Phosphoproteomics of patient-derived xenografts identifies targets and markers associated with sensitivity and resistance to EGFR blockade in colorectal cancer

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.

Automated summary

This study used proteomics and phosphoproteomics to investigate the molecular basis of response to EGFR blockade in metastatic colorectal cancer (mCRC). The research found distinctive response signatures in the proteome and phosphoproteome, with sensitive tumors showing increased pathway activity related to MAPK inhibition and abundant tyrosine phosphorylation of cell junction proteins, while resistant tumors exhibited epithelial-mesenchymal transition and increased MAPK and AKT signaling. The analysis also revealed high kinase activity of ERBB2, EGFR, MET, Src, and ephrin kinase family members in cetuximab-resistant tumors. Inhibition of these hyperactive kinases resulted in growth inhibition of ex vivo and in vivo models. These findings highlight the potential value of phosphoproteomics in improving our understanding of anti-EGFR treatment and response prediction in mCRC, and identifying alternative drug targets in cetuximab-resistant tumors.