Investigation of ecDNA as a driver of intratumoral heterogeneity and treatment resistance in high-risk medulloblastoma

该提案的总体目标是研究环状染色体外DNA（ecDNA）作为一种潜在的 髓母细胞瘤中肿瘤内异质性和治疗抵抗的驱动因素，最常见的儿科 恶性脑瘤肿瘤内异质性是治疗耐药的主要决定因素之一 和治疗失败，是癌症患者总体生存率低的主要原因之一。但 ecDNA作为髓母细胞瘤中肿瘤异质性和治疗抗性驱动因素的功能相关性 几乎没有被研究过。分析ecDNA在不同分子亚组中的临床影响， 在髓母细胞瘤的研究中，我们收集了来自468个国家的多机构全基因组测序数据， 髓母细胞瘤患者样本。使用新的计算方法来检测和重建 我们在82名患者（18%）中发现了ecDNA，并观察到ecDNA的存在与 更糟糕的结果。此外，我们发现个别髓母细胞瘤肿瘤往往具有多个 ecDNA的变体，每个变体含有不同的扩增的癌基因沿着与共扩增的非编码 调节DNA（“增强子”）。基于我们的初步结果，我们提出了核心假设，即ecDNA 导致高危髓母细胞瘤患者的瘤内异质性和治疗抵抗。中央 假设将通过以下三个具体目标进行测试：调查的分子进化 ecDNA作为治疗耐药性的潜在驱动因素（目的1）;评估靶向的组合疗法 针对ecDNA形成和聚集的机制，以减少治疗抗性（目的2）;探索 神经管母细胞瘤肿瘤依赖性的功能抑制的编码和非编码调控DNA共 在ecDNA上扩增（Aim 3）。本申请中提出的研究具有技术性、概念性和 生物学创新，包括使用新型成像技术在单细胞水平上分析ecDNA， 髓母细胞瘤肿瘤和患者来源的异种移植物中的多组单核测序方法 (PDX)模型这项拟议中的研究意义重大，因为目前对儿童的标准治疗 髓母细胞瘤引起发育障碍、神经损伤和继发性转移。小说 迫切需要治疗方法。我们的方法将测试标准治疗的影响 在ecDNA的分子进化和功能测试新的组合治疗针对 ecDNA发生和聚类。这些临床前研究有可能揭示新的机制， ecDNA有助于髓母细胞瘤的发病机制，并确定新的科学线索， 改进治疗方法。我们希望我们的研究能够揭示ecDNA的贡献 突变体对治疗抗性的出现，揭示了它们的选择优势，验证了最近描述的 ecDNA的性质及其治疗敏感性，并确定新的肿瘤依赖性基因 在一些最具侵袭性的髓母细胞瘤肿瘤中的ecDNA扩增。

The overall objective of this proposal is to investigate circular extrachromosomal DNA (ecDNA) as a potential driver of intratumoral heterogeneity and treatment resistance in medulloblastoma, the most common pediatric malignant brain tumor. Intratumoral heterogeneity is one of the leading determinants of therapeutic resistance and treatment failure and one of the main reasons for poor overall survival in cancer patients. However, the functional relevance of ecDNA as a driver of tumor heterogeneity and treatment resistance in medulloblastoma has hardly been studied. To analyze the clinical impact of ecDNA in the different molecular subgroups of medulloblastoma, we assembled a multi-institutional cohort of Whole Genome Sequencing data from 468 medulloblastoma patient samples. Using novel computational methods for the detection and reconstruction of ecDNA, we found ecDNA in 82 patients (18%) and observe that the presence of ecDNA is associated with significantly poorer outcomes. In addition, we find that individual medulloblastoma tumors often harbor multiple variants of ecDNA, each containing different amplified oncogenes along with co-amplified non-coding regulatory DNA (‘enhancers’). Based on our preliminary results, we propose the central hypothesis that ecDNA drives intratumoral heterogeneity and treatment resistance in high-risk medulloblastoma patients. The central hypothesis will be tested through the following three specific aims: To investigate the molecular evolution of ecDNA as a potential driver of treatment resistance (Aim 1); To evaluate combinatorial therapies targeted against mechanisms of ecDNA formation and clustering to reduce treatment resistance (Aim 2); To probe medulloblastoma tumor-dependencies by functional inhibition of coding and non-coding regulatory DNA co- amplified on ecDNA (Aim 3). The research proposed in this application has technical, conceptual, and biological innovations, including the analysis of ecDNA on the single-cell level using novel imaging and multiome single-nucleus sequencing methods in medulloblastoma tumors and in patient-derived xenograft (PDX) models. The proposed research is significant, because the current standard treatment for children with medulloblastoma causes developmental disorders, neurological damage, and secondary metastases. Novel therapeutic approaches are urgently needed. Our approach will test the impact of standard-of-care treatments on the molecular evolution of ecDNA and functionally test novel combination treatments targeted against ecDNA genesis and clustering. These preclinical studies have the potential to uncover novel mechanisms by which ecDNA contributes to the pathogenesis of medulloblastoma and to identify new scientific leads for the development of improved treatments. We expect that our studies will expose the contribution of ecDNA variants to the emergence of therapy resistance, reveal their selection advantages, validate recently described properties of ecDNA and their therapeutic susceptibilities, and identify novel tumor-dependency genes amplified on ecDNA in some of the most aggressive medulloblastoma tumors.