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.

抽象的尤因肉瘤的生存在数十年中没有改善目的地的奇异驾驶躯体知识。突变，有害的EWS-FLI1融合癌蛋白。作为异常转录因子EWS-FLI1改变表达成千上万的基因制定了对大多数细胞类型有毒的复杂程序的表达，以至于细胞尤因肉瘤的起源仍然是一个辩论问题。尤因肉瘤的发生率较高（大约10-- 与非洲初级血统相比，欧洲血统的孩子的折叠），拉丁裔儿童比白人儿童大约有⅔和亚洲儿童½的风险。祖先实际上是尤因肉瘤的最强已知危险因素，但是这些风险差异的分子基础已经是只有很少的调查。随着EWS-FLI1与基因组相互作用的机制已成为很明显，可以使用适当的模型来研究基因组血统通常是如何在特定基因座的调节EWS-FLI1活性，包括其对表观遗传和转录程序的下游影响。到我们已经设计了一种在诱导多能干细胞（IPSC）衍生物中引入EWS-FLI1的策略。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无访问变体阵列数据，我们确定了bank iPSC系列来自拥有一系列欧洲，非洲和阿米林族人的个人。我们将介绍EWS-FLI1 与尤因肉瘤发生相关的发育中间阶段的表达 - 即神经rest细胞和间充质干细胞。功能公差和分子状态的度量将被比较来自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表现出与尤林表达的相似性与欧洲的比例祖先百分比。 EWS-FLI1的全基因组染色质占用率将进行介绍，并与其关系使用长阅读测序定义的本地血统。受EWS-FLI1的影响不同的基因 “允许的”（欧洲血统），“适度允许”（Amerind Ancestry）和“无通常”（非洲人祖先）基因组将被视为潜在治疗价值的靶标，并将使用 CRISPR/CAS9基因组工程和功能测定。由此产生的数据将代表第一个也是据我们所知研究EWS-FLI1结合和下游效应。此外，我们将提供基因组规模数据由我们的研究生产和免费分发了我们的IPSC模型，以供Ewing肉瘤研究人员更广泛使用。