A pan-cancer transcriptome analysis to identify the molecular mechanism of prexasertib resistance [microarray]. A pan-cancer transcriptome analysis to identify the molecular mechanism of prexasertib resistance [microarray]

The combined influence of oncogenic drivers, genomic instability, and/or DNA damage repair deficiencies increases replication stress in cancer. Cells with high replication stress rely on the upregulation of checkpoints like those governed by CHK1 for survival. Previous studies of the CHK1 inhibitor prexasertib demonstrated activity across multiple cancer types. Therefore, we sought to (1) identify markers of prexasertib sensitivity and (2) define the molecular mechanism(s) of intrinsic and acquired resistance using preclinical models representing multiple tumor types. Our findings indicate that while cyclin E dysregulation is a driving mechanism of prexasertib response, biomarkers associated with this aberration lack sufficient predictive power to render them clinically actionable for patient selection. Transcriptome analysis of a pan-cancer cell line panel and in vivo models revealed an association between expression of E2F target genes and prexasertib sensitivity and identified innate immunity genes associated with prexasertib resistance. Functional RNAi studies supported a causal role of replication fork components as modulators of prexasertib response. Mechanisms which protect cells from oncogene-induced replication stress may safeguard tumors from such stress induced by a CHK1 inhibitor, resulting in acquired drug resistance. Furthermore, resistance to prexasertib may be shaped by innate immunity. We included microarray data here as part of the study. RNAseq data was uploaded separately. Overall design: Prexasertib resistant cell lines from different cancer types were generated using a long-term drug concentration escalation protocol. With at least 3 biological replicates in both resistant and parental lines, Affymetrix microarrays were used to identify differentially expressed genes, which helped to understand molecular mechanism of acquired resistance.

致癌驱动因子、基因组不稳定以及DNA损伤修复缺陷的协同作用，会加剧癌症中的复制应激（replication stress）。存在高水平复制应激的癌细胞，依赖CHK1调控的细胞周期检查点通路的上调以维持存活。
既往针对CHK1抑制剂prexasertib的研究显示，其在多种癌症类型中均具有抗肿瘤活性。
因此，本研究旨在（1）鉴定prexasertib的敏感性标志物，（2）通过涵盖多种肿瘤类型的临床前模型，阐明prexasertib固有耐药与获得性耐药的分子机制。
本研究结果显示，尽管细胞周期蛋白E（cyclin E）失调是影响prexasertib应答的核心机制，但与该异常相关的生物标志物的预测效能不足，无法用于临床指导患者筛选。
对泛癌细胞系组合（pan-cancer cell line panel）与体内模型的转录组分析显示，E2F靶基因的表达水平与prexasertib敏感性呈正相关，并鉴定出与prexasertib耐药相关的固有免疫基因。
功能性RNA干扰（RNAi）实验证实，复制叉（replication fork）相关组分可作为prexasertib应答的调控因子。
能够保护细胞免受致癌因子诱导的复制应激的机制，可使肿瘤免受CHK1抑制剂诱导的同类应激，进而导致获得性耐药。此外，prexasertib的耐药性可能受固有免疫通路调控。
本研究已将微阵列（microarray）数据纳入本文数据集，RNA测序（RNAseq）数据则另行上传。
实验整体设计：通过长期药物浓度递增法，构建了不同癌症类型的prexasertib耐药细胞系。本研究在耐药株与亲本株中均设置至少3次生物学重复，采用Affymetrix微阵列芯片筛选差异表达基因，以阐明获得性耐药的分子机制。