Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib

Abstract

Non-small cell lung cancers (NSCLCs) with activating mutations in the kinase domain of the epidermal growth factor receptor (EGFR) demonstrate dramatic, but transient, responses to the reversible tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Some recurrent tumors have a common secondary mutation in the EGFR kinase domain, T790M, conferring drug resistance, but in other cases the mechanism underlying acquired resistance is unknown. In studying multiple sites of recurrent NSCLCs, we detected T790M in only a small percentage of tumor cells. To identify additional mechanisms of acquired resistance to gefitinib, we used NSCLC cells harboring an activating EGFR mutation to generate multiple resistant clones in vitro. These drug-resistant cells demonstrate continued dependence on EGFR and ERBB2 signaling for their viability and have not acquired secondary EGFR mutations. However, they display increased internalization of ligand-activated EGFR, consistent with altered receptor trafficking. Although gefitinib-resistant clones are cross-resistant to related anilinoquinazolines, they demonstrate sensitivity to a class of irreversible inhibitors of EGFR. These inhibitors also show effective inhibition of signaling by T790M-mutant EGFR and killing of NSCLC cells with the T790M mutation. Both mechanisms of gefitinib resistance are therefore circumvented by irreversible tyrosine kinase inhibitors. Our findings suggest that one of these, HKI-272, may prove highly effective in the treatment of EGFR-mutant NSCLCs, including tumors that have become resistant to gefitinib or erlotinib.

Fig. 1.

EGFR sequence analysis in recurrent metastatic lesions from two NSCLC patients with acquired gefitinib resistance. (A) Case 1. The T790M mutation in EGFR is present in a recurrent liver lesion after the development of clinical gefitinib resistance. (Left) The mutation was not detected in the primary lung lesion at the time of diagnosis. (Right) Both the primary lung tumor and the recurrent liver lesion harbor the L858R gefitinib-sensitizing mutation. Of note, the L858R mutation is present in the expected ratio for a heterozygous mutation in both primary and recurrent lesions, whereas T790M is detectable at low levels compared with the wild-type allele. A polymorphism (G/A) is shown in the same tracing to demonstrate equivalent representation of the two alleles in the uncloned PCR product. (B) Case 2. The T790M mutation is present within a small minority of gefitinib-resistant cells. (Left) The T790M mutation was undetectable either in the lung primary tumor or in eight recurrent liver lesions from this case by sequencing uncloned PCR products. Heterozygosity at an adjacent polymorphism (G/A) confirms amplification of both EGFR alleles from these specimens. The heterozygous gefitinib-sensitizing mutation, L861Q, was detected at the expected ratio within the primary lung tumor as well as each of the eight recurrent liver lesions.

Fig. 2.

Acquired resistance to gefitinib in bronchoalveolar cancer cell lines and persistent sensitivity to irreversible ERBB family inhibitors. (A) Inhibition by tyrosine kinase inhibitors of proliferation of bronchoalveolar cancer cell lines with wild-type EGFR (NCI-H1666), the activating delE746-A750 mutation in EGFR (NCI-H1650), or two representative gefitinib-resistant subclones of NCI-H1650 (G7 and C11). The effect of the reversible inhibitor gefitinib is compared with that of the irreversible inhibitor HKI-357. Comparable results were observed with the other irreversible inhibitors. Cell numbers were measured by crystal violet staining, after culture in 5% FCS, with 100 ng/ml EGFR, at 72 h after exposure to indicated drug concentrations. Each data point represents the mean of four samples. (B) Chemical structure of gefitinib, a reversible inhibitor of EGFR; EKB-569, an irreversible inhibitor of EGFR; and HKI-272 and HKI-357, two irreversible dual inhibitors of EGFR and ERBB2. (C) Generation of drug-resistant NCI-H1650 cells after treatment with varying concentrations of gefitinib or the irreversible ERBB inhibitor EKB-569. Colonies were stained after 12 days in culture in the presence of inhibitors.

Fig. 3.

Persistent dependence on EGFR and ERBB2 signaling in gefitinib-resistant cells, and altered receptor trafficking. (A) Cell viability after siRNA-mediated knockdown of EGFR and ERBB2 in bronchoalveolar cell lines with wild-type EGFR (NCI-H1666), compared with cells with the activating delE746-A750 mutation in EGFR (NCI-H1650) and two gefitinib-resistant derivatives (G7 and C11). Viable cells were counted 72 h after treatment with double-stranded RNA and are shown as a fraction relative to cells treated with nonspecific siRNA, with standard deviations based on triplicate samples. (B) Inhibition of EGFR autophosphorylation (Y1068) and phosphorylation of downstream effectors AKT and MAPK (ERK) in cells treated with increasing concentrations of gefitinib or the irreversible inhibitor HKI-357, followed by a 2-h pulse with EGF. The parental cell line NCI-H1650 is compared with a representative gefitinib-resistant line, G7. Total AKT and MAPK are shown as controls; tubulin is used as loading control for total EGFR levels, which are at the lower limit of detection in these cells. (C) Altered EGFR internalization in gefitinib-resistant NCI-H1650 (G7) cells, compared with the sensitive NCI-H1650 parental cell line. Rhodamine-tagged EGF is used to label EGFR at 5 and 20 min, after addition of ligand. The increased internalization of EGFR in NCI-H1650 (G7) cells is most evident at 20 min. (Zeiss microscope, ×63 magnification). (D) Immunoblotting of internalized EGFR from NCI-H1650 parental cells and the resistant derivative G7 after pulse labeling of cell surface proteins by biotinylation and chase over 20 min. The increased intracellular EGFR in NCI-H1650 (G7) cells is compared with the unaltered transferrin receptor (TR) internalization.

Fig. 4.

Effectiveness of irreversible ERBB inhibitors in suppressing the T790M EGFR mutant. (A) Comparison of gefitinib and two irreversible inhibitors, HKI-357 and HKI-272, in their ability to suppress EGFR autophosphorylation (Y1068) and phosphorylation of downstream effectors AKT and MAPK (ERK) in the NCI-H1975 bronchoalveolar cell line, harboring both a sensitizing mutation (L858R) and the resistance-associated mutation (T790M). Total EGFR, AKT, and MAPK are shown as loading controls. (B) Suppression of proliferation in NCI-H1975 cells harboring the L858R and T790M mutations by the three irreversible ERBB family inhibitors, compared with gefitinib.