Abstract B77: Erk phosphorylation predicts synergism between gemcitabine and EGFR inhibitor AG1478

Abstract

Abstract Clinical trials combining epidermal growth factor receptor (EGFR) inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly KRAS mutation. It has been hypothesised that this antagonism relates to G1 arrest caused by EGFR inhibition. This study aimed to explore the relationship between synergy/antagonism, G1 arrest and phospho-ERK, as a measure of mitogen-activated protein kinase (MAPK) inhibition. Two NSCLC cell lines, A549 (mutant KRAS) and H322 (wildtype KRAS), were treated with gemcitabine and the EGFR inhibitor AG1478 alone and concurrently. Cytotoxicity was assessed using SRB assay and cytotoxic interaction was calculated using median effect analysis. Cell cycle distribution was assessed using flow cytometry and ERK phosphorylation was determined by ELISA. Cytotoxic interaction between gemcitabine and AG1478 were dose dependent with antagonism at high dose AG1478, particularly in the KRAS mutant cell line. Under conditions in which gemcitabine caused S phase arrest, antagonism was related to the G1 arrest effects of AG1478. However, synergy was also observed in the presence of gemcitabine-induced g1 arrest. Furthermore, gemcitabine increased ERK phosphorylation, the suppression of which by AG1478 was related to antagonism particularly in KRAS wildtype cell line. In the KRAS mutant cell line, synergy/antagonism was observed without a changed in phosphorylation of ERK. Our findings are consistent with a model in which gemcitabine causes cell cycle arrest and increased ERK phosphorylation. Low dose AG1478 is synergistic possibly due to suppression of EGFR-related survival signals. However, anti-proliferative effect of high dose AG1478 is antagonistic as it interferes with gemcitabine-induced cell cycle arrest as well as reduces ERK phosphorylation, which may be an important death signal. This data confirms the importance of EGFR inhibition-related G1 arrest in antatogism but also identifies that synergy can still occur in the presence of G1 arrest and highlights an important potential role for phospho-ERK in determining cell death. In summary, this study suggests that ERK phosphorylation is more predictive of synergism/antagonism than cell cycle changes when gemcitabine is combined with an EGFR inhibitor. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B77.