Activity of tarloxotinib‐E in cells with EGFR exon‐20 insertion mutations and mechanisms of acquired resistance

Background Approximately 10% of non‐small cell lung cancers (NSCLCs) that harbor epidermal growth factor receptor ( EGFR ) gene mutations have in‐frame insertions in exon 20 of the EGFR gene. These tumors do not usually respond to currently available EGFR‐tyrosine kinase inhibitors (TKIs). Tarloxotinib is a novel hypoxia‐activated prodrug that releases a potent, irreversible pan‐ERBB TKI (tarloxotinib‐E) under solid tumor hypoxia. Methods We examined the efficacy of tarloxotinib‐E against several types of Ba/F3 cells with introduced EGFR exon 20 mutations ( EGFR A763insFQEA, V769insASV, D770insSVD, H773insH and H773insNPH mutations). We assayed growth inhibition for tarloxotinib (prodrug), tarloxotinib‐E (active form), poziotinib, afatinib, and osimertinib in Ba/F3 cells with each EGFR exon 20 mutation. We also explored acquired resistance mechanisms to tarloxotinib‐E by establishing cells with resistance to tarloxotinib‐E via chronic drug exposure after N‐ethyl‐N‐nitrosourea mutagenesis treatment. Results Among all tested Ba/F3 cell lines, IC 50 was ≥72.1 times higher for tarloxotinib than for tarloxotinib‐E, which implies a wide therapeutic window with this prodrug strategy. Tarloxotinib‐E was efficacious against all tested Ba/F3 cells except for H773insH, which was less sensitive to all tested EGFR‐TKIs. As acquired resistance mechanisms to tarloxotinib‐E, we identified either T790M or C797S secondary mutations, depending on the original EGFR exon 20 mutation. Conclusions These findings indicate that tarloxotinib‐E could be effective for NSCLC with EGFR exon 20 mutations. Our results also show that T790M or C797S mutations can confer acquired resistance to tarloxotinib‐E; and suggest that resistance mechanisms are influenced by the baseline EGFR exon 20 mutations.