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

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.

The EGFR is a membrane-bound receptor tyrosine kinase that belongs to a subfamily of 4 closely related receptors: HER1/EGFR/ERBB1, HER2/NEU/ERBB2, HER3/ERBB3, and HER4/ERBB4. Upon binding to extracellular ligands, the receptors undergo conformational changes that facilitate homo- or heterodimerization. Receptor dimerization leads to activation of downstream signaling pathways that regulate cell proliferation and survival (1).

Epithelial tumors often display aberrant expression of EGFR. Thus, a major focus of recent anticancer drug development has centered on agents that target the receptor (2). Current clinically available anti-EGFR therapies include antibodies that bind to the extracellular domain of the protein (e.g., cetuximab or panitumumab) or small-molecule tyrosine kinase inhibitors (TKIs; e.g., gefitinib or erlotinib) that selectively inhibit the kinase activity of the receptor. These agents have been FDA approved for use against colorectal, head and neck, and lung cancers. Notably, both antibodies and TKIs were originally developed to target wild-type EGFR.

In 2004, we and others reported that lung adenocarcinomas sensitive to gefitinib and erlotinib often harbor somatic mutations in exons encoding the tyrosine kinase domain of EGFR (3–5). Nearly 90% of these mutations occur as either multi-nucleotide in-frame deletions in exon 19 that eliminate 4 amino acids (LREA) or as single missense mutations that result in substitution of arginine for leucine at position 858 (L858R) (6). Both mutations lead to constitutive activation of the kinase. Expression of either mutant allele in mouse lung epithelia leads to the formation of lung tumors (7, 8). Mutant receptors also display increased affinity for drug and decreased affinity for ATP (9–11). The hypothesis that EGFR mutations are predictive of increased benefit from EGFR TKIs was recently validated in a phase III, randomized, open-label, first-line study of gefitinib versus chemotherapy (carboplatin/paclitaxel) in East Asian patients with advanced non–small cell lung cancer (NSCLC) (i.e., the IRESSA Pan Asia Study, also known as IPASS). Those with EGFR mutant tumors experienced longer progression-free survival (PFS) with gefitinib, and those without mutations had longer PFS with chemotherapy (EGFR mutation positive, hazard ratio (HR) 0.48 [95% CI, 0.36–0.64]; P < 0.0001 [favors gefitinib]; EGFR mutation negative, HR 2.85 [95% CI, 2.05–3.980]; P < 0.0001 [favors chemotherapy]) (12).

Unfortunately, after about 1 year on therapy, patients with drug-sensitive EGFR mutations whose tumors initially respond to gefitinib or erlotinib eventually develop acquired resistance (13, 14). In about half of the cases, tumors biopsied after disease progression contain a second-site mutation in the EGFR kinase domain (15–20). The most common (>90%) alteration involves a C→T change at nucleotide 2369 in exon 20, which results in substitution of methionine for threonine at position 790 (T790M). This substitution is analogous to the BCR-ABL T315I change found in patients with chronic myelogenous leukemias, who have developed acquired resistance to imatinib (Gleevec) (21). Based upon crystal structure analyses, the EGFR^T790M substitution may impair binding of either gefitinib or erlotinib to the EGFR ATP-binding pocket (22). The change could also alter the relative affinity of ATP versus drug (23). An alternative mechanism of resistance — amplification of the gene encoding the MET tyrosine kinase — occurs in about 20% of patients with acquired resistance (24). MET amplification occurs independent of T790M status (25).

Like other drug-sensitizing EGFR mutations, the T790M change by itself has been shown to increase kinase activity and oncogenic potential when compared with wild-type protein (26). Induced expression of EGFR^T790M in mouse lung epithelia leads to the formation of lung adenocarcinomas (27). Although somatic T790M mutations in patients who never received gefitinib or erlotinib are rarely detected by conventional mutational analyses (i.e., dideoxynucleotide sequencing), they can occasionally be found in tumors with primary drug resistance (28), and they exist at low frequency in untreated patients with metastatic disease (29). Certain cases of inherited susceptibility to lung cancer may also be associated with a germ line T790M mutation (30).

Preclinical studies have suggested that second-generation EGFR inhibitors may be able to overcome T790M-mediated resistance, at least in vitro (18, 31). Unlike gefitinib and erlotinib, which compete with ATP in a “reversible” manner, many of these new compounds form a covalent bond with EGFR and are thus considered “irreversible” inhibitors. Agents under evaluation include HKI-272 (32), BIBW-2992 (20, 33), and PF00299804 (34). However, no targeted agents have been clinically approved for use in patients with acquired resistance to current EGFR TKIs. The efficacy of anti-EGFR antibodies in EGFR mutant tumors also remains to be established.

To study further the biology of EGFR mutant lung tumors, our groups previously generated mouse tumor models that develop lung adenocarcinomas driven by EGFR^L858R (8), EGFR^T790M, or EGFR^L858R+T790M (27). These models employ a tetracycline–inducible (tet-inducible) system, involving bitransgenic animals. One transgene carries a tet transactivator in lung epithelia (i.e., Clara cell secretory protein – reverse tetracycline transactivator [CCSP-rtTA], herein referred to as “C” mice). The 3 relevant strains are referred to as C/L858R, C/T790M, and C/L+T, respectively. As expected, tumors harboring EGFR^L858R are sensitive to erlotinib, while tumors expressing EGFR^T790M are resistant. Here, we used “clinical trials” in the animal models in conjunction with EGFR mutant cell lines, various anti-EGFR therapies, and multiple molecular biological techniques to identify a strategy to overcome T790M-mediated resistance. Surprisingly, we found that only dual targeting of EGFR with both an antibody (cetuximab) and a second-generation EGFR TKI (BIBW-2992) was effective at targeting T790M-driven tumors. These studies have immediate therapeutic implications for lung cancer patients. Moreover, these data provide new insights into the development of agents against EGFR that could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.

Effect of BIBW-2992 in EGFR mutant mouse models of lung cancer. BIBW-2992 is one of several promising new irreversible EGFR inhibitors in clinical development. Enzymatic assays using recombinant human wild-type EGFR and HER2 indicate that the IC₅₀ values are 0.5 and 14 nmol/l, respectively (20). The agent has been shown in patients to induce regressions of lung cancers with EGFR drug-sensitizing mutations (35) and has displayed modest activity against erlotinib-resistant EGFR^T790M-harboring mouse lung tumor models (33). To confirm and extend reported results, we treated C/L858R, C/L+T and C/T790M animals with BIBW-2992. Mice were administered 25 mg/kg/d, the maximum tolerated dose (data not shown). Within days of treatment, 4 of 4 C/L858R mice displayed complete responses (CRs), as shown by a greater than 80% reduction in tumor volume on MRI (see Methods) after therapy (Figure 1 and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI38746DS1). By contrast, 0 of 7 C/L+T animals displayed CRs to the same drug; 6 showed stable disease (SD) and 1 showed progressive disease (PD). Only 1 mouse could be treated for 4 weeks; this mouse showed PD. The 6 additional mice had to be sacrificed, because they showed modest signs of respiratory distress. Two C/T790M animals treated with BIBW-2992 also showed PD (Supplemental Figure 1 and Supplemental Table 2). Upon histological examination, all T790M mice that were treated with BIBW-2992 showed viable tumor (Figure 1 and Supplemental Tables 1 and 2).

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.

Lung tumors from C/L+T mice express higher levels of the EGFR ligands, amphiregulin and epiregulin, compared with normal lung. Since BIBW-2992 displayed limited activity against lung tumors in C/L+T and C/T790M animals, we sought to identify genes regulated by expression of mutant EGFRs whose products could potentially serve as additional targets for therapy. We performed mRNA expression profiling of lung tumors from C/L858R, C/T790M, and C/L+T mice and from corresponding normal lung tissue. Unsupervised clustering showed that tumors could readily be distinguished from normal lung (Figure 2A). A more detailed analysis of the mRNA profiles from these tumors and additional ones driven by other oncogenes will be presented elsewhere (K.A. Politi et al., unpublished observations).

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.

Relevant to this study, genes encoding the EGFR ligands, amphiregulin (Areg) and epiregulin (Ereg), were among those most highly expressed in tumors compared with normal lungs (Table 1 and Supplemental Table 3). Other genes did not appear to be obviously linked to EGFR signaling. In tumors from C/L+T mice, Ereg displayed approximately 91-fold higher levels of expression (false discovery rate [FDR] 5.61 × 10^–9), while Areg levels were approximately 29-fold higher (FDR 1.33 × 10^–7) (Table 1). These findings were grossly confirmed by RT-PCR (Figure 2B). Similar results were obtained when comparing tumors from C/L858R and C/T790M animals to normal lung (Table 1 and Figure 2B). ELISAs further confirmed that levels of mouse Ereg and Areg proteins were higher in tumors compared with normal tissue (Figure 2C). Thus, induction of mutant EGFRs in lung epithelia leads to increased EGFR ligand expression, irrespective of the specific EGFR genotype.

Table 1

Two of the most highly expressed genes in tumors versus normal lung tissue include Ereg and Areg

To determine whether EGFR mutant lung adenocarcinoma cells alone might be the source of EREG and AREG, we interrogated 2 other existing microarray datasets on relevant human cell lines: our own dataset comparing erlotinib-sensitive H3255 (EGFR^L858R) cells after treatment with control (DMSO) or erlotinib (Y. Gong and W. Pao, unpublished observations) and a published dataset comparing erlotinib-resistant H1975 (EGFR^L858R+T790M) cells after treatment with control or another irreversible EGFR inhibitor, CL-387-785 (36). After 12 hours of treatment with erlotinib, compared with control-treated cells, H3255 cells displayed approximately 7- and approximately 16-fold less AREG and EREG, respectively (data not shown), while after 24 hours, analogously treated H1975 cells displayed 4.85- and 4.71-fold less AREG and EREG, respectively. These data support the possibility that EGFR ligands are derived from tumor cells themselves and act in an autocrine manner. However, the results do not exclude the possibility that Ereg and Areg are also derived from surrounding cells and play a role in paracrine signaling. For example, EREG is highly expressed in peripheral blood leukocytes, which are often recruited to tumors as part of an inflammatory response (37).

Effect of cetuximab in EGFR mutant mouse models of lung cancer. EGFR kinase domain mutants are constitutively activated but remain sensitive to further stimulation with EGFR ligands (3, 38). Increased expression of Areg and Ereg in the mouse lung tumors raised the possibility that these factors additionally contribute to EGFR mutant lung tumorigenesis. Cetuximab is a human-murine chimeric IgG1 monoclonal anti-EGFR antibody that has been shown to inhibit competitively the binding of EGF, TGF-α, and potentially other ligands to EGFR (39). To determine whether interfering with EGFR ligand binding might have a therapeutic effect in the EGFR mutant tumor models, we treated various lung tumor-bearing mice with the antibody. Cetuximab also induces downregulation and internalization of both wild-type (40) and mutant receptors (41).

Tumors from 5 of 5 C/L858R mice completely responded to cetuximab within 2 weeks (1 mg i.p. every 3 days; Figure 3A, Figure 4, and Supplemental Table 4). Histologic analysis of lung tissue confirmed a lack of viable tumor in all 5 treated mice (Figure 3A and Supplemental Table 4). By contrast, among 7 C/L+T animals treated with the same regimen of cetuximab, no CRs were observed using MRI; 2 mice displayed partial responses (PRs) and 5 animals showed SD (Figure 3B, Figure 4, and Supplemental Table 4). Additionally, 2 C/T790M animals treated with the same regimen of cetuximab displayed SD (Figure 3B, Supplemental Figure 1, and Supplemental Table 2). Histological analysis of lung tissue from the 9 mice carrying the T790M mutation and treated with cetuximab showed viable tumor (Figure 3B and Supplemental Tables 2 and 4).

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).

Reasons for the discrepant responses observed with EGFR^L858R- and EGFR^L858R+T790M-driven lung tumors are currently unclear. We confirmed via immunoprecipitation of tumor lysates using cetuximab that the antibody is able to bind to both types of mutant receptors (Supplemental Figure 2). Further experiments to elucidate mechanistic differences are under investigation and outside the scope of this study (see Discussion).

Effect of combination treatment with BIBW-2992 and cetuximab in EGFR mutant models. Previously, investigators have shown that AG1478, an experimental EGFR TKI, synergistically inhibits the growth of tumors overexpressing EGFR, when used in combination with the EGFR-specific mAb 806 (42). mAb 806, in preclinical development, binds only a transitional form of the receptor after it untethers but before forming the back-to-back, ligated, active oligomer. To determine whether analogous synergy could be achieved with BIBW-2992 and cetuximab, we treated tumor-bearing C/L+T and C/T790M animals with both drugs together for a maximum of 4 weeks. Eight of eight C/L+T animals displayed tumor shrinkage. Remarkably, 7 of these were CRs (Figure 3C, Figure 4, and Supplemental Table 1). Three of three C/T790M animals similarly showed CRs (Figure 3C, Supplemental Figure 1, and Supplemental Table 2). Histological analysis of lungs from animals displaying CRs after treatment showed either scant or no viable tumor cells (Figure 3C and Supplemental Tables 1 and 2). CRs were observed regardless of which drug was administered first as a single agent. By contrast, combinations of erlotinib plus cetuximab did not result in any CRs in C/L+T mice (Figure 4 and Supplemental Table 4). Such dramatic responses were not observed with any other attempted drug regimen, including with chemotherapy (pemetrexed or paclitaxel) (Figure 4 and Supplemental Table 5). Mice were not treated for longer periods of time or observed for tumor recurrence.

In vivo antitumor activity of BIBW-2992 with cetuximab against H1975 xeno­grafts. To assess the efficacy of the cetuximab/BIBW-2992 combination in a separate in vivo model, we treated mice bearing xenografts of H1975 cells. These lung adenocarcinoma cells harbor the EGFR L858R and T790M mutations in cis and are resistant to erlotinib in vitro (19). Consistent with results obtained from the mouse lung tumor model, the combination of cetuximab and BIBW-2992 was superior to either agent alone in 3 independent experiments (Figure 5).

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.

Effect of the cetuximab/BIBW-2992 combination on EGFR^L858R+T790M. To assess how cetuximab and BIBW-2992 could overcome T790M-mediated resistance, we examined the status of EGFR in tumor lysates derived from C/L+T mice, briefly treated with either drug alone or the combination. Compared with placebo-treated samples, cetuximab-treated lungs displayed decreased levels of total EGFR, likely due to degradation induced by the antibody (Figure 6A). By contrast, BIBW-2992–treated lungs showed decreased levels of phospho-EGFR. When cetuximab and BIBW-2992 were used together, levels of both phospho-EGFR and total EGFR were markedly depleted (Figure 6A).

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.

To confirm and extend these data, we examined the effect of single and combination drug against H1975 xenografts. Again, compared with tumors treated with vehicle control or either drug alone, tumors treated with the combination of BIBW-2992 plus cetuximab displayed dramatic inhibition of both phospho-EGFR and total EGFR (Figure 6B).

Finally, we studied the effect of the drugs against mutant EGFR in vitro. NR6 mouse fibroblasts, an NIH 3T3 line devoid of EGFR (43), were stably transduced with retroviruses carrying EGFR^L858R+T790M and treated with cetuximab, BIBW-2992, or both. NR6 transfectants treated with cetuximab showed degradation of EGFR, while cells treated with BIBW-2992 displayed lower levels of phosphorylated EGFR. The combination of cetuximab and BIBW-2992 enhanced depletion of both phosphorylated and total EGFR levels (Figure 6C). Collectively, these data suggest that CRs were induced by the double combination of anti-EGFR therapies in mice bearing EGFR^T790M-driven tumors, because only the combination could achieve sufficient levels of EGFR inactivation.

The second-site EGFR^T790M mutation is found in about half of patients whose EGFR mutant tumors develop acquired resistance to gefitinib or erlotinib. Many second-generation irreversible EGFR inhibitors, such as HKI-272 (32), BIBW-2992 (33), and PF00299804 (34), are being developed to overcome T790M-mediated resistance. However, by modeling acquired resistance in vitro, others have shown that HKI-272 can overcome T790M-mediated resistance only at suprapharmacologic concentrations (44), and we have obtained analogous results with BIBW-2992 (J. Chmielecki and W. Pao, unpublished observations). Consistent with these findings, in mice bearing tumors with EGFR^L858R+T790M that were treated with BIBW-2992, we and others (33) have not observed any CRs. Moreover, in a clinical trial of BIBW-2992 in patients with acquired resistance to an EGFR TKI, only modest activity has been observed (35). Thus, use of these agents alone may not be as effective as originally anticipated.

To find additional targets for therapy, we compared the mRNA expression profiles of lung tumors from EGFR^L858R+T790M mutant transgenic animals versus normal lung. We found that the EGFR ligands, Areg and Ereg, were more highly expressed in tumors from C/L+T mice as well as from C/T790M and C/L858R animals, at both the mRNA and protein levels, when compared with expression of control animals. Notably, our animal models were generated with transgenes encoding human EGFRs, and at least murine Ereg has been shown to bind readily to human EGFR (45).

To determine whether interfering with the binding of such ligands might have an antitumor effect, we treated tumor-bearing mice with cetuximab. This anti-EGFR antibody has been approved by the FDA for use in treating colorectal and head and neck cancers, respectively (46, 47), and addition of the antibody to systemic chemotherapy conferred a modest but superior survival over chemotherapy alone in the treatment of chemo-naive patients with unselected non–small cell lung cancer (48). The effect of cetuximab against EGFR mutant tumors is less clear. In preclinical models, although most EGFR mutant lung cancer cell lines are sensitive to treatment with EGFR TKIs, only 1 line (with an exon 19 deletion) thus far has been shown to be sensitive to treatment with the antibody (49, 50). In mouse models of lung cancer driven by EGFR^L858R, others have found, similar to data presented here, that cetuximab can induce dramatic tumor regressions (7). Data derived from patients is still relatively scant. In a report of 4 patients with tumors bearing EGFR exon 19 deletions, cetuximab induced only SD (50). After developing disease progression, all patients had PRs on treatment with gefitinib. To our knowledge, no response data to cetuximab have yet been reported for patients whose tumors harbor EGFR^L858R.

In the animals treated with cetuximab in this study, we did observe some antitumor activity. In a total of 9 mice bearing T790M mutant-positive tumors (2 C/T790M and 7 C/L+T), 7 displayed SD and 2 showed PRs. However, we did not observe any CRs. This result suggests that ligands contribute to EGFR mutant lung tumorigenesis but that antibody treatment alone is insufficient to induce substantial tumor shrinkage in the majority of cases. Consistent with this finding, some of us have found that the presence of EGFR ligands can raise the threshold for drug sensitivity of EGFR mutant lung cancer cells (I. Vivanco et al., unpublished observations).

Because dual targeted therapy against EGFR has been shown to be synergistic, at least with preclinical compounds (i.e., AG1478 with mAb806 [ref. 42]), we next tested the combination of BIBW-2992 with cetuximab in the mouse models. Remarkably, the administration of both drugs together induced CRs in 7 of 8 C/L+T animals and 3 of 3 C/T790M animals. This difference versus the effect of cetuximab (0 of 9) or BIBW-2992 (0 of 8) alone was statistically significant (P = 0.003 and P = 0.0007, respectively; Fisher’s exact test). No other treatments tested were effective at overcoming T790M-mediated resistance, including the combination of erlotinib with cetuximab. BIBW-2992 plus cetuximab was similarly synergistic in a separate relevant xenograft model.

Analysis of 3 separate biological systems (primary mouse lung tumors, human xenografts, and NR6 transfectants) revealed that the drug combination overcomes T790M-mediated resistance by targeting the mutant receptor more effectively than either agent alone. While the antibody (cetuximab) induces receptor degradation, it is insufficient to inhibit the ligand-independent activity of the mutant receptors. The kinase inhibitor (BIBW-2992) inhibits phospho-EGFR activity but only incompletely at the doses administered. Only the combination of both agents together induced depletion of both phosphorylated and total EGFR, resulting in the induction of CRs. Multiple mechanisms could explain this observation. One possibility is that BIBW-2992 increases binding of cetuximab to the cell surface. Consistent with this, AG1478 increases binding of mAb 806 to the cell surface through 2 distinct mechanisms: an immediate effect on the conformation of EGFR and a longer-term increase in cell surface underglycosylated EGFR, an event known to increase mAb 806 reactivity (51). As a consequence of increased binding, EGFR could be degraded more efficiently. A second possibility is that cetuximab and BIBW-2992 target different receptor pools. Consistent with this, cetuximab alone induces degradation of total EGFR without significantly affecting levels of phospho-EGFR, while BIBW-2992 dephosphorylates EGFR without inducing degradation of the receptor. The combination allows BIBW-2992 to inhibit more efficiently any residual kinase activity. A third possibility, in vivo at least, is that cetuximab binding leads to enhanced antibody-dependent cellular cytotoxicity (52).

At this juncture, we cannot explain why tumors in C/L858R animals respond to single-agent cetuximab, while tumors in C/L+T mice remain mostly stable. One explanation is that cetuximab-induced receptor downregulation is different for EGFR^L858R versus EGFR^L858R+T790M. Others have demonstrated that cetuximab in vitro degrades mutant EGFRs to a greater degree in lung tumor cells harboring drug-sensitive mutations than in cells harboring the double mutation (53). Interestingly, mice bearing tumors driven by EGFR^T790M alone also did not radiographically respond to single-agent cetuximab (n = 2) but did display CRs after treatment with BIBW-2992/cetuximab (n = 3). This result suggests that the difference in responses may be in part due to the T790M change itself and may not be a property of the double-mutant EGFR. Perhaps the T790M change induces conformational changes within the receptor that lead to differential partnering of mutant receptor, either with itself (homodimers) or with other EGFR-related family members (heterodimers). Another possibility is that although both mutant receptors are constitutively active, the L858R single mutant is less active but more responsive to ligand activation, while the double mutant is more active but less responsive to ligands. In this context, cetuximab is able to lower the threshold for EGFR activation in the single but not the double mutant. Finally, it is possible that the T790M change alters the amount of mutant receptor that reaches the cell surface compared with that with the L858R mutant alone. Although our immunoprecipitation studies with cetuximab showed that the antibody bound to either mutant receptor (Supplemental Figure 2), the studies were qualitative, not quantitative. We plan in future studies to address this issue in more detail.

Based upon the preclinical data shown here, we believe a trial is warranted of BIBW-2992 in combination with cetuximab for patients with EGFR mutant tumors and acquired resistance to gefitinib or erlotinib. The animals treated with both drugs appeared to tolerate the regimen without difficulty (data not shown). However, in humans we acknowledge that such treatment could lead to excess skin toxicity. Dual targeting of EGFR will also likely enrich for EGFR-independent mechanisms of acquired resistance, such as MET amplification (24, 25). Thus, future studies will need to address how to overcome resistance that develops due to both T790M and MET amplification together.

Finally, mutant receptor tyrosine kinases have served as tractable substrates for targeted cancer therapy. The dual targeting approach presented here, with both a TKI and an antibody, could serve as an important model for targeting other receptor tyrosine kinases activated in various human cancers.

View Supplemental data

We thank Mihaela Lupu and Dov Winkelman for assistance with animal MRI, Dennis Grossano for providing chemotherapeutic agents, and Gregory Riely for critical reading of the manuscript. This work was supported by the American Cancer Society (to K.A. Politi); the Labrecque Foundation (to K.A. Politi); NIH National Cancer Institute (NCI) grants K08-CA097980 (to W. Pao), R01-CA121210 (to W. Pao), K99-CA131488 (to K.A. Politi), and R01-CA120247 (to H. Varmus for K.A. Politi); Joan’s Legacy: The Joan Scarangello Foundation to Conquer Lung Cancer (to Y. Gong and W. Pao); the MSKCC Experimental Therapeutics Core (to W. Pao); the MSKCC Translational and Integrative Medicine Research Fund (to W. Pao); the Elaine Terner Cooper Fellowship (to W. Pao for L. Regales); the Doris Duke Foundation (to I.K. Mellinghoff); the Sontag Foundation (to I.K. Mellinghoff); the Sidney Kimmel Foundation (to I.K. Mellinghoff); and the Golfers against Cancer Foundation (I.K. Mellinghoff). Services provided by the Genomics Core Facility were partially supported by an NCI CCSG award to MSKCC (P30-CA008748). W. Pao received additional support from Vanderbilt’s Specialized Program of Research Excellence in Lung Cancer grant (CA90949) and the VICC Cancer Center Core grant (P30-CA68485).

Address correspondence to: William Pao, Vanderbilt-Ingram Cancer Center, 2220 Pierce Avenue, 777 Preston Research Building, Nashville, Tennessee 37232, USA. Phone: (615) 936-3831; Fax: (615) 343-7602; E-mail: william.pao@vanderbilt.edu.

Conflict of interest: The rights to a patent application on the testing of EGFR T790M mutations have been licensed on behalf of K.A. Politi, W. Pao, and others by MSKCC to MolecularMD.

Reference information: J. Clin. Invest.119:3000–3010 (2009). doi:10.1172/JCI38746.

William Pao’s present address is: Vanderbilt-Ingram Cancer Center, Nashville, Tennessee, USA.

1. Yarden, Y., Sliwkowski, M.X. 2001. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2:127-137.
   View this article via: CrossRef PubMed Google Scholar
2. Mendelsohn, J., Baselga, J. 2000. The EGF receptor family as targets for cancer therapy. Oncogene. 19:6550-6565.
   View this article via: CrossRef PubMed Google Scholar
3. Lynch, T.J., et al. 2004. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 350:2129-2139.
   View this article via: CrossRef PubMed Google Scholar
4. Paez, J.G., et al. 2004. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304:1497-1500.
   View this article via: CrossRef PubMed Google Scholar
5. Pao, W., et al. 2004. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc. Natl. Acad. Sci. U. S. A. 101:13306-13311.
   View this article via: CrossRef PubMed Google Scholar
6. Pao, W., Miller, V.A. 2005. Epidermal growth factor receptor mutations, small-molecule kinase inhibitors, and non-small-cell lung cancer: current knowledge and future directions. J. Clin. Oncol. 23:2556-2568.
   View this article via: CrossRef PubMed Google Scholar
7. Ji, H., et al. 2006. The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies. Cancer Cell. 9:485-495.
   View this article via: CrossRef PubMed Google Scholar
8. Politi, K., et al. 2006. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev. 20:1496-1510.
   View this article via: CrossRef PubMed Google Scholar
9. Carey, K.D., et al. 2006. Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib. Cancer Res. 66:8163-8171.
   View this article via: CrossRef PubMed Google Scholar
10. Yun, C.H., et al. 2007. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell. 11:217-227.
    View this article via: CrossRef PubMed Google Scholar
11. Zhang, X., Gureasko, J., Shen, K., Cole, P.A., Kuriyan, J. 2006. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell. 125:1137-1149.
    View this article via: CrossRef PubMed Google Scholar
12. Mok, T.S., et al. 2009. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N. Engl. J. Med.Online publication ahead of print.
    View this article via: PubMed Google Scholar
13. Jackman, D.M., et al. 2006. Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib. Clin. Cancer Res. 12:3908-3914.
    View this article via: CrossRef PubMed Google Scholar
14. Riely, G.J., et al. 2006. Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin. Cancer Res. 12:839-844.
    View this article via: CrossRef PubMed Google Scholar
15. Balak, M.N., et al. 2006. Novel D761Y and common secondary T790M mutations in EGFR-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors. Clin. Cancer Res. 12:6494-6501.
    View this article via: CrossRef PubMed Google Scholar
16. Kobayashi, S., et al. 2005. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 352:786-792.
    View this article via: CrossRef PubMed Google Scholar
17. Kosaka, T., et al. 2006. Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin. Cancer Res. 12:5764-5769.
    View this article via: CrossRef PubMed Google Scholar
18. Kwak, E.L., et al. 2005. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc. Natl. Acad. Sci. U. S. A. 102:7665-7670.
    View this article via: CrossRef PubMed Google Scholar
19. Pao, W., et al. 2005. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2:e73.
    View this article via: PubMed Google Scholar
20. Bean, J., et al. 2008. Acquired resistance to epidermal growth factor receptor kinase inhibitors associated with a novel T854A mutation in a patient with EGFR-mutant lung adenocarcinoma. Clin. Cancer Res. 14:7519-7525.
    View this article via: CrossRef PubMed Google Scholar
21. Gorre, M.E., et al. 2001. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science. 293:876-880.
    View this article via: CrossRef PubMed Google Scholar
22. Clark, J., Cools, J., Gilliland, D.G. 2005. EGFR inhibition in non-small cell lung cancer: resistance, once again, rears its ugly head. PLoS Med. 2:e75.
    View this article via: PubMed Google Scholar
23. Yun, C.H., et al. 2008. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl. Acad. Sci. U. S. A. 105:2070-2075.
    View this article via: CrossRef PubMed Google Scholar
24. Engelman, J.A., et al. 2007. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039-1043.
    View this article via: CrossRef PubMed Google Scholar
25. Bean, J., et al. 2007. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc. Natl. Acad. Sci. U. S. A. 104:20932-20937.
    View this article via: CrossRef PubMed Google Scholar
26. Godin-Heymann, N., et al. 2007. Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation. Cancer Res. 67:7319-7326.
    View this article via: CrossRef PubMed Google Scholar
27. Regales, L., et al. 2007. Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors. PLoS ONE. 2:e810.
    View this article via: PubMed Google Scholar
28. Inukai, M., et al. 2006. Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res. 66:7854-7858.
    View this article via: CrossRef PubMed Google Scholar
29. Maheswaran, S., et al. 2008. Detection of mutations in EGFR in circulating lung-cancer cells. N. Engl. J. Med. 359:366-377.
    View this article via: CrossRef PubMed Google Scholar
30. Bell, D.W., et al. 2005. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat. Genet. 37:1315-1316.
    View this article via: CrossRef PubMed Google Scholar
31. Carter, T.A., et al. 2005. Inhibition of drug-resistant mutants of ABL, KIT and EGF receptor kinases. Proc. Natl. Acad. Sci. U. S. A. 102:11011-11016.
    View this article via: CrossRef PubMed Google Scholar
32. Li, D., et al. 2007. Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy. Cancer Cell. 12:81-93.
    View this article via: CrossRef PubMed Google Scholar
33. Li, D., et al. 2008. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 27:4702-4711.
    View this article via: CrossRef PubMed Google Scholar
34. Engelman, J.A., et al. 2007. PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib. Cancer Res. 67:11924-11932.
    View this article via: CrossRef PubMed Google Scholar
35. .
36. Kobayashi, S., et al. 2006. Transcriptional profiling identifies cyclin D1 as a critical downstream effector of mutant epidermal growth factor receptor signaling. Cancer Res. 66:11389-11398.
    View this article via: CrossRef PubMed Google Scholar
37. Toyoda, H., Komurasaki, T., Uchida, D., Morimoto, S. 1997. Distribution of mRNA for human epiregulin, a differentially expressed member of the epidermal growth factor family. Biochem. J. 326:69-75.
    View this article via: PubMed Google Scholar
38. Greulich, H., et al. 2005. Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants. PLoS Med. 2:e313.
    View this article via: PubMed Google Scholar
39. Graham, J., Muhsin, M., Kirkpatrick, P. 2004. Cetuximab. Nat. Rev. Drug Discov. 3:549-550.
    View this article via: CrossRef PubMed Google Scholar
40. Sunada, H., Magun, B.E., Mendelsohn, J., MacLeod, C.L. 1986. Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 83:3825-3829.
    View this article via: CrossRef PubMed Google Scholar
41. Doody, J.F., et al. 2007. Inhibitory activity of cetuximab on epidermal growth factor receptor mutations in non small cell lung cancers. Mol. Cancer Ther. 6:2642-2651.
    View this article via: CrossRef PubMed Google Scholar
42. Johns, T.G., et al. 2003. Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478. Proc. Natl. Acad. Sci. U. S. A. 100:15871-15876.
    View this article via: CrossRef PubMed Google Scholar
43. Di Fiore, P.P., et al. 1987. Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell. 51:1063-1070.
    View this article via: CrossRef PubMed Google Scholar
44. Godin-Heymann, N., et al. 2008. The T790M “gatekeeper” mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor. Mol. Cancer Ther. 7:874-879.
    View this article via: CrossRef PubMed Google Scholar
45. Toyoda, H., et al. 1995. Epiregulin. A novel epidermal growth factor with mitogenic activity for rat primary hepatocytes. J. Biol. Chem. 270:7495-7500.
    View this article via: CrossRef PubMed Google Scholar
46. Bonner, J.A., et al. 2006. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N. Engl. J. Med. 354:567-578.
    View this article via: CrossRef PubMed Google Scholar
47. Jonker, D.J., et al. 2007. Cetuximab for the treatment of colorectal cancer. N. Engl. J. Med. 357:2040-2048.
    View this article via: CrossRef PubMed Google Scholar
48. Pirker, R., et al. 2009. Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX): an open-label randomised phase III trial. Lancet. 373:1525-1531.
    View this article via: CrossRef PubMed Google Scholar
49. Amann, J., et al. 2005. Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer. Cancer Res. 65:226-235.
    View this article via: PubMed Google Scholar
50. Mukohara, T., et al. 2005. Differential effects of gefitinib and cetuximab on non-small-cell lung cancers bearing epidermal growth factor receptor mutations. J. Natl. Cancer Inst. 97:1185-1194.
    View this article via: PubMed Google Scholar
51. Gan, H.K., et al. 2007. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 increases the formation of inactive untethered EGFR dimers. Implications for combination therapy with monoclonal antibody 806. J. Biol. Chem. 282:2840-2850.
    View this article via: CrossRef PubMed Google Scholar
52. Kurai, J., et al. 2007. Antibody-dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines. Clin. Cancer Res. 13:1552-1561.
    View this article via: CrossRef PubMed Google Scholar
53. Perez-Torres, M., Guix, M., Gonzalez, A., Arteaga, C.L. 2006. Epidermal growth factor receptor (EGFR) antibody down-regulates mutant receptors and inhibits tumors expressing EGFR mutations. J. Biol. Chem. 281:40183-40192.
    View this article via: CrossRef PubMed Google Scholar
54. Tichelaar, J., Lu, W., Whitsett, J. 2000. Conditional expression of fibroblast growth factor-7 in the developing and mature lung. J. Biol. Chem. 275:11858-11864.
    View this article via: CrossRef PubMed Google Scholar
55. Solca, F. 2008. Method for treating cancer harboring EGFR mutations. World International Property Organization patent WO/2008/034776, filed September 14, 2007, and issued March 27, 2008.