Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer

Abstract

EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.

Figure 1. BIBW-2992 induces radiographic CRs in lung tumor-bearing C/L858R but not C/L+T transgenic animals.

MRI images of lungs from tumor-bearing C/L858R and C/L+T mice pretreatment and after treatment with BIBW-2992 for 2 weeks (2W) and 4 weeks. Corresponding H&E-stained sections of lungs from treated mice (right panels) (original magnification, ×40). BIBW, BIBW-2992; H, heart.

Figure 2. EGFR mutant lung tumors display higher levels of the EGFR ligands, Areg and Ereg, compared with normal lungs.

(A) Unsupervised clustering of tumors from C/L858R, C/T790M, and C/L+T animals and normal lungs from control littermates fed a dox-containing diet form 2 separate groups. “Normal-1” lungs were derived from animals on a pure FVB background, and “normal-2” lungs were from animals on a mixed genetic background (see Methods for details). (B) RT-PCR for Ereg and Areg (and actin) was performed in the presence or absence of reverse transcriptase on mRNA extracted from 3 individual normal lungs and 3 separate macrodissected tumors from C/L+T, C/T790M, and C/L858R mice, respectively. h, human; m, mouse; NI, individual normal lung. (C) Levels of mouse epiregulin and amphiregulin were measured using ELISAs (see Methods) in lysates derived from 3 individual normal lungs and 3 separate macrodissected tumors from C/L+T, C/L858R, and C/T790M mice, respectively. Data represent mean ± SEM.

Figure 3. The combination of cetuximab and BIBW-2992 induces tumor regressions of mouse lung tumors driven by EGFR^L858R+T790M.

(A) MRI images of lungs from a tumor-bearing C/L858R mouse pretreatment and after treatment with cetuximab for 2 weeks. H&E-stained section from treated C/L858R mouse (right panel) (original magnification, ×40). (B) MRI images of lungs from tumor-bearing C/L+T (top panels) and C/T790M (bottom panels) mice, pretreatment, after 6 days of erlotinib (erloti), and then after 4 weeks of cetuximab. H&E-stained section from treated C/L+T and C/T790M mice (right panels) (original magnification, ×40). (C) MRI images of lungs from tumor-bearing C/L+T and C/T790M mice, pretreatment, after treatment with either cetuximab for 2 weeks or BIBW-2992 for 2 weeks, and after treatment with cetuximab (cetux) and BIBW-2992 for 4 weeks. H&E-stained sections of lungs from mice treated with the drug combination (right panels) (original magnification, ×40). Representative images are shown from all studies.

Figure 4. Change in radiographic tumor volume from baseline by treatment for individual lung tumor-bearing C/L858R and C/L+T animals.

Graphed is the percentage change in tumor volume, calculated for individual animals pretreatment and after treatment with paclitaxel (pacli), pemetrexed (peme), erlotinib, cetuximab, BIBW-2992, or combinations of erlotinib or BIBW-2992 with cetuximab. Cutoffs of 20% growth, 30% shrinkage, and 80% shrinkage (dotted lines) are shown to delineate PD, PR, and CR, respectively. Mice that displayed less than 20% growth and less than 30% shrinkage in tumor volume were considered to have SD (see Methods for details.) Statistical significance (calculated using Fisher’s exact test) of BIBW-2992/cetuximab–induced CRs in C/L+T animals versus paclitaxel (P = 0.0047), pemetrexed (P = 0.01), erlotinib (P = 0.02), cetuximab (P = 0.001), and cetuximab/erlotinib (P = 0.01).

Figure 5. The combination of cetuximab and BIBW-2992 induces regression of H1975 cell xenografts.

Athymic nude mice bearing established H1975 tumor cell xenografts were treated with placebo, cetuximab, BIBW-2992, or cetuximab with BIBW-2992 for 1 month. Five mice were treated in each group. Statistical analysis by a repeated measures ANOVA model is as follows: cetuximab versus control, P = 0.01; BIBW-2992 versus control, P = 0.09; BIBW-2992 plus cetuximab versus control, P = 0.006; BIBW-2992 versus cetuximab, P = 0.66; BIBW-2992 versus BIBW-2992 plus cetuximab, P = 0.13; cetuximab versus BIBW-2992 plus cetuximab, P = 0.02. Shown are representative results from 3 independent xenograft experiments. Data represent mean ± SEM.

Figure 6. Effect of cetuximab plus BIBW-2992 on EGFR^L858R+T790M.

(A) Tumor (T) lysates from C/L+T mice treated for 1 week with cetuximab (C), BIBW-2992 (B), or BIBW-2992 plus cetuximab (B+C) were probed by immunoblot analyses, using antibodies against the indicated phospho- (p-) or total (t) proteins and actin. Lysates from normal lung (N) were also examined. (B) Tumor lysates from H1975 xenograft models, treated as above for 1 week, were harvested for analogous immunoblot analyses. (C) NR6 mouse fibroblasts were stably transfected with cDNAs encoding EGFR^L858R+T790M and then treated with cetuximab (20 μg/ml), BIBW-2992 (50 nmol), or the combination of drugs as indicated. Corresponding cell lysates were subjected to immunoblotting with the indicated antibodies.