Regulation and retention of extrachromosomal oncogene amplifications in cancer

癌症中染色体外癌基因扩增的调节和保留

• 批准号: 10529994
• 负责人: King L. Hung
• 金额: $ 4.11万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10529994
• 关键词: Affect; Binding Proteins; CRISPR screen; Cancer cell line; Cell Nucleus; Cell Separation; Cell Survival; Cell division; Cells; Centromere; ChIP-seq; Chemicals; Chromatin; Chromosomes; DNA; Data; Data Set; Daughter; Dependence; Development; Elements; Epigenetic Process; Episome; Flow Cytometry; Gene Expression Profile; Gene Expression Regulation; Genetic; Genetic Screening; Genetic Transcription; Genetic screening method; Goals; Image; Interphase; Knock-out; Libraries; Link; Maintenance; Malignant Neoplasms; Mediating; Mediator of activation protein; Methods; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular Conformation; Neurosphere; Nuclear Proteins; Oncogene Activation; Oncogenes; Patient-Focused Outcomes; Patients; Phase; Postdoctoral Fellow; Prevalence; Proteins; Proteomics; RNA; Regulation; Research; Sampling; Scientist; Shotguns; Structure; Techniques; Therapeutic; Training; Tumor-Derived; Up-Regulation; Work; anticancer research; base; cancer cell; cancer genome; cancer type; career; chromosome conformation capture; epigenetic profiling; epigenome; epigenomics; experimental study; extrachromosomal DNA; genetic element; genetic profiling; insight; live cell imaging; novel; overexpression; pre-doctoral; segregation; transcriptome; transcriptomics; tumor growth; tumor progression; tumorigenesis; whole genome

PROJECT SUMMARY Oncogene amplification is a key driver of cancer progression and can either occur within chromosomes or via formation of circular extrachromosomal DNA (ecDNA). ecDNA is detected in a quarter of cancer samples and half of all cancer types, and is associated with poor patient outcomes. Despite the prevalence of ecDNA- mediated oncogene amplification in cancer, we have a limited understanding of how oncogene expression is regulated on ecDNAs and how these molecules are maintained in cancer cells. My goal is to elucidate how oncogenes are amplified and dysregulated on ecDNA in cancer using multiplexed genetic perturbations, epigenomic profiling and novel genetic screening methods in cancer cell line models. In the F99 phase, I will systematically identify unique transcriptional dependencies of ecDNA-harbored oncogenes. ecDNA is linked to high levels of oncogene overexpression and accessible chromatin, suggesting that oncogene expression on ecDNA may be uniquely regulated. In my dissertation work so far, I have used imaging, chromatin conformation and epigenetic perturbation approaches to discover a novel mechanism by which ecDNAs cluster with one another in the interphase nucleus and engage in cooperative, intermolecular oncogene activation. These observations suggest that oncogene expression is regulated differently on extrachromosomal oncogene amplicons compared to chromosomal loci. I hypothesize that differential regulation of gene expression on ecDNA depends on unique transcriptional regulators. I will use a combination of flow cytometry, CRISPR screening, single-cell transcriptomics by Perturb-seq, and bulk epigenomic profiling to identify unique transcriptional regulators of oncogenes amplified on ecDNA in a panel of cancer cell lines and patient-derived neurospheres. In the K00 phase, I will elucidate the mechanism of retention of ecDNA-harbored oncogenes in cancer cells. ecDNA lacks centromeres and is uncoupled from the mitotic spindle during cell division. Therefore, ecDNA segregates randomly between daughter nuclei. Nevertheless, ecDNA is retained, and even selected for, during tumorigenesis, suggesting an uncharacterized mechanism for ecDNA segregation. Surprisingly, live cell imaging during mitosis showed strong colocalization of ecDNA with chromosomes, suggesting that ecDNAs may be able to co-opt chromosomal segregation mechanisms despite lacking centromeres. I hypothesize that specific genetic elements on ecDNA enable hitchhiking onto chromosomes in order to partition into daughter nuclei during cancer cell division. I propose to identify DNA elements and protein mediators that enable retention of ecDNAs using a shotgun episome-based genetic screen, mitotic chromatin conformation capture, proximity proteomics, CRISPR screening and integration of epigenomic datasets. Together, elucidating the mechanisms of extrachromosomal oncogene upregulation and amplicon retention in cancer cells will reveal potential therapeutic opportunities for ecDNA+ cancers. It will also provide important insights into how organization of the cancer genome and altered epigenome affect regulation of oncogene expression and ultimately tumor growth.

项目摘要 癌基因扩增是癌症进展的关键驱动因素，可以发生在染色体内或通过染色体上。 环状染色体外DNA（ecDNA）的形成。在四分之一的癌症样本中检测到ecDNA， 占所有癌症类型的一半，并与患者预后不良相关。尽管ecDNA的流行- 虽然我们对癌基因的表达是如何影响癌细胞增殖的了解有限， 以及这些分子如何在癌细胞中维持。我的目标是阐明 癌基因在癌症中的ecDNA上使用多重遗传扰动被扩增和失调， 表观基因组分析和新的遗传筛选方法在癌细胞系模型。在F99阶段，我将 系统地鉴定具有ecDNA的癌基因的独特转录依赖性。ecDNA与 高水平的癌基因过表达和可接近的染色质，表明癌基因表达在 ecDNA可能受到独特的调控。到目前为止，在我的论文工作中，我已经使用了成像，染色质构象， 和表观遗传扰动方法来发现一种新的机制，通过这种机制， 另一个位于间期核中，参与合作的分子间癌基因激活。这些 观察结果表明，染色体外癌基因的表达受到不同的调节， 与染色体基因座相比。我假设ecDNA上基因表达的差异调节 依赖于独特的转录调节因子我将联合使用流式细胞术，CRISPR筛选， 通过Perturb-seq的单细胞转录组学和批量表观基因组分析来鉴定独特的转录 在一组癌细胞系和患者来源的神经球中，在ecDNA上扩增的癌基因的调节子。 在K 00阶段，我将阐明ecDNA携带的癌基因在癌细胞中保留的机制。 ecDNA缺乏着丝粒，并且在细胞分裂期间与有丝分裂纺锤体解偶联。因此，ecDNA 在子核之间随机分离。尽管如此，ecDNA被保留，甚至被选择， 肿瘤发生，这表明一个未知的机制ecDNA分离。令人惊讶的是，活细胞成像 在有丝分裂过程中显示出强烈的ecDNA与染色体共定位，这表明ecDNAs可能能够 尽管缺乏着丝粒，但仍采用染色体分离机制。我假设特定的基因 ecDNA上的元件能够搭便车到染色体上，以便在癌症期间划分成子细胞核。 细胞分裂我建议使用一种新的方法来鉴定能够保留ecDNAs的DNA元件和蛋白质介质。 基于鸟枪附加体的遗传筛选、有丝分裂染色质构象捕获、邻近蛋白质组学、CRISPR 筛选和整合表观基因组数据集。总之，阐明了染色体外 癌细胞中癌基因上调和扩增子保留将揭示潜在的治疗机会， ecDNA+癌症。它还将为癌症基因组的组织和改变提供重要的见解。 表观基因组影响癌基因表达调节并最终影响肿瘤生长。