Drug resistance poses a major challenge for targeted cancer therapy. To be able to functionally screen large randomly mutated target gene libraries for drug resistance mutations, we developed a biochemically defined high-throughput assay termed PhosphoFlowSeq. Instead of selecting for proliferation or resistance to apoptosis, PhosphoFlowSeq directly analyzes the enzymatic activities of randomly mutated kinases, thereby reducing the dependency on the signaling network in the host cell. Moreover, simultaneous analysis of expression levels enables compensation for expression-based biases on a single cell level. Using EGFR and its kinase inhibitor erlotinib as a model system, we demonstrate that the clinically most relevant resistance mutation T790M is reproducibly detected at high frequencies after four independent PhosphoFlowSeq selection experiments. Moreover, upon decreasing the selection pressure, also mutations which only confer weak resistance were identified, including T854A and L792H. We expect that PhosphoFlowSeq will be a valuable tool for the prediction and functional screening of drug resistance mutations in kinases.

耐药性对靶向癌症治疗提出了重大挑战。为了能够在功能上筛选大型随机突变靶基因文库的耐药性突变，我们开发了一种生化定义的高通量测定，称为 PhosphoFlowSeq。PhosphoFlowSeq 不是选择增殖或抗凋亡，而是直接分析随机突变激酶的酶活性，从而减少对宿主细胞中信号网络的依赖。此外，同时分析表达水平可以补偿单个细胞水平上基于表达的偏差。使用 EGFR 及其激酶抑制剂厄洛替尼作为模型系统，我们证明，在四次独立的 PhosphoFlowSeq 选择实验后，临床上最相关的抗性突变 T790M 可重复检测到高频。此外，在降低选择压力后，还鉴定出仅赋予弱抗性的突变，包括 T854A 和 L792H。我们预计 PhosphoFlowSeq 将成为预测和功能筛选激酶耐药性突变的宝贵工具。