Quantitative Notch Signaling in Hematopoiesis

造血过程中的定量缺口信号传导

• 批准号: 9126157
• 负责人: IRWIN D BERNSTEIN
• 金额: $ 14.08万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9126157
• 关键词: Agonist; Angiopoietins; Antibodies; Blood; Blood Cells; Bypass; CD34 gene; CD34+ precursor; Cell Count; Cell Survival; Cell surface; Cells; Clinical Trials; Development; Effectiveness; Engineering; Engraftment; Evolution; Extracellular Domain; Generations; Goals; Grant; Growth; Hematopoietic; Human; Laboratories; Ligands; Mediating; Methods; Modality; Notch Signaling Pathway; Notch signaling in hematopoiesis; Outcome; Patients; Population; Population Heterogeneity; Predisposition; Property; Proteins; Protocols documentation; Receptor Activation; Regulation; Safety; Signal Pathway; Signal Transduction; Stem cells; Testing; Therapeutic; Umbilical Cord Blood; Umbilical Cord Blood Transplantation; Variant; base; cell growth; cell type; cellular engineering; cost effective; design; in vivo; insight; interest; neglect; notch protein; novel; novel strategies; paralogous gene; prevent; receptor; receptor expression; receptor function; self-renewal; stem; stem cell population

PROJECT SUMMARY Our laboratory has developed novel culture methods using engineered Notch ligands to increase the number of CD34+ precursors, including those able to provide rapid hematopoietic engraftment in patients undergoing cord blood (CB) transplantation. Clinical trials have determined the safety and effectiveness of this approach, but indicate the need to derive greater hematopoietic stem and progenitor cell (HSPC) numbers for a more potent, economically feasible therapy. Consequently, the focus of this grant is to elucidate new strategies to regulate Notch-induced signals to maximize HSPC growth versus differentiation. To this point, our CB CD34+ expansion protocol has focused on regulating Notch signal strength by controlling ligand availability, while largely neglecting the contribution of variation in cell-surface Notch paralog expression and susceptibility to activation. Recent observations compel us to investigate the use of paralog-specific activating antibodies to tightly regulate the level of Notch signal strength generated during the evolution of cell surface Notch expression that accompanies ex vivo culture (Aim 1). These studies also suggest that paralog-specific antibodies raised against the amino-terminus of the Notch extracellular domain induce Notch activation in stem cell populations insusceptible to Notch ligand-induced activation by overcoming the inhibition of Notch activation due to endogenous Notch ligand expression (cis-inhibition). We will confirm that differential agonist potency in promoting Notch activation stems from ligand-mediated cis- inhibition and determine whether use of Notch antibody agonists to overcome this suppression enhances the generation of engraftable HSPC (Aim 2). These studies will provide insight into the cell-autonomous mechanisms regulating Notch receptor function, an outcome of conceptual and practical interest to those focused on manipulating cell-fate decisions in Notch-dependent stem cell types. Lastly, we will combine our findings maximizing Notch-induced HSPC self- renewal while preventing differentiation with the activation of signaling pathways promoting HSPC survival, allowing further expansion of HSPC (Aim 3). Together, these aims advance us toward our long-term goal of developing novel strategies for stem cell engineering, with a focus on achieving a substantial increase in HSPC expansion for therapeutic purposes.

项目概要 我们的实验室开发了使用工程Notch配体的新型培养方法 增加 CD34+ 前体的数量，包括那些能够提供快速造血功能的前体 接受脐带血（CB）移植的患者的植入。临床试验有 确定了该方法的安全性和有效性，但表明需要获得更大的 造血干细胞和祖细胞 (HSPC) 数量更有效、更经济 可行的疗法。因此，这笔赠款的重点是阐明监管的新策略 Notch 诱导信号可最大限度地促进 HSPC 生长与分化。至此，我们的CB CD34+扩展协议的重点是通过控制配体来调节Notch信号强度 可用性，同时很大程度上忽略了细胞表面Notch旁系同源变异的贡献 表达和激活的敏感性。最近的观察迫使我们调查 使用旁系同源特异性激活抗体严格调节Notch信号强度水平 在细胞表面Notch表达进化过程中产生，伴随离体 文化（目标 1）。这些研究还表明，针对旁系同源物产生的特异性抗体 Notch胞外结构域的氨基末端诱导干细胞中的Notch激活 通过克服Notch配体诱导的抑制作用，群体不易受到Notch配体诱导的激活的影响 由于内源性Notch配体表达（顺式抑制）而导致Notch激活。我们将确认 促进Notch激活的不同激动剂效力源于配体介导的顺式- 抑制并确定是否使用Notch抗体激动剂来克服这种抑制 增强可移植 HSPC 的生成（目标 2）。这些研究将提供深入了解 调节Notch受体功能的细胞自主机制，概念的结果 以及那些专注于在Notch依赖性中操纵细胞命运决定的人的实际兴趣 干细胞类型。最后，我们将结合我们的发现最大化 Notch 诱导的 HSPC 自 通过激活信号通路促进更新，同时防止分化 HSPC 存活，允许 HSPC 进一步扩展（目标 3）。这些目标共同推动我们前进 实现我们开发干细胞工程新策略的长期目标，重点是 实现用于治疗目的的 HSPC 扩增的大幅增加。