In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer

Abstract

EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the "therapeutic window" of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations.

Keywords: EGFR exon 20 insertion mutations; EGFR mutation; EGFR tyrosine kinase inhibitors; in vitro modeling; lung cancer.

Figure 1. Sensitivity of Ba/F3 cells harboring EGFR mutations to EGFR-TKIs

A. MTS assay for Ba/F3 cells harboring EGFR exon 19 deletion and L858R. The proportional cell viability is shown. B. MTS assay for Ba/F3 cells harboring EGFR exon 19 deletion+T790M and L858R+T790M. The proportional cell viability is shown. C. MTS assay for Ba/F3 cells harboring wild type EGFR. The proportional cell viability is shown. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. Error bars indicate standard deviation.

Figure 2. Inhibition of the phosphorylation of EGFR and downstream proteins by EGFR-TKIs in BaF3 cells harboring EGFR mutations

The results of immunoblotting for Ba/F3 cells with EGFR exon 19 deletion, L858R, exon 19 deletion+T790M, and L858R+T790M are shown. The cells were treated with the indicated concentrations of EGFR-TKIs for 4 h. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. pEGFR, pAKT, and pERK indicate the phosphorylated form of EGFR, AKT, and ERK, respectively. Actin was used as a loading control.

Figure 3. IC50 values and in vitro modeling

A. IC50 values (nM) of EGFR-TKIs for wild type and mutated EGFR are shown. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. B. Calculated values of the selectivity index (SI) for EGFR mutations, exon 19 deletion, L858R, exon 19 deletion+T790M, and L858R+T790M. *; SI index >1.

Figure 4. Sensitivity of lung cancer cell lines to EGFR-TKIs

A. MTS assay for PC-9, H3255, PC9-ER, and H1975 cells. The IC50 values (nM) for EGFR-TKIs are shown. Error bars indicate standard deviation. B. The results of immunoblotting for PC-9, H3255, PC9-ER, and H1975 cells are shown. The cells were treated with the indicated concentrations of EGFR-TKIs for 4 h. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. pEGFR, pAKT, and pERK indicate the phosphorylated form of EGFR, AKT, and ERK, respectively. Actin was used as a loading control.

Figure 5. Sensitivity of Ba/F3 cells harboring EGFR exon 20 insertion mutations to EGFR-TKIs

A. MTS assay for Ba/F3 cells harboring EGFR exon 20 insertion mutations. The mutations studied include A763_Y764insFQEA, Y764_V765insHH, A767_V769dupASV, and D770_N771insNPG. The proportional cell viability is shown. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. Error bars indicate standard deviation. B. IC50 values (nM) of EGFR-TKIs for EGFR exon 20 insertion mutations. C. The calculated values of the selectivity index (SI) for EGFR exon 20 insertion mutations are shown. *; SI index > 1.

Figure 6. Efficacy of EGFR-TKIs for EGFR exon 20 insertion mutations

A. MTS assay (left) and immunoblotting (right) for BID007 (EGFR A763_Y764insFQEA) cells. Error bars indicate standard deviation. B. Results of immunoblotting for Ba/F3 cells with EGFR exon 20 insertion mutations. The cells were treated with the indicated concentrations of EGFR-TKIs for 4 h. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. pEGFR, pAKT, and pERK indicate the phosphorylated form of EGFR, AKT, and ERK, respectively. Actin was used as a loading control. C. Apoptosis assay using cytometry. Ba/F3 cells harboring wild type EGFR and EGFR D770_N771insNPG (NPG) were treated with EGFR-TKIs for 48 h, subsequently the cells were stained with propidium iodide and annexin V-APC. The numbers indicate the proportion of annexin V-positive and/or propidium iodide-positive cells.