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.

摘要 尤文肉瘤的生存率几十年来没有提高，尽管长期以来人们都知道它的单一驱动体细胞 EWS-FLI 1融合癌蛋白。作为一种异常转录因子，EWS-FLI 1改变了 数千个基因的表达制定了一个复杂的程序，对大多数细胞类型有毒，以至于细胞 尤文肉瘤的起源仍有争议。尤文肉瘤的发生率明显较高（大约10- 与主要是非洲血统的儿童相比，欧洲血统的儿童增加了一倍），而拉丁裔儿童则增加了一倍 亚裔儿童患尤文肉瘤的风险大约是白色儿童的一半。事实上， 最强的已知风险因素尤文肉瘤，但这些差异的分子基础的风险已被 只是谨慎地调查。由于EWS-FLI 1与基因组相互作用的机制已经成为 显然，使用适当的模型来研究基因组祖先在一般情况下和特定基因座上是可行的， 调节EWS-FLI 1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-FLI 1引入诱导多能干细胞（iPSC）的衍生物中， 以表征EWS-FLI 1表达的下游分子和功能后果。使用 我们从几个大型干细胞库中获得了可访问的变体阵列数据， 来自欧洲、非洲和美洲血统的个体。我们将介绍EWS-FLI 1 在与尤文肉瘤发生相关的中间发育阶段表达-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 来自仅具有欧洲血统的受试者的相应样本。在全球范围内，我们将研究基因是否 表达和染色质状态表现出与Ewing表达特征的相似性， 祖先百分比将分析EWS-FLI 1的全基因组染色质占有率，并分析其与 使用长读测序定义的本地祖先。EWS-FLI 1在小鼠中差异影响的基因 “宽容”（欧洲血统）、“适度宽容”（美洲血统）和“宽容”（非洲血统） 祖先）基因组将被视为具有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。得到的数据将代表第一个也是唯一一个 努力，据我们所知，利用已知的风险差异尤文肉瘤的祖先， 研究EWS-FLI 1结合和下游效应。此外，我们将提供基因组规模的数据， 并免费分发我们的iPSC模型，供尤文肉瘤研究人员更广泛地使用。