Simple Summary Non-small-cell lung carcinoma (NSCLC) covers for almost 85% of all
lung cancers and a major contributor to the overall cancer death rate. Erlotinib is
used to treat NSCLC via inhibition of epithelial growth factor receptor (EGFR) kinase
activity. Despite its high efficacy, recurrence can occur in patients who become resistant
to the drug. We performed DARTS LC-MS/MS with SWATH of DIA analysis and identified
a novel binding protein of Erlotinib that may underlie NSCLC resistance. Our study
indicated that Erlotinib binds POLA2 in addition to EGFR. This was confirmed by DARTS
and CETSA results. Importantly, POLA2 expression levels in four NSCLC cell lines were
positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation
coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary
target protein of Erlotinib, and could clinically provide validity as a surrogate
marker for drug resistance in patients with NSCLC. Erlotinib inhibits epithelial growth
factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer
(NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant
to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated
additional mechanisms related to mode-of-drug-action, by multiple protein-binding
interactions, besides EGFR by using drug affinity responsive target stability (DARTS)
and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib.
DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein
that was validated by the DARTS platform, complemented with cellular thermal shift
assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the
drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas
the overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive
cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in
four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib
efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that
POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide
validity as a surrogate marker for drug resistance in patients with NSCLC.
