Figure S1 Synergistic efficacy of afatinib and cetuximab combination therapy Figure S2 Efficacy of EGFR-TKIs and cetuximab combination therapy Figure S3 Effect of EGFR tyrosine kinase inhibitors on EGFR monomer dimer equilibrium Figure S4 Dimerization of EGFR by EGF Figure S5 Proliferation of Ba/F3 cells with indicated EGFR genotypes in vitro Figure S6 Detection of monomer or dimer of EGFR in NIH-3T3 cells by immunoblotting Figure S7 Synergistic efficacy of dacomitinib and cetuximab combination therapy Figure S8 Monomer preference of dacomitinib Figure S9 Effect of the combination therapy in vivo model Figure S10 Evaluation of the cell surface EGFR expression level after drug treatment by flow cytometric analysis Figure S11 Efficacy of the afatinib and cetuximab combination therapy in osimertinib resistant cell lines
ARTICLE ABSTRACT
EGFR-mutated lung cancer is a significant subgroup of non–small cell lung cancer. To inhibit EGFR-mediated signals, multiple EGFR tyrosine kinase inhibitors (EGFR-TKI) have been developed; however, approximately one third of patients with EGFR-mutated lung cancer do not respond to EGFR-TKIs. More effective inhibition of EGFR-mediated signals is therefore necessary. For cancers expressing mutated EGFR, including EGFR T790M, which confers resistance to first- (gefitinib and erlotinib) and second- (afatinib) generation EGFR-TKIs, the synergistic efficacy of afatinib and cetuximab combination therapy has been reported in preclinical and clinical studies; however, the mechanisms underlying this effect remain elusive. In this study, we evaluated the effects of multiple EGFR-TKIs on the EGFR monomer–dimer equilibrium by inducing dimerization-impairing mutations in cells expressing EGFR. Interestingly, we found that afatinib and dacomitinib exhibit a monomer preference: cells expressing dimerization-impaired EGFR mutants exhibited increased sensitivity to afatinib and dacomitinib relative to those with dimerization-competent EGFR mutants. Although EGFR-TKIs themselves induce dimerization of EGFR, the inhibition of dimerization by cetuximab overcame EGFR-TKI–induced dimerization. By shifting the monomer–dimer equilibrium toward monomer dominance using cetuximab, the effectiveness of afatinib and dacomitinib improved significantly. We report a novel and clinically relevant phenomenon, the monomer preference of EGFR-TKIs, which can explain the mechanism underlying the synergism observed in afatinib and cetuximab combination therapy. In addition, we propose the novel concept that monomer–dimer equilibrium is an important factor in determining EGFR-TKI efficacy. These findings provide novel insights into treatment strategies for EGFR-TKI–refractory non–small cell lung cancer.
