Deconvoluting the Ewing sarcoma genetic program using ancestry-informed human iPSC modeling

使用基于血统的人类 iPSC 模型对尤文肉瘤遗传程序进行解卷积

基本信息

批准号：
10562800
负责人：
Beau Richard Webber
金额：
$ 62.51万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-07 至 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10562800
关键词：
ATAC-seq Adolescent African African American African ancestry Age Asian Automobile Driving Binding Biological Assay Bone neoplasms CRISPR/Cas technology Candidate Disease Gene Cell Line Cells Child Chromatin Communities Complex Data Data Set Development Disparity EWS-FLI1 fusion protein Engineering Epigenetic Process Etiology European European ancestry Ewings sarcoma Exhibits Fusion Oncogene Proteins Gene Expression Gene Expression Profile Gene Expression Profiling Genes Genetic Genetic Engineering Genetic Transcription Genome Genome engineering Genomics Heterozygote Human Hybrids In Vitro Incidence Individual Intervention Investigation Knock-out Knowledge Latino Link Measures Mesenchymal Stem Cells Methods Modeling Molecular Neural Crest Neural Crest Cell Pathway interactions Pattern Pediatric Neoplasm Phenotype Play Population Genetics Protocols documentation Regulatory Element Research Research Personnel Risk Risk Factors Role Sampling Somatic Mutation System Technology Therapeutic Validation Variant Xenograft Model candidate identification cell repository cell type experimental study genome-wide improved in vivo induced pluripotent stem cell mesenchymal stromal cell metaplastic cell transformation novel novel therapeutics overexpression permissiveness pharmacologic programs public health relevance response stem cell model stem cells transcription factor transcriptome transcriptome sequencing virtual

项目摘要

Abstract Ewing sarcoma survival has not improved in decades despite long knowledge of its singular driving somatic mutation, the pernicious EWS-FLI1 fusion oncoprotein. As an aberrant transcription factor EWS-FLI1 alters expression of thousands of genes enacting a complex program toxic to most cell types, so much so that the cell of origin for Ewing sarcoma is still a matter of debate. Ewing sarcoma occurs at starkly higher rates (roughly 10- fold) in children of European ancestry compared to those with primarily African ancestry, while Latino children have roughly ⅔ and Asian children ½ the risk of Ewing sarcoma than do white children. Ancestry is in fact the strongest known risk factor for Ewing sarcoma, but the molecular basis of these differences in risk have been investigated only sparingly. As the mechanisms by which EWS-FLI1 interacts with the genome have become clear, it is feasible with an appropriate model to investigate how genomic ancestry in general and at specific loci modulates EWS-FLI1 activity including its downstream effects on epigenetic and transcriptional programs. To this end we have devised a strategy to introduce EWS-FLI1 in derivatives of induced pluripotent stem cells (iPSC) in order to characterize downstream molecular and functional consequences of EWS-FLI1 expression. Using accessible variant array data from several large stem cell repositories we identified banked iPSC lines derived from individuals with a range of European, African, and Amerind ancestry. We will introduce EWS-FLI1 expression at intermediate stages of development relevant to Ewing sarcomagenesis—namely neural crest cells and mesenchymal stem cells. Measures of functional tolerance and molecular state will be compared to corresponding samples from subjects with solely European ancestry. Globally we will examine whether gene expression and chromatin state exhibits similarity to the Ewing expression signature in proportion to European ancestry percentage. Genome-wide chromatin occupancy of EWS-FLI1 will be profiled and its relationship to local ancestry defined using long-read sequencing. Genes that are differentially influenced by EWS-FLI1 in “permissive” (European ancestry), “moderately permissive” (Amerind ancestry) and “impermissive” (African ancestry) genomes will be considered targets of potential therapeutic value and will undergo validation using CRISPR/Cas9 genome engineering and functional assays. The resulting data will represent the first and only effort, to our knowledge, to take advantage of the known differences in risk for Ewing sarcoma by ancestry to study EWS-FLI1 binding and downstream effects. In addition, we will make available the genome-scale data produced by our study and freely distribute our iPSC models for wider use by Ewing sarcoma researchers.

抽象的尽管人们长期以来了解尤文肉瘤独特的驱动体细胞，但其存活率几十年来并未改善突变，有害的 EWS-FLI1 融合癌蛋白作为异常转录因子 EWS-FLI1 发生改变。数千个基因的表达，执行一个对大多数细胞类型有毒的复杂程序，以至于细胞尤文肉瘤的起源仍然是一个有争议的问题，尤文肉瘤的发生率明显更高（大约 10-10）。欧洲血统的儿童与主要是非洲血统的儿童相比，拉丁裔儿童事实上，亚裔儿童患尤文肉瘤的风险大约是白人儿童的 ⅔ 和 1/2。尤文肉瘤已知的最强风险因素，但这些风险差异的分子基础是由于 EWS-FLI1 与基因组相互作用的机制已被很少研究。显然，使用适当的模型来研究基因组祖先在一般情况下和在特定位点上的情况是可行的调节 EWS-FLI1 活性，包括其对表观遗传和转录程序的下游影响。为此，我们设计了一种策略，将 EWS-FLI1 引入诱导多能干细胞 (iPSC) 的衍生物中为了表征 EWS-FLI1 表达的下游分子和功能后果。来自几个大型干细胞存储库的可访问变体阵列数据，我们确定了衍生的 iPSC 系库来自具有欧洲、非洲和美洲血统的个体，我们将介绍 EWS-FLI1。与尤文肉瘤发生相关的发育中间阶段的表达，即神经嵴细胞功能耐受性和分子状态的测量将与间充质干细胞进行比较。我们将在全球范围内检查来自仅具有欧洲血统的受试者的相应样本是否具有基因。表达和染色质状态与尤因表达特征相似，与欧洲人成比例将分析 EWS-FLI1 的全基因组染色质占用率及其与使用长读长测序定义的本地血统受到 EWS-FLI1 的差异影响。 “宽容”（欧洲血统）、“适度宽容”（美洲血统）和“不宽容”（非洲血统）祖先）基因组将被视为具有潜在治疗价值的目标，并将使用以下方法进行验证 CRISPR/Cas9 基因组工程和功能测定结果将代表第一个也是唯一的数据。据我们所知，努力利用已知的尤文肉瘤风险差异研究 EWS-FLI1 结合和下游效应此外，我们将提供基因组规模的数据。我们的研究制作并免费分发我们的 iPSC 模型，供尤文肉瘤研究人员更广泛使用。