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.

项目概要/摘要 胶质母细胞瘤（GBM）是最常见的原发性恶性脑肿瘤，患者生存率几乎没有改善 尽管采取了积极的治疗方法，但在过去的几十年里。更好地理解底层机制 GBM 对于设计更有效的治疗策略是必要的。染色体外 DNA (ecDNA) 是 促进肿瘤快速生长的众所周知的癌基因扩增机制。虽然被发现 几十年前，最近的技术进步已经更详细地描述了 ecDNA 的特征，并表明 它们在许多癌症类型中都很常见。在一项泛癌分析中，患有肿瘤的患者 发现 ecDNA 扩增的患者的生存期明显短于肿瘤具有其他扩增的患者 放大类型。最近的一项研究使用 GBM 患者肿瘤组织、其衍生细胞系和原位 从这些品系生成的异种移植小鼠模型表明，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 GBM 肿瘤含有 ecDNA，使其成为 ecDNA 患病率最高的癌症。使 ecDNA 生成的机制 GBM 如此高的发病率已经开始显现；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，我们将利用耐药性 允许 ecDNA 生成从原代患者来源的 GBM 细胞发展出克隆系的机制 模型 ecDNA 生成。这些系统将是同基因的（即，没有/之前和有/之后 ecDNA 生成） 和克隆以尽量减少 ecDNA 水平的细胞间异质性。克隆前后的采购 ecDNA 生成，以及能够以这种方法生成 ecDNA 的克隆知识，结合 对感兴趣的基因进行所需的操作可以评估这些基因在一代中的因果关系 过程。使用这些开发的模型系统，我们将测试目标 2 中的假设，即 DNA 修复机制是 具有控制该过程的特定 DNA 修复基因的 ecDNA 生成是必需的。有越来越多 证据表明 ecDNA 生成中存在 DNA 修复。此外，DNA修复作为目标正在积极研究 用于 GBM 治疗，因为它通过修复 GBM 在抵抗 GBM 标准治疗方面发挥着重要作用 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 诱导的这些基因抑制对 ecDNA 生成的影响。我们期望 该项目的研究结果将有助于确定 ecDNA 生成过程和治疗潜力 靶向 GBM 中的 ecDNA 对其他具有高 ecDNA 患病率的癌症具有影响。