Crizotinib sensitizes the erlotinib resistant HCC827GR5 cell line by influencing lysosomal function.

Nele Van Der Steen,Christian Rolfo,Patrick Pauwels,René J P Musters,Ietje Kathmann,Letizia Porcelli,Henk Dekker,Richard J Honeywell,Elisa Giovannetti,Daniel S K Liu,Adam E Frampton,Johan Van Meerloo,Godefridus J Peters,Rob Ruijtenbeek,Kaylee Keller

doi:10.1002/jcp.29463

Abstract

In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live‐cell microscopy. We observed additive effects in EBC‐1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR‐inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live‐cell microscopy with a pH‐sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib–erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions.

Highlights

Non‐small cell lung cancer (NSCLC) consists of three histological subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, and has a very poor 5‐year survival rate of around 10% (Travis, Brambilla, Burke, Marx, & Nicholson, 2015)
The parental HCC827 cell line is a NSCLC adenocarcinoma cell line harboring an epidermal growth factor receptor (EGFR) exon 19 deletion, which is a sensitizing mutation of EGFR. This cell line has been made resistant to gefitinib through prolonged exposure with increasing dosages, which resulted in cMET amplification (Engelman et al, 2007), and combined treatment with the EGFR inhibitor gefitinib and the cMET inhibitor PHA‐665752, which resulted in substantial growth inhibition (Engelman et al, 2007)
We have shown a strong synergism with two compounds commonly used in the clinical setting, and determined, for the first time, the potential role of lysosomes in this pharmacological interaction

Summary

| INTRODUCTION

Non‐small cell lung cancer (NSCLC) consists of three histological subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, and has a very poor 5‐year survival rate of around 10% (Travis, Brambilla, Burke, Marx, & Nicholson, 2015). ABC transporters, such as PgP, multidrug resistance‐associated proteins (MRPs) and BCRP can mediate resistance by effluxing drugs out of the cells These efflux pumps are located on the cellular membranes, have high expression in gut epithelium and their primary role is to remove xenobiotics from the body (Sharom, 2008). The parental HCC827 cell line is a NSCLC adenocarcinoma cell line harboring an EGFR exon 19 deletion, which is a sensitizing mutation of EGFR This cell line has been made resistant to gefitinib through prolonged exposure with increasing dosages, which resulted in cMET amplification (Engelman et al, 2007), and combined treatment with the EGFR inhibitor gefitinib and the cMET inhibitor PHA‐665752, which resulted in substantial growth inhibition (Engelman et al, 2007). We have shown a strong synergism with two compounds commonly used in the clinical setting (erlotinib and crizotinib), and determined, for the first time, the potential role of lysosomes in this pharmacological interaction

| MATERIALS AND METHODS

| RESULTS

| DISCUSSION

| CONCLUSION

Findings

DATA AVAILABILITY STATEMENT