Characterization of the Role of Nemo-like Kinase in Normal and Diamond Blackfan Anemia Models of Erythropoiesis.

总结/摘要 Diamond Blackfan贫血（DBA）是一种先天性骨髓衰竭综合征， 早期红系祖细胞的生理畸形和缺陷。超过80%的患者携带一种突变， 超过20个核糖体基因，导致单倍不足和缺陷的全球核糖体生物合成，但 这导致红细胞生成缺陷的机制知之甚少。我观察到， 激酶（NLK）在核糖体不足的红系祖细胞中被激活，与驱动无关。 核糖体突变抑制NLK促进造血干细胞和祖细胞的红系扩增 细胞（HSPC）从DBA患者和小鼠模型在体外。 该提案的总体目标是确定NLK在DBA发病机制中的作用， 确定NLK的新型上游调节剂和下游底物。通过学习新技能， 我的指导团队开创了最先进的技术，我预见到成功解决 提出的研究目标以及建立我的研究所需的技能和初步数据的发展 独立的研究计划。在目标1中，我将使用CRISPR/Cas9敲除核糖体中的NLK- 供体HSPC不足，并检查移植到受体小鼠后的植入。由于NLK是 在DBA模型中激活，无论驱动突变如何，这代表了一种基因治疗方法， 自体干细胞移植具有治愈疾病的血液学影响的潜力。在Aim中 2、我将在DBA中识别和表征NLK的下游底物。在目标3中，我将确定和 表征DBA中NLK激活上游的失调蛋白。最后两个目标包括 初步候选物的表征，通过全基因组筛选补充以鉴定新因子。 总的来说，这些研究有可能确定新的治疗靶点，改善DBA的预后。 患者 这项拟议的工作也将为我提供必要的工具和专业知识， 过渡到独立职业。我将获得的实验室技能包括小鼠干细胞移植， CRISPR/Cs 9介导的干细胞、核糖体和线粒体生物合成、翻译 分析和全基因组激酶组分析。涵盖实验室技能的课程（例如RNA生物学，应用 计算工具和生物信息学）和职业发展（如导师制、人事管理 我的导师团队将为我提供指导。坂本博士有一个典型的 在培养领导者方面有着良好的记录，并致力于在我获得更多自主权的过程中继续提供指导。博士 坂本和斯坦福大学对我的职业发展表现出了非凡的承诺 在我的奖学金和教练培训，我毫不怀疑他们对我们的持续支持， 共同的目标是发展一个世界级的独立研究计划，致力于了解 非恶性血液病如DBA的发病机制。

Summary / Abstract Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with physical malformations and defects in early erythroid progenitors. Over 80% of patients carry mutations in one of over twenty ribosomal genes, leading to haploinsufficiency and defective global ribosome biogenesis, but the mechanism by which this leads to erythropoiesis defects is poorly understood. I observed that Nemo-like Kinase (NLK) is activated in erythroid progenitors with ribosome-insufficiency, irrespective of the driving ribosomal mutation. Suppression of NLK improves erythroid expansion of hematopoietic stem and progenitor cells (HSPCs) from DBA patients and mouse models in vitro. The overarching goal of this proposal is to define the role of NLK in the pathogenesis of DBA and identify novel upstream regulators and downstream substrates of NLK. Through the acquisition of new skills in state-of-the-art technologies pioneered by my mentoring team, I foresee the successful resolution of the proposed research aims and the development of the skillset and preliminary data necessary to establish my own independent research program. In Aim 1, I will use CRISPR/Cas9 to knock out NLK in ribosome- insufficient donor HSPCs and examine engraftment after transplantation into recipient mice. As NLK is activated in DBA models irrespective of the driving mutation, this represents a gene therapy approach for autologous stem cell transplantation with the potential to cure the hematological impacts of the disease. In Aim 2, I will identify and characterize downstream substrates of NLK in DBA. In Aim 3, I will identify and characterize deregulated proteins upstream of NLK activation in DBA. The last two aims include characterization of preliminary candidates, complimented by genome wide screens to identify novel factors. Collectively, these studies have the potential to identify new therapeutic targets and improve outcomes for DBA patients. This proposed work will also provide me with the necessary tools and expertise to successfully transition to an independent career. Bench skills I will acquire include mouse stem cell transplantation, CRISPR/Cs9-mediated gene therapy of stem cells, ribosome and mitochondrial biogenesis, translational analysis and genome-wide kinome analysis. Coursework covering bench skills (e.g. RNA biology, applied computational tools, and bioinformatics) and career development (e.g. mentorship, personnel management and faculty transitioning) will compliment guidance from my mentoring team. Dr. Sakamoto has an exemplary track record of producing leaders and is committed to continued guidance as I take on more autonomy. Dr. Sakamoto and Stanford have demonstrated exceptional commitment to my professional development throughout my fellowship and instructor training and I have no doubt their continued support towards our shared goal of developing a world class independent research program dedicated to understanding the pathogenesis of nonmalignant hematological disorders such as DBA.