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

摘要/摘要 钻石黑粉贫血(DBA)是一种先天性骨髓衰竭综合征，与 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 20多个核糖体基因，导致单倍体不足和全球核糖体生物发生缺陷，但 这导致红细胞生成缺陷的机制还知之甚少。我观察到类似尼莫的东西 核糖体缺乏性红系祖细胞中的激酶(NLK)被激活，与驱动因素无关 核糖体突变。抑制NLK促进造血干/祖细胞红系扩增 DBA患者和小鼠模型体外培养细胞(HSPC)。 这项建议的首要目标是确定NLK在DBA和DBA的发病机制中的作用。 确定NLK的新型上游调节剂和下游底物。通过获得新的技能 由我的指导团队开创的最先进的技术，我预见到成功解决 建议的研究目标和技能的发展以及建立我的研究计划所需的初步数据 拥有独立的研究项目。在目标1中，我将使用CRISPR/Cas9敲除核糖体中的NLK- 供者的HSPC不足，并检查移植到受体小鼠体内后的植入情况。就像NLK 在DBA模型中激活，而不考虑驱动突变，这代表了一种基因治疗方法 自体干细胞移植有可能治愈这种疾病对血液学的影响。在AIM 2、我将在DBA中鉴定和鉴定NLK的下游底物。在目标3中，我将确定和 鉴定DBA中NLK激活上游的非调控蛋白。最后两个目标包括 初步候选的特征，加上全基因组筛选，以确定新的因素。 总的来说，这些研究有可能确定新的治疗靶点并改善DBA的预后 病人。 这项拟议的工作还将为我提供必要的工具和专业知识，以便成功 过渡到独立的职业生涯。我将获得的技能包括小鼠干细胞移植， CRISPR/Cs9介导的干细胞、核糖体和线粒体生物发生的基因治疗，翻译 分析和全基因组的基因组分析。涵盖板凳技能的课程作业(例如，应用的RNA生物学 计算工具和生物信息学)和职业发展(例如导师、人事管理 和教师过渡)将对我的指导团队的指导表示赞赏。Sakamoto博士有一个典范 我有生产领导者的记录，并致力于在我获得更多自主权时继续指导。Dr。 Sakamoto和Stanford对我的职业发展表现出了非凡的承诺 在我的团契和讲师培训期间，我相信他们会继续支持我们的 共同的目标是开发一个世界级的独立研究计划，致力于了解 非恶性血液病的发病机制，如DBA。

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.