NOTCH SIGNALING IN MYELOID DEVELOPMENT

描述（由申请人提供）：该项目描述了一个为期5年的职业发展计划，旨在促进血液学研究和血液病理学的学术生涯。拟议的研究计划将建立在现有的知识基金的基础上，利用华盛顿大学的独特资源，该大学在培养医生科学家方面有着良好的记录。Timothy Ley博士是骨髓发育和癌症基因组学专家，曾获得美国血液学学会导师奖，他将担任研究导师。本提案的目标是获得科学方法、技术技能、资助写作和科学出版方面的实践和/或教学培训，以建立一个专注于造血发育的独立研究项目。拟议的研究将集中在Notch途径，以确定Notch信号改变骨髓发育的机制。我们最近的研究表明，表达PML-RARA癌基因的髓系祖细胞（使用Ctsg-PML-RARA小鼠模型）具有增强的自我更新，这可以通过Notch抑制在体外被废除。这些观察将在体内使用已建立的Notch失活小鼠模型（条件性DN-MAML-GFP）进行扩展，目的如下：具体目的1：我们将研究Notch信号传导在体内自我更新和骨髓发育中的作用。Notch阻断在正常和Ctsg-PML-RARA衍生的造血干/祖细胞（HSPC）中的影响将通过体内方法评估，以评估祖细胞适应性和信号传导，以及骨髓肿瘤的长期发展;具体目标2：我们将鉴定早期骨髓祖细胞中的Notch靶基因和表观遗传变化。DN-MAML 1-GFP融合体上的GFP部分将被靶向用于染色质免疫沉淀，然后进行Illumina配对末端测序（ChIP-Seq），以确定c-Kit阳性细胞（富含骨髓干细胞/祖细胞）中Notch激活的基因组靶标。这些研究将与转录谱分析和额外的ChIP-Seq实验配对，以监测组蛋白修饰。这些研究将确定Notch途径影响骨髓发育的机制，并将确定骨髓特异性Notch功能，这可能最终指导骨髓疾病的新治疗方法。

DESCRIPTION (provided by applicant): This project describes a 5-year career development program designed to foster an academic career in hematologic research and hematopathology. The proposed research program will build on an existing fund of knowledge using the unique resources available at Washington University, which has a proven track record of developing physician scientists. Dr. Timothy Ley, an expert in myeloid development and cancer genomics and a previous recipient of the American Society of Hematology Mentor Award, will serve as the research mentor. The goals of this proposal are to obtain practical and/or didactic training in scientific methodology, technical skills, grant writing, and scientific publication for the purposeof establishing an independent research program focused on hematopoietic development. The proposed research will focus on the Notch pathway to determine the mechanism(s) by which Notch signaling alters myeloid development. Our recent studies have shown that myeloid progenitors expressing the PML-RARA oncogene (using the Ctsg-PML-RARA mouse model) have enhanced self-renewal, which can be abrogated in vitro by Notch inhibition. These observations will be extended in vivo using an established mouse model of Notch inactivation (conditional DN-MAML-GFP) in the following Aims: Specific Aim 1: We will investigate the role of Notch signaling in self-renewal and myeloid development in vivo. The impact of Notch blockade in both normal and Ctsg-PML-RARA-derived hematopoietic stem/progenitor cells (HSPCs) will be assessed by in vivo methods to assess progenitor fitness and signaling, as well as the long-term development of myeloid neoplasms; and Specific Aim 2: We will identify Notch target genes and epigenetic changes in early myeloid progenitors. The GFP moiety on the DN-MAML1-GFP fusion will be targeted for chromatin immunoprecipitation followed by Illumina paired-end sequencing (ChIP-Seq) to determine the genomic targets of Notch activation in c-Kit positive cells (enriched in myeloid stem/progenitors). The studies will be paired with transcriptional profiling and additional ChIP-Seq experiments to monitor histone modifications. These studies will define the mechanisms by which the Notch pathway influences myeloid development and will identify myeloid-specific Notch functions, which may ultimately guide novel therapeutic approaches for myeloid diseases.