Investigating the roles of oncogenic extrachromosomal circular DNAs in cancer

摘要 焦点扩增介导的癌基因表达增加是癌基因的常见机制 在人类癌症中的激活。导致癌基因扩增的两种主要机制已被描述： 染色体扩增和非染色体扩增。后一种机制的特点是 存在被认为起源于碎片的多个环状DNA拷贝，以及 随后染色体片段的循环。这些“染色体外环状DNA”(EcDNA)有 长期以来，因为它们出现在中期伸展和缺乏着丝粒而被称为“双分钟”。 序列。 在过去的几年里，人们对这类与癌症相关的染色体重排重新产生了兴趣 由于技术进步和认识到，由于它们在有丝分裂时的随机分离，ecDNA 可以加速肿瘤演变，调节耐药性，并通常促进更具侵袭性的表型。 然而，尽管取得了实质性进展，但关于ecDNA的生物学、其动态的几个关键问题 在肿瘤形成的早期阶段，以及它们对肿瘤发生和发展的贡献仍然存在 无人接听。这在一定程度上是由于缺乏有效的手段来设计和跟踪正常细胞和 在模型生物体中。 我们的团队在生殖系和体细胞小鼠模型的产生和特征方面拥有广泛的专业知识 我们已经率先使用体细胞基因组编辑来设计染色体 小鼠体内的重排。在这项拨款申请中，我们描述了一种由强大的初步数据支持的新颖的 在细胞和小鼠中模拟ecDNA的一般策略。 我们已经产生了三个新的基因工程小鼠品系，在这些品系中，ecDNA的形成 含有在人类癌症中最常见扩增的癌基因，可以在时间上和 空间控制的方式。使用类似的策略，我们也产生了细胞系，其中形成了 利用荧光报告分子和可选择的标记可以非侵入性地诱导和跟踪特定的ecDNA。 我们建议使用这些创新工具和试剂来解决以下关键问题： 1)致癌的ecDNA能否在体内启动肿瘤的形成和/或加速肿瘤的进展和转移？ 2)致癌的ecDNA如何响应细胞内外环境的变化？ 3)在原代细胞中是否有阻止ecDNA形成和繁殖的机制？ 4)癌细胞中ecDNA的存在是否与独特的治疗上可操作的脆弱性有关？ 5)ecDNA能在细胞间水平传递吗？

ABSTRACT Increased oncogene expression mediated by focal amplifications is a common mechanism for oncogene activation in human cancers. Two major mechanisms leading to oncogene amplification have been described: chromosomal amplification and non-chromosomal amplification. The latter mechanism is characterized by the presence of multiple copies of circular DNAs that are thought to originate following the fragmentation and subsequent circularization of pieces of chromosomes. These “extrachromosomal circular DNAs” (ecDNAs) have long been known as “double minutes” for their appearance in metaphase spreads and by the lack of centromeric sequences. In the past few years renewed interest in this class of cancer-associated chromosomal rearrangements has been fueled by technological advances and by the realization that, due to their random segregation at mitosis, ecDNAs can accelerate tumor evolution, mediate drug resistance, and generally promote a more aggressive phenotype. Despite substantial progress, however, several key questions regarding the biology of ecDNAs, their dynamics during the early stages of tumor formation, and their contribution to tumor initiation and progression, remain unanswered. This is in part due to the lack of effective means to engineer and track ecDNAs in normal cells and in model organisms. Our group has extensive expertise in the generation and characterization of germline and somatic mouse models of human cancers, and we have pioneered the use of somatic genome editing to engineer chromosomal rearrangements in mice. In this grant application, supported by strong preliminary data, we describe a novel general strategy to model ecDNAs in cells and in mice. We have already generated three new genetically engineered mouse strains in which the formation of ecDNAs containing the oncogenes most commonly amplified in human cancers can be induced in a temporally and spatially controlled manner. Using a similar strategy, we have also generated cell lines in which formation of specific ecDNAs can be induced and tracked non-invasively using fluorescent reporters and selectable markers. We propose to use these innovative tools and reagents to address the following key questions: 1) Can oncogenic ecDNAs initiate tumor formation and/or accelerate tumor progression and metastasis in vivo? 2) How do oncogenic ecDNAs respond to changes in intracellular and extracellular environment? 3) Are there mechanisms preventing ecDNA formation and propagation in primary cells? 4) Is the presence of ecDNAs in cancer cells associated with unique therapeutically actionable vulnerabilities? 5) Can ecDNAs be transmitted horizontally between cells?