Extrachromosomal DNA as a Targetable Mechanism in Glioblastoma

项目总结/摘要 本项目的目标是确定染色体外DNA（ecDNA）对肿瘤演变的影响 和抗性，并验证基于ecDNA抑制的潜在治疗策略。的存在 ecDNA最初是由亚瑟斯普里格斯在50多年的开创性细胞病理学研究中认识到的 前我们知道，ecDNAs在很大程度上是肿瘤细胞的一种现象，可以在近一半的实体中检测到。 癌症，并经常表达癌基因，使它们成为癌基因表达扩增的载体 以及随后的肿瘤发生。我们在胶质母细胞瘤中的研究扩展了这一知识，表明ecDNAs是 且显著增加肿瘤内基因组异质性。尽管有这些知识， 对ecDNA驱动的肿瘤发生机制的研究基本上不存在。为了推进ecDNA 为了使研究达到下一个层次的理解，我们现在必须确定其起源，复制机制， 有针对性的治疗策略。然而，目前，此类研究存在重大障碍，例如 缺乏分析技术和分子工具来准确检测和表征这些 致癌元素这里提出的工作将消除这些阻碍进展的障碍。我们假设 ecDNA驱动肿瘤发生超出其作为癌基因激活剂作用， 形成和复制可以作为潜在的治疗靶点。为了验证我们的假设，我们将开发 可视化和追踪活细胞中ecDNA的方法，并使用这些工具来定义ecDNA的机制 肿瘤内的定位和转移。我们将从功能上评估ecDNA介导的顺式调节是否 可以调节非ecDNA基因转录以改变肿瘤细胞行为。最后，我们将探讨 药物消耗ecDNA是否影响细胞和动物中的肿瘤生长和维持 模型系统我们的具体目标是：1）开发基于CRISPR的跟踪工具，用于实时成像， ecDNA动力学; 2）ecDNA介导的癌基因激活的发现和功能询问;和3） 通过抑制核糖核苷酸还原酶（RNR）靶向ecDNA，RNR是负责产生 脱氧核糖核苷酸三磷酸是常染色体和染色体外DNA的结构单元。 影响：该项目基于ecDNAs是癌症特异性的概念创新， 使基因组发生改变。尽管几十年前就认识到了ecDNA，但这些结构的功能 以及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新的见解的特点， 行为和复制机制，并将评估ecDNA是否是一个未开发的癌细胞 脆弱性，可以治疗利用病人的利益。

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.