Chemical Proteomics Uncovers EPHA2 as a Mechanism of Acquired Resistance to Small Molecule EGFR Kinase Inhibition

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.