Extrachromosomal DNA as a Targetable Mechanism in Glioblastoma

项目摘要/摘要 该项目的目标是确定染色体外dna(Ecdna)对肿瘤进化的影响。 并验证基于ecDNA抑制的潜在治疗策略。的存在 EcDNA是由Arthur Spriggs在五十多年的时间里通过开创性的细胞病理学研究首次发现的 那是以前的事了。我们知道ecDNA在很大程度上是一种肿瘤细胞现象，可以在近一半的固体中检测到 癌症，并频繁表达癌基因，使其成为放大癌基因表达的载体 以及随之而来的肿瘤形成。我们对胶质母细胞瘤的研究扩展了这一知识，表明ecDNA是 高度普遍，并显著增加肿瘤内基因组的异质性。尽管有这样的知识， 对ecDNA驱动的肿瘤发生机制的研究基本上是不存在的。推进ecDNA 研究到下一个层次的理解，我们现在必须定义它的起源、复制机制和 将其作为治疗目标的策略。然而，目前这类研究面临着重大障碍，例如 缺乏分析技术和分子工具来准确地检测和表征这些 致癌元素。这里提出的工作将消除这些取得进展的障碍。我们假设 EcDNA推动肿瘤发生超越了其作为癌基因激活剂的作用，以及ecDNA的机制 形成和复制可作为潜在的治疗靶点。为了检验我们的假设，我们将开发 方法在活细胞中可视化和示踪ecDNA，并使用这些工具来确定ecDNA的作用机制 肿瘤内的定位和转移。我们将从功能上评估ecDNA介导的顺式调控 可以通过调控非ecDNA基因转录来改变肿瘤细胞的行为。最后，我们将探索 药物耗尽ecDNA是否影响细胞和动物肿瘤的生长和维持 模型系统。我们的具体目标是：1)开发基于CRISPR的实时成像跟踪工具 EcDNA动力学；2)ecDNA介导的癌基因激活的发现和功能询问；3) 通过抑制核糖核苷酸还原酶(RNR)来靶向ecDNA，RNR是负责产生 脱氧核糖核酸三磷酸是常染色体和染色体外DNA的组成成分。 影响：该项目基于ecDNA是癌症特异性和癌症的概念创新- 使基因组改变成为可能。尽管几十年前就认识到ecDNA，但这些结构的功能 而如何有效地瞄准它们的起源，实际上是一个未知的领域。除了开发新的、广泛的 适用于ecDNA研究的工具，这项工作的结果将提供对特征的新见解， EcDNA的行为和复制机制，并将评估ecDNA是否是未开发的癌细胞 可以从治疗上利用的脆弱性，以造福患者。

PROJECT SUMMARY/ABSTRACT The goal of this project is to determine the influence of extrachromosomal DNA (ecDNA) on tumor evolution and resistance and to validate a potential therapeutic strategy based on ecDNA inhibition. The existence of ecDNA was first recognized through pioneering cytopathology studies by Arthur Spriggs over five decades ago. We know that ecDNAs are largely a phenomenon of tumor cells, can be detected in nearly half of all solid cancers, and frequently express oncogenes, making them vehicles for amplification of oncogene expression and consequent tumorigenesis. Our studies in glioblastoma extend this knowledge, showing that ecDNAs are highly pervasive and significantly increase intra-tumoral genomic heterogeneity. Despite this knowledge, research into the mechanisms of ecDNA-driven tumorigenesis is largely non-existent. To advance ecDNA research to the next level of understanding, we must now define its origins, replication mechanisms, and strategies to target it for treatment. At this time, however, there are significant barriers to such studies, such as the absence of profiling technologies and molecular tools to accurately detect and characterize these oncogenic elements. The work proposed here will remove these barriers to progress. We hypothesize that ecDNAs drive tumorigenesis beyond their role as oncogene activators, and that mechanisms of ecDNA formation and replication could serve as potential therapeutic targets. To test our hypothesis, we will develop methods to visualize and trace ecDNA in live cells and use these tools to define the mechanisms of ecDNA localization and transfer within a tumor. We will functionally evaluate whether ecDNA mediated cis-regulation of non-ecDNA gene transcription can be modulated to change tumor cell behavior. Finally, we will explore whether pharmacological depletion of ecDNA affects tumor growth and maintenance in cellular and animal model systems. Our Specific Aims are to: 1) Develop CRISPR-based tracing tools for real-time imaging of ecDNA dynamics; 2) Discovery and functional interrogation of ecDNA mediated oncogene activation; and 3) Target ecDNA through inhibition of ribonucleotide reductase (RNR), the enzyme responsible for generating the deoxyribonucleotide triphosphates that are the building blocks of autosomal and extrachromosomal DNA. Impact: This project is based on the conceptual innovation that ecDNAs are cancer-specific and cancer- enabling genomic alterations. Despite the recognition of ecDNA decades ago, the function of these structures and how to effectively target their genesis, is virtually uncharted territory. In addition to developing new, broadly applicable, tools for ecDNA studies, the results of this work will provide novel insights into the characteristics, behavior and replicative mechanism of ecDNA, and will assess whether ecDNA is an unexploited cancer cell vulnerability that can be therapeutically exploited for patient benefit.