2018年10月22日 摘要 造血干细胞（HSC）移植是治疗各种恶性肿瘤的唯一选择， 少数非恶性疾病。成功的结果取决于注入足够数量的 功能活性动员的造血祖细胞（HPC）。患者活动不充分 显示对当前动员剂的不良反应仍然是临床问题。此外，本发明还提供了一种方法， 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 失败因此，开发更有效和低风险的HPC动员方案和策略将 大大改善了患者的治疗效果。我们最近发现了Notch作为细胞表面的一种新作用， 粘附以及信号分子将干细胞保留在骨髓微环境中。凹口 是一种保守的细胞表面受体，调节干性、生长和分化。凹口 反式激活是Notch受体与Notch配体功能性接合的结果， 依赖于Notch受体与O-岩藻糖的翻译后修饰， Notch细胞外结构域的表皮生长因子（EGF）模块。结构研究已经 证明O-岩藻糖连接到核心配体结合区的特异性苏氨酸残基上 Notch EGF重复序列作为替代氨基酸发挥作用，与表皮生长因子进行特异性和功能性接触， Notch配体DLL 4和JAG 1。将O-岩藻糖延长为二糖（GlcNAcβ1- 3岩藻糖）进一步增加了Notch与一些Notch配体的结合亲和力。在上一次融资中， 循环，我们确定了保守的Notch-配体粘附相互作用及其修饰O- 岩藻糖基化用于HSC静止和生态位维持。我们提供了有力的证据 O-岩藻糖基化的缺乏导致HSC静止和HSC与骨的粘附减少 骨髓巢细胞，并增加HSC从骨髓中的排出。我们还报道说， DLL 4或JAG 1以及阻断Notch 2显著增强HSC向外周的流出。更多， 最近，我们发现短暂的Notch 2阻断导致上级移植和造血干细胞移植。 动员的HSPC的恢复与HSPC活性增强和ER应激减弱相关 activation.此外，我们发现Fringe修饰的携带核心配体的重组Notch肽 结合EGF重复序列作为Notch配体的诱饵，诱导HSC和祖细胞流出， 成骨细胞球体因此，我们假设Notch 2与DLL 4或JAG 1的相互作用调节了 配体和小生境特异性HSPC保留。我们进一步假设Notch 2靶向动员 方案可以通过减轻ER应激反应激活来改善HCT结果。我们将测试这个 三个目标的假设。在目的1中，我们将研究表达的JAG 1和DLL 4的不同作用。 通过内皮细胞和未成熟的成骨细胞调节HSC的龛位保持， 位置和动员。在目标2中，我们将评估配体结合亲和力和免疫调节的效率。 Fringe修饰的Notch肽作为诱饵以增强HSPC从HSC小生境中排出。在目标3中，我们 研究阻断Notch 2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新的 Notch信号在HSC生态位生物学和ER应激调节中的方面， 了解Notch信号传导在应激造血过程中控制HSC活性的资源， 这对确定改善HCT结果的方法具有意义。

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.