Chromothripsis: unraveling the tumor evolution in vivo, from initiating tumor cells to therapy resistance

Chromothripsis：揭示体内肿瘤进化，从起始肿瘤细胞到治疗耐药

• 批准号: 512935482
• 负责人: Privatdozentin Dr. Aurélie Ernst
• 金额: --
• 依托单位: DKFZ-Juniorgruppe Genominstabilität in Tumoren
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/512935482?language=en
• 关键词: Chromothripsis; unraveling; tumor; evolution; vivo

Chromothripsis is a form of genome instability, by which a single catastrophic event generates extensive genome rearrangements of one chromosome. Generally considered as an early event in tumor development, this phenomenon is now recognized as frequent in cancer and linked with poor prognosis. However, direct evidence of initiating copy-number alterations occurring in vivo and how genetic clones become selected are lacking. Furthermore, the potential use of chromothripsis detection in liquid biopsies and how this may contribute to developing early detection tools or guiding treatment decisions has not been explored.First we will characterize the preneoplastic landscape of chromothriptic tumors in vivo. Nearly all patients with Li-Fraumeni syndrome (TP53 germline mutations) develop cancer, and these tumors show a close to 100% chromothripsis prevalence. We will dissect early-stage genomic alterations preceding tumor diagnosis in available blood cells from Li-Fraumeni syndrome individuals enrolled in surveillance programs (n=25). We will do single-cell DNA and RNA sequencing i) to identify clones with initiating copy-number alterations and cell types prone to such early copy-number alterations ii) to integrate early events with the genomic landscape of advanced stage cancers. Second we will use functional genomics to dissect the molecular effects of factors identified in Goal 1 and known candidate genes that potentially promote chromothripsis or lead to clonal expansion in chromothriptic tumors. These “factors” represent the specific signal transduction and transcriptional regulatory machinery leading to the development of chromothriptic tumors. We will perform a CRISPR-pooled screen of 15 candidate genes identified as putative promoters of clonal expansion, followed by the analysis of phenotypic features in the engineered lines (e.g. proliferation, apoptosis). In addition to candidates from Goal 1, we will dissect the mechanistic role of key players in the p53 pathway in chromothripsis. Third we will benchmark the use of chromothripsis detection in liquid biopsies. As part of the preliminary data for this proposal, we showed chromothriptic rearrangements in cell-free DNA from cancer patients. We will analyze the evolution of chromothriptic patterns in longitudinal liquid biopsies and identify fusion genes generated by chromothripsis by whole-genome and RNA sequencing of cell-free DNA, tumor tissue and matched germline controls (n=20 cases per tumor type, all samples are available). Finally, we will characterize extrachromosomal circular DNAs in tumors and matched cell-free DNA to get insights into the role of these structures in oncogene activation.By a multi-disciplinary approach using single-cell genomics, bioinformatics and functional studies, we will obtain a better understanding of the biological processes leading to chromothripsis, as a prerequisite for laying the basis for the development of novel surveillance and treatment strategies.

染色体碎裂是基因组不稳定的一种形式，单个灾难性事件会导致一条染色体发生广泛的基因组重排。通常认为这种现象是肿瘤发展的早期事件，但现在人们认为这种现象在癌症中很常见，并且与不良预后有关。然而，缺乏体内发生拷贝数改变以及基因克隆如何被选择的直接证据。此外，尚未探索染色体碎裂检测在液体活检中的潜在用途，以及这如何有助于开发早期检测工具或指导治疗决策。首先，我们将描述体内染色体碎裂肿瘤的癌前景观。几乎所有 Li-Fraumeni 综合征（TP53 种系突变）患者都会罹患癌症，并且这些肿瘤的染色质碎裂患病率接近 100%。我们将在参与监测计划的李法美尼综合征个体 (n=25) 的可用血细胞中剖析肿瘤诊断前的早期基因组改变。我们将进行单细胞 DNA 和 RNA 测序 i) 识别具有起始拷贝数改变的克隆和容易发生这种早期拷贝数改变的细胞类型 ii) 将早期事件与晚期癌症的基因组景观整合起来。其次，我们将使用功能基因组学来剖析目标 1 中确定的因素和已知候选基因的分子效应，这些基因可能促进染色体碎裂或导致染色体碎裂肿瘤的克隆扩张。这些“因子”代表了导致染色质肿瘤发生的特定信号转导和转录调节机制。我们将对 15 个候选基因进行 CRISPR 筛选，这些候选基因被确定为克隆扩增的推定启动子，然后分析工程系的表型特征（例如增殖、凋亡）。除了目标 1 中的候选者之外，我们还将剖析染色质碎裂中 p53 途径关键参与者的机制作用。第三，我们将对液体活检中染色体碎裂检测的使用进行基准测试。作为该提案初步数据的一部分，我们展示了癌症患者游离 DNA 中的染色体碎裂重排。我们将分析纵向液体活检中染色体碎裂模式的演变，并通过对游离 DNA、肿瘤组织和匹配种系对照的全基因组和 RNA 测序来鉴定染色体碎裂产生的融合基因（每种肿瘤类型 n = 20 例，所有样本均可用）。最后，我们将表征肿瘤中染色体外环状DNA和匹配的无细胞DNA，以深入了解这些结构在癌基因激活中的作用。通过利用单细胞基因组学、生物信息学和功能研究的多学科方法，我们将更好地了解导致染色体碎裂的生物过程，作为开发新型监测技术奠定基础的先决条件 和治疗策略。