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

• 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
• 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

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 会改变 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- 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 与白人儿童相比，亚洲儿童患尤文肉瘤的风险大约为 ⅔，亚洲儿童则为 1/2。血统实际上是 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 只进行了少量调查。随着 EWS-FLI1 与基因组相互作用的机制变得 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。到 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。使用 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 和间充质干细胞。功能耐受性和分子状态的测量将与 来自纯欧洲血统的受试者的相应样本。在全球范围内，我们将检查基因是否 表达和染色质状态与尤因表达特征相似，与欧洲人成比例 血统百分比。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本地血统。受 EWS-FLI1 不同影响的基因 “宽容”（欧洲血统）、“适度宽容”（美洲人血统）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。结果数据将代表第一个也是唯一一个 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。