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

胶质母细胞瘤染色体外 DNA 生成研究模型系统的开发和应用

• 批准号: 10647263
• 负责人: Amit Gujar
• 金额: $ 8.38万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-18 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647263
• 关键词: Biological Models; CRISPR interference; Cell Line; Cells; Circular DNA; Communities; Cryopreservation; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Inhibition; DNA Repair Pathway; DNA amplification; DNA analysis; DNA biosynthesis; Dependence; Development; Diagnosis; Dihydrofolate Reductase; Disease; Drug resistance; Etiology; Future; Gene Amplification; Generations; Genes; Glioblastoma; Goals; Growth; Heterogeneity; Knowledge; Malignant Neoplasms; Malignant neoplasm of brain; Methotrexate; Modeling; Molecular; Monitor; Normal Cell; Normal tissue morphology; Oncogenes; Pathway interactions; Patient-Focused Outcomes; Patients; Play; Prevalence; Process; Proliferating; Protocols documentation; Repression; Research; Resistance; Resources; Role; System; Technology; Testing; Therapeutic; Transcription Repressor; Tumor Tissue; Xenograft procedure; aggressive therapy; biobank; brain cell; cancer cell; cancer therapy; cancer type; design; extrachromosomal DNA; gene induction/repression; genetic manipulation; improved; insight; interest; loss of function; mouse model; neoplastic cell; novel; repaired; resistance mechanism; response; therapeutic target; therapeutically effective; tool; tumor; tumor growth

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.

项目摘要/摘要 胶质母细胞瘤（GBM）是最常见的原发性恶性脑肿瘤，患者存活率几乎没有改善 在过去的几十年中，尽管有积极的治疗选择。更好地理解机制 GBM对于设计更有效的治疗策略是必要的。外染色体DNA（ECDNA）是一个 促进肿瘤快速生长的癌基因扩增的机制。虽然被发现 几十年前，最近的技术进步已经用细节来表征ECDNA，并表明 在许多癌症类型中，它们是常见的情况。在泛伴侣分析中，肿瘤患者 发现ECDNA扩增的生存率明显短于肿瘤具有其他的患者 扩增的类型。最近使用GBM患者肿瘤组织，其衍生细胞系和原位的一项研究 从这些线产生的异种移植小鼠模型表明，大多数癌基因在 这些系统是外染色体。实际上，研究表明，大约60％的GBM肿瘤包含 ECDNA，使其成为ECDNA患病率最高的癌症。使eCDNA生成的机制 GBM中如此高的比率开始揭示。但是，目前尚无因果系统可用的模型系统 分析特定基因在其一代中的作用。在AIM 1中，我们将使用耐药性 允许eCDNA产生从主要患者衍生的GBM细胞发展的克隆线的机制 模型ECDNA产生。这些系统将是同等基因的（即，没有/之前和之后ecdNA产生的情况） 克隆以最大程度地减少ECDNA水平的细胞间异质性。在之前和之后采购克隆 ECDNA的产生以及能够在这种方法中产生ecdnas的克隆的知识，并结合 所需的感兴趣基因的操纵允许评估这些基因的因果关系 过程。使用这些模型系统，我们将在AIM 2中检验一个假设，即DNA修复机械是 使用该过程的特定DNA修复基因产生ECDNA所必需的。有在增加 涉及DNA生成中DNA修复的证据。此外，DNA修复正在积极研究为目标 GBM治疗是由于其在抗GBM的标准治疗中有据可查的作用 治疗引起的DNA损伤。将分析关键DNA修复基因在ECDNA生成中的因果作用 通过确定CRISPRI诱导的这些基因对ECDNA产生的影响。我们期望这一点 该项目的发现将有助于定义eCDNA生成过程和治疗潜力 靶向GBM中的ECDNA，对其他高ECDNA患病率的癌症的影响。