Development and Application of Model Systems to Study Extrachromosomal DNA Generation in Glioblastoma

项目摘要/摘要 胶质母细胞瘤(GBM)是最常见的原发恶性脑肿瘤，对患者的生存影响甚微。 在过去的几十年里，尽管有积极的治疗选择。更好地理解潜在的机制 GBM对于设计更有效的治疗策略是必要的。染色体外DNA(EcDNA)是一种 众所周知的癌基因扩增促进肿瘤快速生长的机制。虽然发现了 几十年前，最近的技术进步已经更详细地表征了ecDNA，并表明 它们在许多癌症类型中都是常见的。在一项泛癌症分析中，肿瘤有 研究发现，ecDNA扩增的患者比其他肿瘤患者的生存期要短得多。 扩增的类型。最近一项利用GBM患者肿瘤组织及其衍生细胞系和原位移植进行的研究 从这些品系产生的异种移植小鼠模型显示，大多数癌基因扩增 这些系统是染色体外的。事实上，研究表明，大约60%的基底膜肿瘤含有 EcDNA，使其成为ecDNA患病率最高的癌症。使ecDNA能够在 GBM中如此高的比率开始被揭示；然而，目前还没有模型系统可用于因果关系 分析特定基因在其世代中的作用。因此，在目标1中，我们将利用抗药性 一种机制，允许ecDNA产生从原代患者来源的GBM细胞发展克隆系到 模型ecDNA生成。这些系统将是等基因的(即，没有/之前和有/之后产生ecDNA) 以及克隆，以最大限度地减少ecDNA水平上的细胞间异质性。在采购克隆之前和之后 EcDNA生成，以及能够在这种方法中生成ecDNA的克隆的知识，与 对感兴趣基因的期望操作允许评估这些基因在世代中的因果关系 进程。使用开发的这些模型系统，我们将检验目标2中的一个假设，即DNA修复机制是 对于具有控制这一过程的特定DNA修复基因的ecDNA生成是必要的。有越来越多的 在ecDNA生成中涉及DNA修复的证据。此外，DNA修复正在被作为一个目标而积极研究 对于GBM的治疗，因为它在通过修复 治疗导致的DNA损伤。将分析关键的dna修复基因在ecdna生成中的因果作用。 通过确定CRISPRi诱导的这些基因的抑制对ecDNA生成的影响。我们期待着 该项目的发现将有助于确定ecDNA的产生过程和治疗潜力。 针对GBM中的ecDNA，以及对其他ecDNA患病率较高的癌症的影响。

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is the most common primary malignant brain tumor with little improvement in patient survival in past few decades despite aggressive treatment options. Better understanding of the mechanisms underlying GBM is necessary to design more effective therapeutic strategies. Extrachromosomal DNAs (ecDNAs) are a well-known mechanism of oncogene amplification that promotes rapid tumor growth. Although discovered decades ago, recent technological advances have characterized ecDNAs in finer detail and have shown that they are a common occurrence in many cancer types. In a pan-cancer analysis, patients whose tumors had ecDNA amplifications were found to have significantly shorter survival than patients whose tumors had other types of amplifications. A recent study using GBM patient tumor tissues, their derivative cell lines, and orthotopic xenograft mouse models generated from these lines, showed that the majority of oncogene amplifications in these systems are extrachromosomal. Indeed, studies have shown that around 60% of GBM tumors contain ecDNAs, making it the cancer with highest ecDNA prevalence. Mechanisms that enable ecDNA generation at such high rates in GBM are starting to be revealed; however, no model systems are currently available to causally analyze the role of specific genes in their generation. In Aim 1, we will therefore utilize a drug resistance mechanism that allows ecDNA generation to develop clonal lines from primary patient-derived GBM cells to model ecDNA generation. These systems will be isogenic (i.e., without/before and with/after ecDNA generation) and clonal to minimize intercellular heterogeneity in ecDNA levels. Procurement of clones before and after ecDNA generation, and knowledge of clones that are able to generate ecDNAs in this approach, combined with desired manipulations of genes of interest permits assessment of causality of those genes in the generation process. Using these model systems developed, we will test a hypothesis in Aim 2 that DNA repair machinery is necessary for ecDNA generation with specific DNA repair genes governing the process. There is increasing evidence implicating DNA repair in ecDNA generation. Moreover, DNA repair is actively being studied as a target for GBM treatment due to its well-documented role in resistance to standard therapy for GBM via repair of therapy-induced DNA damage. The causal role of key DNA repair genes in ecDNA generation will be analyzed by determining the effects of CRISPRi-induced repression of these genes on ecDNA generation. We expect that the findings from this project will help define the ecDNA generation process and the therapeutic potential of targeting ecDNA in GBM with implications for other cancers with high ecDNA prevalence.