Tyrosine kinase inhibitors (TKIs) have become an important therapeutic option 
for treating several forms of cancer. Gefitinib, an inhibitor of the epidermal 
growth factor receptor (EGFR), is in clinical use for treating non-small cell 
lung cancer (NSCLC) harboring activating EGFR mutations. However, despite high 
initial response rates, many patients develop resistance to gefitinib. The 
molecular mechanisms of TKI resistance often remain unclear. Here, we describe a 
chemical proteomic approach comprising kinase affinity purification (kinobeads) 
and quantitative mass spectrometry for the identification of kinase inhibitor 
resistance mechanisms in cancer cells. We identified the previously described 
amplification of MET and found EPHA2 to be more than 10-fold overexpressed (p < 
0.001) in gefitinib-resistant HCC827 cells suggesting a potential role in 
developing resistance. siRNA-mediated EPHA2 knock-down or treating cells with 
the multikinase inhibitor dasatinib restored sensitivity to gefitinib. Of all 
dasatinib targets, EPHA2 exhibited the most drastic effect (p < 0.001). In 
addition, EPHA2 knockdown or ephrin-A1 treatment of resistant cells decreased 
FAK phosphorylation and cell migration. These findings confirm EPHA2 as an 
actionable drug target, provide a rational basis for drug combination 
approaches, and indicate that chemical proteomics is broadly applicable for the 
discovery of kinase inhibitor resistance.