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.

项目总结/摘要 胶质母细胞瘤（GBM）是最常见的原发性恶性脑肿瘤， 在过去的几十年里，尽管有积极的治疗选择。更好地了解其内在机制 GBM是设计更有效的治疗策略所必需的。染色体外DNA（ecDNAs）是一种 众所周知的致癌基因扩增机制，促进肿瘤快速生长。虽然发现 几十年前，最近的技术进步已经更详细地描述了ecDNAs，并表明， 它们在许多癌症类型中是常见的。在一项泛癌症分析中， 发现ecDNA扩增的患者的生存期明显短于肿瘤有其他扩增的患者。 放大的类型。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 从这些细胞系产生的异种移植小鼠模型显示， 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 这使其成为ecDNA患病率最高的癌症。使ecDNA产生的机制 GBM的这种高比率开始被揭示;然而，目前没有模型系统可以因果地 分析特定基因在它们产生过程中的作用。因此，在目标1中，我们将利用耐药性 这一机制允许ecDNA生成从原代患者来源的GBM细胞发展克隆系， 模型ecDNA生成。这些系统将是同基因的（即，无/前和有/后ecDNA生成） 和克隆以最小化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的克隆的知识，结合 对感兴趣的基因的期望操作允许评估这些基因在产生中的因果关系 过程使用这些模型系统开发，我们将测试目标2中的假设，即DNA修复机制是 这是ecDNA生成所必需的，具有控制该过程的特定DNA修复基因。人们日益 证据表明，DNA修复涉及ecDNA的产生。此外，DNA修复作为一个目标正在积极研究 对于GBM治疗，由于其在通过修复GBM的标准治疗中的耐药性方面的作用得到了充分的证明， 治疗引起的DNA损伤关键DNA修复基因在ecDNA生成中的因果作用将被分析 通过确定CRISPRi诱导的这些基因的阻遏对ecDNA生成的影响。我们预计 该项目的发现将有助于确定ecDNA的产生过程和治疗潜力， 靶向GBM中的ecDNA，并对具有高ecDNA患病率的其他癌症产生影响。