Regulation and retention of extrachromosomal oncogene amplifications in cancer

项目总结 癌基因扩增是癌症进展的关键驱动因素，它可以发生在染色体内，也可以通过 形成环状染色体外DNA(EcDNA)。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，并与不良的患者预后有关。尽管ecDNA很普遍- 在癌症中介导的癌基因扩增，我们对癌基因表达的了解有限 对ecDNA的调控以及这些分子在癌细胞中的维持方式。我的目标是阐明 癌基因在癌症中利用多重遗传扰动在ecDNA上被扩增和失调， 癌细胞系模型中的表观基因组图谱和新的基因筛选方法。在F99阶段，我将 系统鉴定携带ecDNA的癌基因的独特转录依赖关系。EcDNA被链接到 高水平的癌基因过度表达和可及染色质，表明癌基因在 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ecDNA与一个 另一种则在间期核内参与协同、分子间癌基因的激活。这些 观察表明，染色体外癌基因的表达受到不同程度的调控。 将扩增产物与染色体基因座进行比较。我假设ecDNA上基因表达的差异调控 取决于独特的转录调控因子。我将结合使用流式细胞术，CRISPR筛查， 通过扰动序列的单细胞转录组学和批量表观基因组图谱鉴定独特的转录 在一组癌细胞和患者来源的神经球中，癌基因的调节因子在ecDNA上放大。 在K00阶段，我将阐明携带ecDNA的癌基因在癌细胞中保留的机制。 EcDNA缺乏着丝粒，在细胞分裂过程中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仍被保留，甚至在 肿瘤发生，表明ecDNA分离的机制尚未确定。令人惊讶的是，活细胞成像 在有丝分裂期间显示了ecDNA与染色体的强烈共定位，这表明ecDNA可能能够 在缺乏着丝粒的情况下，选择染色体分离机制。我假设特定的基因 EcDNA上的元件能够在癌症过程中搭便车到染色体上，以便分割成子核 细胞分裂。我建议确定DNA元件和蛋白质介体，使ecDNA能够保留使用 基于Episome的遗传筛选、有丝分裂染色质构象捕获、邻近蛋白质组学、CRISPR 表观基因组数据集的筛选和整合。共同阐明了染色体外染色体异常的发生机制 癌基因上调和扩增子在癌细胞中的滞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的调节，最终影响肿瘤的生长。

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.