Network-based elucidation of colon cancer drug resistance by phosphoproteomic time-series analysis

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies over the past decade we developed VESPA—an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations—and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases. Overall design: This experiment represents a large-scale, pooled CRISPR knockout (CRISPRko) screen validtion experiment, targeting all annotated human kinases, phosphatases and E3 ligases (Methods) of two human colorectal cancer cell lines HCT-15 and NCI-H508, perturbed with two drug compounds (linsitib, trametinib). Data was measured with four different guides per target.

信号通路活性异常是肿瘤发生与进展的标志性特征，三十余年来，该特征一直是靶向抑制剂研发的重要指导依据。然而，由快速、情境特异性的信号网络重编程所介导的适应性耐药机制，仍持续制约着肿瘤治疗的疗效。得益于近十年来蛋白质组学技术与网络生物学方法的进步，我们开发了VESPA算法——该算法旨在阐明细胞对药物扰动的应答与适应机制——并利用其分析了结直肠癌细胞经临床相关抑制剂及对照培养基处理后的7个时间点磷酸化蛋白质组时序数据。通过解析肿瘤特异性的酶-底物相互作用，基于底物磷酸化状态的推断结果可精准推算激酶与磷酸酶的活性，有效解决了信号串扰与磷酸化蛋白质组覆盖度不足的问题。该分析不仅阐明了各药物扰动下信号通路的时序应答特征，更重要的是，其揭示的细胞适应性应答与网络重编程现象已通过CRISPR基因敲除（CRISPRko）实验验证，表明该方法在癌症及其他多种疾病中具有广泛的应用前景。实验整体设计：本实验为大规模混合式CRISPR基因敲除（CRISPRko）筛选验证实验，针对两株人结直肠癌细胞系HCT-15与NCI-H508的全部已注释人类激酶、磷酸酶及E3泛素连接酶开展靶向干预（详见方法部分），并使用两种药物化合物（linsitib、trametinib）进行扰动处理。每个靶标采用4条不同的向导序列完成数据检测。