Does inappropriate DNA replication provide a mechanism for acquisition of drug resistance?

该项目将在PC9细胞中获得对AZ药物Osimertinib的耐药性，Osimertinib是同类中最好的EGFR抑制剂，作为模型系统。I)我们将首先在Osimertinib治疗期间应用scRNAseq来表征治疗期间的基因表达。单细胞方法将揭示单个复制细胞的状态，这可能是异质的，因为细胞在延长的处理下不能统一反应。ii)我们开发了一种最先进的方法来检测复制压力和DNA损伤，Trael-seq，它以高灵敏度描述DNA复制模式。Trael-seq将用于复制细胞，以了解药物治疗造成的复制应激。iii)复制应激可引起染色体外环状DNA(EcDNA)，它经常含有导致耐药性的基因扩增。将DTP中与药物相关的复制压力直接与ecDNA的形成联系起来，这将是特别令人兴奋的，我们将使用循环DNA测序进行测试。

This project will use the acquisition of resistance to the AZ drug Osimertinib, the best-in-class EGFR inhibitor, in PC9 cells as a model system. i) We will first apply scRNAseq during Osimertinib treatment to characterise gene expression during treatment. A single cell approach will reveal the status of individual replicating cells, which is likely to be heterogeneous as cells do not react uniformly under extended treatment.ii) We have developed a state-of-the-art method for detecting replication stress and DNA damage, TrAEL-seq, which profiles DNA replication patterns with high sensitivity. TrAEL-seq will be used to replicating cells to understand replication stress caused by drug treatment.iii) Replication stress can give rise to extrachromosomal circular DNA (ecDNA), which frequently harbours gene amplifications that bestow drug resistance. It would be particularly exciting to connect drug-assocatied replication stress in DTPs directly to formation of ecDNA, which we will test using circular DNA sequencing.