表皮生长因子受体（EGFR）是转移性结直肠癌（mCRC）中一个被充分利用的治疗靶点。不幸的是，并非所有患者都能从当前的EGFR抑制剂中受益。我们在30例基因组学和药理学特征化的mCRC患者源性异种移植物（PDX）上进行了基于质谱的蛋白质组学和磷酸化蛋白质组学研究，以研究对EGFR阻断的分子基础和识别克服耐药性的替代药物靶点。鉴于敏感性肿瘤，酪氨酸和全局磷酸化蛋白质组以及蛋白质组都具有独特的反应特征。我们发现，与增加的酪氨酸激酶抗活化蛋白激酶（MAPK）通路活性和细胞结合蛋白相关的丰富酪氨酸磷酸化（例如CXADR和CLDN1/3）相关的应对敏感的肿瘤，而上皮间充质转化和增加的MAPK和AKT信号对应于耐药性肿瘤更为普遍。此外，单个样本中激酶活性的排序确认了ERBB2、EGFR和MET在曲妥珠单抗耐药肿瘤中的驱动活性。此分析还揭示出Src和ephrin激酶家族的几个成员在2个基因组学未解释耐药性的CRC PDX模型中具有高激酶活性。这些高度活化的激酶的抑制, 无论是单独还是与曲妥珠单抗联合应用, 都导致外源PDX衍生器官样体外组织和PDX内的生长抑制。总的来说，这些发现突显了磷酸化蛋白质组学在改善我们对mCRC抗EGFR治疗和反应预测的理解方面的潜在价值，并将曲妥珠单抗耐药肿瘤中的替代药物靶点置于前沿。

Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry-based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient-derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance. Both the tyrosine and global phosphoproteome as well as the proteome harbored distinctive response signatures. We found that increased pathway activity related to mitogen-activated protein kinase (MAPK) inhibition and abundant tyrosine phosphorylation of cell junction proteins, such as CXADR and CLDN1/3, in sensitive tumors, whereas epithelial-mesenchymal transition and increased MAPK and AKT signaling were more prevalent in resistant tumors. Furthermore, the ranking of kinase activities in single samples confirmed the driver activity of ERBB2, EGFR, and MET in cetuximab-resistant tumors. This analysis also revealed high kinase activity of several members of the Src and ephrin kinase family in 2 CRC PDX models with genomically unexplained resistance. Inhibition of these hyperactive kinases, alone or in combination with cetuximab, resulted in growth inhibition of ex vivo PDX-derived organoids and in vivo PDXs. Together, these findings highlight the potential value of phosphoproteomics to improve our understanding of anti-EGFR treatment and response prediction in mCRC and bring to the forefront alternative drug targets in cetuximab-resistant tumors.