Dissecting chromothripsis, one step at a time

解剖染色体碎裂，一次一步

• 批准号: 460595631
• 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/460595631?language=en
• 关键词: Dissecting; chromothripsis; step; time

Chromothripsis is a recently identified form of genome instability, by which a presumably single catastrophic event generates extensive genome rearrangements of one or a few chromosome(s). Widely considered as an early event in tumor development, this phenomenon plays a causative role in tumor onset. Chromothripsis is now recognized as frequent in cancer, occurring in more than 20% of cases in numerous tumor types. Importantly, chromothripsis is linked with poor prognosis. Even though chromothriptic events play a major role in tumor development, direct evidence of what predisposes cells to chromothripsis and what is the exact sequence of events in this process are lacking. Furthermore, it remains unclear how chromothriptic cells come to dominate tumor cell populations. We recently characterized a unique model system allowing to directly capture each step of the chromothriptic process. Primary cultures from patients with Li-Fraumeni syndrome (germline mutation in TP53) display no major rearrangement at early passages but show spontaneous chromothripsis occurrence at late passages. In Goal 1, we will identify what key alterations lead to chromothripsis, longitudinally map the clonal dynamics of how chromothriptic cells arise and come to dominate cell populations, and identify key transition points to mechanistically test. Starting from cultured patient-derived cells with no complex rearrangement and going up to spontaneous chromothripsis occurrence, we will dissect clonal evolution by single-cell genomics and phenotypic profiling. We will capture the process by longitudinal single-cell DNA sequencing and quantify over time mechanistic features linked with chromothripsis (e.g. telomere hallmarks, micronuclei formation, replication stress) to explain cellular processes associated with the appearance of dominant chromothriptic clones. In Goal 2, we will dissect the mechanistic role of SETD2 in chromothripsis. We identified the SETD2 methyltransferase as strongly linked with chromothripsis and SETD2 inactivation as an early event in medulloblastomas with chromothripsis. We will characterize the specific signal transduction and transcriptional regulatory machinery promoting chromothripsis downstream of SETD2. We will perform CRISPR gene editing followed by the analysis of complex genome rearrangements and phenotypic features linked with chromothripsis in the engineered lines. Beyond SETD2, we will analyze other critical factors that potentially control chromothripsis (identified in Goal 1) using functional genomics. Through a multi-disciplinary approach combining single-cell genomics, bioinformatics and functional studies, and using unique models that we established, we will decipher the genetic and cellular mechanisms giving rise to chromothripsis. A better understanding of the underlying biological processes leading to chromothripsis will be a prerequisite for laying the basis for the development of novel therapeutic strategies.

染色体断裂是最近发现的一种基因组不稳定形式，可能是由于单一的灾难性事件导致一个或几个染色体的广泛基因组重排。这种现象被广泛认为是肿瘤发展的早期事件，在肿瘤发病中起着致病作用。现在认为染色体断裂在癌症中很常见，在许多肿瘤类型中超过20%的病例发生。重要的是，脊柱裂与预后不良有关。尽管嗜色分裂事件在肿瘤发展中起着重要作用，但缺乏直接证据表明是什么使细胞易发生嗜色分裂，以及这一过程中事件的确切顺序是什么。此外，尚不清楚嗜色细胞是如何支配肿瘤细胞群的。我们最近描述了一种独特的模型系统，可以直接捕获色素分解过程的每个步骤。来自Li-Fraumeni综合征（TP53种系突变）患者的原代培养在早期传代时没有重大的重排，但在传代后期出现自发的染色体裂解。在目标1中，我们将确定导致嗜色细胞分裂的关键改变，纵向绘制嗜色细胞如何产生并主导细胞群体的克隆动力学，并确定进行机械测试的关键转折点。从培养的没有复杂重排的患者来源的细胞开始，直到自发的染色体断裂发生，我们将通过单细胞基因组学和表型谱分析来解剖克隆进化。我们将通过纵向单细胞DNA测序来捕捉这一过程，并随着时间的推移量化与染色体分裂相关的机制特征（例如端粒标记、微核形成、复制压力），以解释与显性染色体分裂克隆出现相关的细胞过程。在目标2中，我们将剖析SETD2在染色体裂解中的机制作用。我们发现SETD2甲基转移酶与染色体断裂密切相关，SETD2失活是髓母细胞瘤伴染色体断裂的早期事件。我们将描述促进SETD2下游染色体裂解的特定信号转导和转录调控机制。我们将进行CRISPR基因编辑，然后分析工程系中与染色体断裂相关的复杂基因组重排和表型特征。除了SETD2之外，我们将使用功能基因组学分析其他可能控制染色体分裂的关键因素（在目标1中确定）。通过结合单细胞基因组学，生物信息学和功能研究的多学科方法，并使用我们建立的独特模型，我们将破译导致染色体分裂的遗传和细胞机制。更好地了解导致染色体碎裂的潜在生物学过程将是为开发新的治疗策略奠定基础的先决条件。