EGFR-mutated lung cancer is a significant subgroup of non–small cell lung cancer. To inhibit EGFR-mediated signals, multiple EGFR tyrosine kinase inhibitors (EGFR-TKI) have been developed; however, approximately one third of patients with EGFR-mutated lung cancer do not respond to EGFR-TKIs. More effective inhibition of EGFR-mediated signals is therefore necessary. For cancers expressing mutated EGFR, including EGFR T790M, which confers resistance to first- (gefitinib and erlotinib) and second- (afatinib) generation EGFR-TKIs, the synergistic efficacy of afatinib and cetuximab combination therapy has been reported in preclinical and clinical studies; however, the mechanisms underlying this effect remain elusive. In this study, we evaluated the effects of multiple EGFR-TKIs on the EGFR monomer–dimer equilibrium by inducing dimerization-impairing mutations in cells expressing EGFR. Interestingly, we found that afatinib and dacomitinib exhibit a monomer preference: cells expressing dimerization-impaired EGFR mutants exhibited increased sensitivity to afatinib and dacomitinib relative to those with dimerization-competent EGFR mutants. Although EGFR-TKIs themselves induce dimerization of EGFR, the inhibition of dimerization by cetuximab overcame EGFR-TKI–induced dimerization. By shifting the monomer–dimer equilibrium toward monomer dominance using cetuximab, the effectiveness of afatinib and dacomitinib improved significantly. We report a novel and clinically relevant phenomenon, the monomer preference of EGFR-TKIs, which can explain the mechanism underlying the synergism observed in afatinib and cetuximab combination therapy. In addition, we propose the novel concept that monomer–dimer equilibrium is an important factor in determining EGFR-TKI efficacy. These findings provide novel insights into treatment strategies for EGFR-TKI–refractory non–small cell lung cancer.

Lung cancer is a leading cause of cancer-related deaths worldwide (1). Somatic mutations of the tyrosine kinase domain of EGFR are found in approximately 20%–40% of non–small cell lung cancer (NSCLC) cases (2–6). These somatic mutations of EGFR promote the active conformation of EGFR by destabilizing its inactive form; hence, they induce ligand-independent constitutive activation of EGFR (7–9). To overcome EGFR-mediated signals, multiple ATP-competitive EGFR tyrosine kinase inhibitors (EGFR-TKI) and anti-EGFR antibodies have been developed. Mutated forms of EGFR, such as those with deletions around the LREA motif, encoded by exon 19, or the point mutation L858R, encoded by exon 21, demonstrate increased affinity for EGFR-TKIs and decreased affinity for ATP compared with wild-type EGFR, which creates a therapeutic window for these ATP-competitive EGFR-TKIs (7, 10).

The development of first- (gefitinib and erlotinib) and second- (afatinib, dacomitinib) generation EGFR-TKIs has led to significant clinical improvements in the outcomes of patients with lung cancer harboring EGFR mutations (11–15). The response rate to these EGFR-TKIs for NSCLC harboring classic EGFR mutations, such as the abovementioned exon 19 deletion or L858R mutation, is approximately 60%–80%. In contrast, the EGFR T790M mutation accounts for about 60% of cases of resistance to first- and second-generation EGFR-TKIs (16, 17); therefore, third-generation EGFR-TKIs have been developed to target the EGFR T790M mutation (18–20). Osimertinib, a third-generation EGFR-TKI, has been clinically demonstrated to be safe and exhibits significant efficacy in the treatment of EGFR T790M–positive or -negative NSCLC (21–23); however, approximately one-third of patients with EGFR-mutated lung cancer do not respond to treatment with this EGFR-TKI, irrespective of EGFR T790M mutation status. The development of new treatment strategies for EGFR-TKI–refractory lung cancers is thus necessary. For such resistant lung cancers, the efficacy of combination therapy with afatinib and cetuximab (Erbitux), a human–mouse chimeric antibody that binds the extracellular domain of EGFR (24), has been reported in preclinical and clinical studies (25–27); however, the mechanistic basis for the observed synergistic effect is unknown. Clarification of this mechanism may provide novel insights helpful for the development of efficient treatment for EGFR-TKI–refractory NSCLC. Recently, the dimerization-independent activation of several EGFR-mutated forms, including those with the exon 19 deletion, exon 20 insertion, and T790M mutation, was reported (28). The authors proposed an association between response to cetuximab and EGFR dimerization. These findings prompted us to evaluate the effect of EGFR-TKIs and cetuximab on the monomer–dimer equilibrium of EGFR.

In this study, we assessed the monomer–dimer equilibrium of EGFR. We found a novel and clinically relevant phenomenon: a monomer preference of EGFR-TKIs, which explains the synergism of afatinib and cetuximab combination therapy. In addition, we propose the novel concept that the monomer–dimer equilibrium is an important factor in determining the efficacy of EGFR-TKIs. These findings provide novel insights relevant to the treatment strategy for refractory NSCLC resistant to EGFR-TKIs.

Cell lines

Six human NSCLC cell lines were used: PC9 [EGFR exon 19 deletion (delE746–A750)]; PC9-ER [EGFR exon 19 deletion (delE746–A750) + T790M]; PC9-AZDR; PC9-COR; A549 [EGFR wild-type]; and H1975 [EGFR L858R + T790M]. PC9 and PC9-ER cells were kindly gifted by Dr. Susumu Kobayashi (Beth Israel Deaconess Medical Center, Boston, MA). PC9-AZDR and PC9-COR were established in our previous work (29). H1975 and A549 cells were purchased from the ATCC. PC9-ER cells became resistant to erlotinib after chronic exposure to this molecule and acquisition of the EGFR T790M mutation. Cell authentication for PC9 and H1975 was performed in June 2015.

Reagents

Erlotinib and afatinib were purchased from LC Laboratories. Osimertinib and dacomitinib were purchased from Selleck Chemicals. Cetuximab was purchased from Keio University Hospital (Tokyo, Japan). Antibodies recognizing total EGFR (#2232), total AKT (#9272), phospho-AKT (S473; D9E; #4060), total p44/42 MAPK (#9102S), phospho-p44/42 MAPK (T202/204; #9101S), and GAPDH (#2118) were purchased from Cell Signaling Technology. The phospho-EGFR (Y1068) antibody (44788G) was purchased from Invitrogen/Life Technologies, and the actin antibody was purchased from Sigma-Aldrich.

Ba/F3 and NIH-3T3 stable cell lines

Ba/F3 cells stably expressing mutated EGFR were created as described previously (30). Ba/F3 cells harboring EGFR mutations were cultured in RPMI1640 growth medium supplemented with 10% FBS at 37°C in a humidified 5% CO2 incubator. NIH-3T3 cells stably expressing mutated EGFR were created as described previously (30) and cultured in DMEM supplemented with 10% FBS at 37°C in a humidified 5% CO2 incubator. The EGFR mutations examined in this study included the following: delL747_P753insS+T790M (exon 19del + T790M); L858R+T790M; exon19del+T790M+L704N, L858R+T790M+L704N, exon19del+T790M+I941R and L858R+T790M+I941R.

Cell proliferation assay

The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was performed as described previously (30). PC9, PC9-ER, A549, and H1975 cells were seeded in 96-well plates. Twenty-four hours after seeding, the appropriate medium, with or without EGFR-TKI and/or cetuximab, was added to each well. Control cells were treated with the same concentration of vehicle DMSO. Seventy-two hours after treatment, absorbance was measured. Ba/F3 cells were seeded with or without EGFR-TKI and/or cetuximab, and absorbance was measured 72 hours after seeding. All experiments were performed at least three times.

Chemical cross-linking of EGFR

Lung cancer Ba/F3 or NIH-3T3 cells harvested using trypsin were washed three times with PBS. Cells were incubated with 2.0 mmol/L bis-(sulfosuccinimidyl) suberate (BS3; Thermo Fisher Scientific) for 1 hour on ice. Reactions were quenched by adding 10 mmol/L Tris for 15 minutes at room temperature and then lysing with Cell Lysis Buffer (Cell Signaling Technology).

Immunoblotting analysis

Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology). Equal amounts of protein were loaded per lane on SDS-PAGE gels. Separated proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated overnight with primary antibodies at 4°C and then with secondary antibodies for 1 hour. For the detection of proteins, membranes were incubated with agitation in LumiGLO reagent and peroxide (Cell Signaling Technology) and then exposed to X-ray film.

Flow cytometric analysis for evaluation of EGFR cell surface expression

Cells were incubated with DMSO, 1 μmol/L of afatinib, 10 μg/mL of cetuximab, or both drugs. Treated cells were trypsinized and incubated with an anti-EGFR antibody (Thermo Fisher Scientific). Cell surface staining was assessed by flow cytometric analysis using a Gallios Flow Cytometer System (Beckman Coulter).

Apoptosis assay

Ba/F3 cells harboring EGFR exon19del+T790M, L858R+T790M, and L858R+T790M+I941R mutations were seeded in 6-well plates. Cells were treated with EGFR-TKIs for 48 hours. Afatinib was used at a concentration of 0.01–0.1 μmol/L. Cetuximab was used at 0.1 μg/mL. Control cells were treated with the same concentration of vehicle (DMSO). We analyzed cell apoptosis using the Annexin V Apoptosis Detection Kit APC (eBioscience), according to the manufacturer's protocol. The proportion of apoptotic cells was evaluated by flow cytometric analysis using a Gallios Flow Cytometer System (Beckman Coulter).

In situ proximity ligation assay

An in situ proximity ligation assay (PLA) for detection of EGFR homodimers was performed with Duolink In Situ Starter Set RED (Sigma Aldrich) according to the manufacturer's protocol. Cells were fixed for 20 minutes with 4% paraformaldehyde in PBS. Fixed cells were incubated overnight at 4°C with rabbit mAbs to EGFR (Cell Signaling Technology). Annealing of the PLUS and MINUS PLA probes occurred when two EGFR monomers were in close proximity. Repeat sequences in the annealed oligonucleotide complexes were amplified and then recognized by a fluorescently labeled oligonucleotide probe. PLA and DAPI signals were detected with a LSM710 Confocal Fluorescence Microscope (Carl Zeiss).

Mouse xenograft experiments

All animal experiments were approved by the Laboratory Animal Center, Keio University School of Medicine (Tokyo, Japan). Female BALB/c-nu mice were purchased from Charles River Laboratories. Mice were anesthetized with ketamine, and H1975 cells were injected subcutaneously in a Matrigel (Corning Inc.,) suspension. Once average tumor volume reached 200 mm3, mice were randomized to receive vehicle, cetuximab (40 mg/kg twice per week, intraperitoneally), afatinib (15 mg/kg daily, orally), or a combination of both. Subcutaneous tumors in these mice were monitored by calipers. Tumor volume (mm3) was calculated as (L × W × H)/2. Animals were humanely sacrificed, and tumor tissues were harvested. Tumor tissue cross-linking experiments were performed as described previously (31). Briefly, tumor tissues were minced with scissors in ice-cold PBS. Then, the tissues were incubated with 2.0 mmol/L BS3 for 30 minutes on ice. Reactions were quenched by adding glycine (100 mmol/L) for 10 minutes and then homogenized.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software, version 4.0 (GraphPad Software). IC50 values were also calculated using GraphPad Prism software. Student t test was used for comparisons. All P values were two-sided; results with values of P < 0.05 were regarded as statistically significant.

Efficacy of afatinib and cetuximab combination therapy in lung cancer cells

First, we performed in vitro experiments to confirm the efficacy of afatinib and cetuximab combination therapy. Combination treatment inhibited the proliferation of EGFR-mutated human lung cancer cell lines H1975 and PC9-ER significantly more than treatment with either agent alone (Fig. 1A; Supplementary Fig. S1A). Similar results were obtained for Ba/F3 cells transduced with mutated EGFR (L858R+T790M and exon 19 deletion+T790M; Fig. 1B; Supplementary Fig. S1B). In contrast, combination therapy with third-generation EGFR-TKIs, osimertinib and cetuximab, did not exhibit any synergistic effect on H1975 and PC9-ER cells (Supplementary Fig. S2A). In addition, first-generation EGFR-TKIs erlotinib and cetuximab did not exhibit any synergistic effect on Ba/F3 cells (Supplementary Fig. S2B and S2C). Next, we evaluated the effect of afatinib and cetuximab combination therapy on the apoptosis of EGFR-mutated cells. Combination therapy induced higher levels of apoptosis of Ba/F3 cells than either treatment alone (Fig. 1C). These data demonstrate the efficacy of second-generation EGFR-TKIs', afatinib, and cetuximab combination therapy on lung cancer cells.

Figure 1.

Synergistic efficacy of afatinib and cetuximab combination therapy. A, MTS assays were conducted in H1975 (n = 3) and PC9-ER (n = 3) cells treated with afatinib and/or cetuximab. Data points represent means ± SD. *, P < 0.05 by t test. B, MTS assays were conducted in Ba/F3 cells expressing EGFR L858R+T790M (n = 3) or exon19del+T790M (n = 3) treated with afatinib and/or cetuximab. Data points represent means ± SD. *, P < 0.05 by t test. C, Apoptosis assays of Ba/F3 cells harboring the indicated EGFR genotypes and treated with afatinib and/or cetuximab for 48 hours prior to staining with propidium iodide and Annexin V-APC, conducted using flow cytometry. Numbers indicate the percentages of cells in the Annexin V– and/or propidium iodide–positive quadrants. A, afatinib; C, cetuximab.

Figure 1.

Synergistic efficacy of afatinib and cetuximab combination therapy. A, MTS assays were conducted in H1975 (n = 3) and PC9-ER (n = 3) cells treated with afatinib and/or cetuximab. Data points represent means ± SD. *, P < 0.05 by t test. B, MTS assays were conducted in Ba/F3 cells expressing EGFR L858R+T790M (n = 3) or exon19del+T790M (n = 3) treated with afatinib and/or cetuximab. Data points represent means ± SD. *, P < 0.05 by t test. C, Apoptosis assays of Ba/F3 cells harboring the indicated EGFR genotypes and treated with afatinib and/or cetuximab for 48 hours prior to staining with propidium iodide and Annexin V-APC, conducted using flow cytometry. Numbers indicate the percentages of cells in the Annexin V– and/or propidium iodide–positive quadrants. A, afatinib; C, cetuximab.

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Effect of EGFR-TKIs and/or cetuximab on EGFR monomer–dimer equilibrium

To evaluate the effect of EGFR-TKIs and/or cetuximab, we performed immunoblotting on cells after BS3 cross-linking. As previously reported (32–34), cetuximab inhibited the dimerization of EGFR in H1975 and PC9 cells (Fig. 2A), indicating that cetuximab directs the monomer–dimer equilibrium of EGFR toward monomer dominance. In contrast, afatinib induced the dimerization of EGFR (Fig. 2A). Although the induction of EGFR dimerization by EGFR-TKIs has been reported previously (35), we found that the effect of afatinib was greater than that of erlotinib (Supplementary Fig. S3A). These data indicate that afatinib influences the monomer–dimer equilibrium of EGFR toward dominance of the dimer conformation. Similarly, dacomitinib and osimertinib induced EGFR dimerization (Supplementary Fig. S3A). Given the opposing effects of afatinib and cetuximab on the monomer–dimer equilibrium of EGFR, we evaluated the effect of afatinib and cetuximab combination therapy on lung cancer cells. Interestingly, afatinib or osimertinib and cetuximab combination therapy decreased EGFR dimerization to levels similar to or below those observed in controls (DMSO-treated; Fig. 2A and Supplementary Fig. S3B). The difference in the phosphorylation levels of EGFR, AKT, and ERK in H1975 and PC9-ER cells between afatinib alone and the afatinib/cetuximab combination was minimal. This may be due to the strong inhibitory effect of afatinib alone that completely blocks any EGFR phosphorylation making it difficult to observe any potentially synergistic effects of the afatinib/cetuximab combination. (Supplementary Fig. S3C). These data indicate that the effect of dimer inhibition induced by cetuximab overcomes that of dimer promotion by afatinib. To visually evaluate the effect of afatinib and/or cetuximab on the monomer–dimer equilibrium of EGFR, we performed PLA experiments. As expected, PLA experiments confirmed the increased level in the homodimerization of EGFR after EGF treatment in H1975 and PC9-ER cells (Supplementary Fig. S4). Interestingly, afatinib clearly induced the homodimerization of EGFR. In contrast, cetuximab monotherapy and afatinib plus cetuximab combination therapy inhibited the homodimerization of EGFR (Fig. 2B), consistent with the above immunoblotting results. Again, these data indicate that the dimer-inhibiting effect of cetuximab overcomes the dimer-promoting effect of afatinib (Fig. 2C) and that EGFR-TKIs and cetuximab exhibit distinctive effects on the EGFR monomer–dimer equilibrium.

Figure 2.

Effect of afatinib and cetuximab on EGFR monomer–dimer equilibrium. A, Detection of EGFR by immunoblotting in H1975 and PC9-ER cells treated with cetuximab (C; 10 μg/mL), afatinib (A; 1 μmol/L), or a combination of both (C+A) for 17 hours prior to cross-linking. β-Actin was used as loading control. B, Representative images of PLA for EGFR homodimerization in H1975 and PC9-ER cells treated with cetuximab (C; 10 μg/mL), afatinib (A; 1 μmol/L), or a combination of both (C+A). Blue, nucleus; red, EGFR homodimerization. Scale bars, 50 μm. C, Proposed model of the effects of afatinib and cetuximab on the EGFR monomer–dimer equilibrium. D, dimer; M, monomer.

Figure 2.

Effect of afatinib and cetuximab on EGFR monomer–dimer equilibrium. A, Detection of EGFR by immunoblotting in H1975 and PC9-ER cells treated with cetuximab (C; 10 μg/mL), afatinib (A; 1 μmol/L), or a combination of both (C+A) for 17 hours prior to cross-linking. β-Actin was used as loading control. B, Representative images of PLA for EGFR homodimerization in H1975 and PC9-ER cells treated with cetuximab (C; 10 μg/mL), afatinib (A; 1 μmol/L), or a combination of both (C+A). Blue, nucleus; red, EGFR homodimerization. Scale bars, 50 μm. C, Proposed model of the effects of afatinib and cetuximab on the EGFR monomer–dimer equilibrium. D, dimer; M, monomer.

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Monomer preference of afatinib and dacomitinib

To evaluate the efficacy of EGFR-TKIs in cells expressing EGFR monomers or dimers, we introduced the mutations L704N and I941R, which impair the dimerization of EGFR, and compared their effects with those of L858R+T790M and exon19del+T790M. Because Ba/F3 cells are pro-B cells whose growth depends on IL3, if transduced mutant EGFRs have transforming ability, Ba/F3 cells will be able to proliferate in the absence of IL3. Ba/F3 cells transduced with L704N, L858R+T790M+L704N, and exon19del+T790M+L704N did not grow without supplementation of IL3 in this experiment; however, Ba/F3 cells transduced with I941R, L858R+T790M+I941R, and exon19del+T790M+I941R were able to grow without IL3 (Supplementary Fig. S5) as previously reported (28), indicating that dimerization is not necessary for the activation of these mutated EGFR proteins. The inhibition of dimerization by I941R was confirmed by immunoblotting (Supplementary Fig. S6). To evaluate the efficacy of EGFR-TKIs on cells expressing dimerization-impaired EGFR, we performed MTS cell proliferation assays on cells treated with erlotinib (first generation), afatinib or dacomitinib (second generation), or osimertinib (third generation). Although no clear difference in the efficacy of erlotinib or osimertinib was observed between cells with and without dimerization-impairing mutations, interestingly, cells treated with afatinib or dacomitinib showed clear and significant differences in sensitivity depending on the presence/absence of dimerization-impairing mutations (Fig. 3A and B). The proliferation of Ba/F3 cells expressing EGFR L858R+T790M+I941R or exon19del+T790M+I941R was inhibited at significantly lower concentrations of afatinib or dacomitinib compared with that of controls. The calculated IC50 values for these experiments are summarized in Table 1. The IC50 values of afatinib and dacomitinib for Ba/F3 cells expressing dimerization-impaired EGFR were approximately 30–1,000-fold lower than those for cells expressing dimerization-competent EGFR mutations. These data indicate that afatinib and dacomitinib exhibit a monomer preference. To examine whether a synergistic effect was observed with dacomitinib and cetuximab combination therapy, we performed MTS assays using H1975 and PC9-ER cells. The synergistic effect of dacomitinib and cetuximab was confirmed in H1975 and PC9-ER cells (Supplementary Fig. S7).

Figure 3.

The monomer preference of afatinib and dacomitinib. A, MTS assays were conducted in Ba/F3 cells expressing EGFR L858R+T790M (LRTM; n = 3) or L858R+T790M+I941R (LRTM+IR; n = 3) treated with the indicated EGFR-TKIs. Data points represent means ± SD. *, P < 0.05 by t test. B, MTS assays were conducted in Ba/F3 cells expressing EGFR exon19del+T790M (DelTM; n = 3) or exon19del+T790M+I941R (DelTM+IR; n = 3) treated with the indicated EGFR-TKIs. Data points represent means ± SD. *, P < 0.05 by t test. C, Effects of EGFR-TKIs on signals downstream of EGFR in Ba/F3 cells expressing the indicated EGFR genotypes. Ba/F3 cells expressing the indicated EGFR genotypes were treated with the indicated concentrations of EGFR-TKIs for 4 hours prior to immunoblotting for the phosphorylated forms of EGFR, AKT, and ERK. β-Actin was used as a loading control. D, Apoptosis assays of Ba/F3 cells harboring the indicated EGFR genotypes treated with afatinib for 48 hours prior to staining with propidium iodide and Annexin V-APC, conducted using flow cytometry. Numbers indicate the percentages of cells in the Annexin V– and/or propidium iodide–positive quadrants. E, Proposed model of the effects of afatinib, dacomitinib, and cetuximab on the EGFR monomer–dimer equilibrium.

Figure 3.

The monomer preference of afatinib and dacomitinib. A, MTS assays were conducted in Ba/F3 cells expressing EGFR L858R+T790M (LRTM; n = 3) or L858R+T790M+I941R (LRTM+IR; n = 3) treated with the indicated EGFR-TKIs. Data points represent means ± SD. *, P < 0.05 by t test. B, MTS assays were conducted in Ba/F3 cells expressing EGFR exon19del+T790M (DelTM; n = 3) or exon19del+T790M+I941R (DelTM+IR; n = 3) treated with the indicated EGFR-TKIs. Data points represent means ± SD. *, P < 0.05 by t test. C, Effects of EGFR-TKIs on signals downstream of EGFR in Ba/F3 cells expressing the indicated EGFR genotypes. Ba/F3 cells expressing the indicated EGFR genotypes were treated with the indicated concentrations of EGFR-TKIs for 4 hours prior to immunoblotting for the phosphorylated forms of EGFR, AKT, and ERK. β-Actin was used as a loading control. D, Apoptosis assays of Ba/F3 cells harboring the indicated EGFR genotypes treated with afatinib for 48 hours prior to staining with propidium iodide and Annexin V-APC, conducted using flow cytometry. Numbers indicate the percentages of cells in the Annexin V– and/or propidium iodide–positive quadrants. E, Proposed model of the effects of afatinib, dacomitinib, and cetuximab on the EGFR monomer–dimer equilibrium.

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Table 1.

IC50 values (nmol/L) of EGFR-TKIs in Ba/F3 cells expressing EGFR mutations

MutationsaErlotinibAfatinibDacomitinibOsimertinib
LRTM >10,000 344 208 15.1 
LRTM-IR 6,730 8.37 8.3 10 
DelTM 3,610 166 137 2.47 
DelTM-IR 3,430 5.7 0.13 1.09 
MutationsaErlotinibAfatinibDacomitinibOsimertinib
LRTM >10,000 344 208 15.1 
LRTM-IR 6,730 8.37 8.3 10 
DelTM 3,610 166 137 2.47 
DelTM-IR 3,430 5.7 0.13 1.09 

aLRTM: L858R+T790M; LRTM-IR: L858R+T790M+I941R; DelTM: exon19del+T790M; DelTM-IR: exon19del+T790M+I941R.

To confirm whether the inhibitory effects of afatinib or dacomitinib on cell proliferation were exerted through the inhibition of EGFR and its downstream signals, we performed immunoblotting experiments. The phosphorylation of EGFR forms with the I941R, L858R+T790M+I941R, and exon19del+T790M+I941R mutations was confirmed (Fig. 3C). The inhibition of EGFR, AKT, and ERK phosphorylation was observed in afatinib-treated, but not erlotinib-treated, cells. Moreover, the inhibition of EGFR, AKT, and ERK phosphorylation was observed at lower concentrations of afatinib in cells expressing dimerization-impaired EGFR. Similar results were observed in dacomitinib-treated cells (Supplementary Fig. S8A). Furthermore, an upregulation in apoptosis was observed after afatinib and dacomitinib treatment of cells expressing dimerization-impaired EGFR (Fig. 3D; Supplementary Fig. S8B). These data indicate that afatinib and dacomitinib exhibit a monomer preference. Shifting the monomer–dimer equilibrium toward dominance of the monomer state thus led to a significant improvement in the efficacy of combined therapy with afatinib/dacomitinib and cetuximab (Fig. 3E).

Effect of afatinib and cetuximab on EGFR monomer–dimer equilibrium in vivo

To confirm the observed effects of afatinib and cetuximab in vivo, we performed mouse xenograft experiments. Interestingly, a clear synergistic effect of afatinib and cetuximab combination therapy was observed in this model (Fig. 4A and B). Afatinib or cetuximab monotherapy did not induce significant regression of tumor volumes; however, combination therapy induced a statistically significant regression of tumors. No clear body weight loss or skin rash was observed (Supplementary Fig. S9A and S9B), indicating tolerance to this combination therapy. To evaluate the effect of afatinib and cetuximab on the EGFR monomer–dimer status in vivo, we performed immunoblotting after cross-linking of harvested tumors. Interestingly, consistent with the in vitro experiments, afatinib promoted the dimerization of EGFR compared with that in the control group. In addition, this enhanced dimerization was abrogated in the cetuximab and combination treatment groups, although intermice variability was observed (Fig. 4C). We also confirmed that combination treatment strongly inhibited EGFR phosphorylation compared with control or afatinib or cetuximab monotherapy by immunoblotting (Supplementary Fig. S9C). These data indicate that the findings of the in vitro and in vivo experiments were consistent.

Figure 4.

Effect of afatinib and/or cetuximab on EGFR monomer–dimer equilibrium in vivo. A, H1975 tumor–bearing mice were randomized into control (n = 5), afatinib (n = 5), cetuximab (n = 4), or afatinib/cetuximab combination (n = 5) treatment groups. Tumor size was measured to calculate tumor volume. Values indicate average tumor volume in each group. *, P < 0.05 by t test for the afatinib/cetuximab combination group versus the control group or mice treated with either alone. Error bars, SD. B, Images of tumors from each group. C, Detection of EGFR by immunoblotting of proteins extracted from the H1975 tumors from each group. β-Actin was used as loading control. D, dimer; M, monomer.

Figure 4.

Effect of afatinib and/or cetuximab on EGFR monomer–dimer equilibrium in vivo. A, H1975 tumor–bearing mice were randomized into control (n = 5), afatinib (n = 5), cetuximab (n = 4), or afatinib/cetuximab combination (n = 5) treatment groups. Tumor size was measured to calculate tumor volume. Values indicate average tumor volume in each group. *, P < 0.05 by t test for the afatinib/cetuximab combination group versus the control group or mice treated with either alone. Error bars, SD. B, Images of tumors from each group. C, Detection of EGFR by immunoblotting of proteins extracted from the H1975 tumors from each group. β-Actin was used as loading control. D, dimer; M, monomer.

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In this study, we identified a novel phenomenon, the monomer preference of EGFR-TKIs, which explains the mechanism underlying the synergistic effect of afatinib and cetuximab combination therapy observed at the preclinical and clinical levels. While the underlying reason that afatinib and dacomitinib manifest a monomer preference, whereas first-generation and third-generation EGFR-TKIs do not, remains elusive, it may involve the similar structure of these second-generation EGFR-TKIs (36). This finding indicates the possibility of differences in the conformation of the tyrosine kinase domains of active monomer and dimer forms of EGFR.

As afatinib exhibits monomer preference, afatinib-induced dimerization of EGFR represents an “escape” mechanism. By inhibiting the escape of EGFR from the monomer to the dimer pool using the antibody cetuximab, which inhibits EGFR dimerization, the efficacy of afatinib is significantly increased. In addition, by inhibiting escape with dimerization-impairing mutations, the efficacy of afatinib and dacomitinib can be increased. To our knowledge, this is the first report providing evidence that the monomer–dimer equilibrium is important for the efficacy of EGFR-TKIs.

Because Watanuki and colleagues reported that internalization and subsequent degradation of EGFR affect drug sensitivity to EGFR-TKIs (37), we evaluated the EGFR expression level on the cell surface after afatinib and/or cetuximab treatment by flow cytometric analysis. We confirmed that the amount of EGFR on the cell surface was similar between DMSO-treated cells and combination-treated cells (Supplementary Fig. S10), indicating that a sufficient amount of EGFR was found on the cell surface after combination treatment. Furthermore, we need to consider the possibility that not only the amount of EGFR on the cell surface but also subcellular localization of EGFR, as reported by several studies (38–40), might have some impact on our models; however, we could not clarify the subcellular localization of dimeric and monomeric EGFR upon treatment with EGFR-TKIs and/or cetuximab.

The efficacy of afatinib and cetuximab combination therapy in EGFR T790M–negative or -positive lung cancers resistant to first- and second-generation EGFR-TKIs has been demonstrated in a clinical trial (26). The response rate for these lung cancers was 29%, indicating that a significant proportion of EGFR T790M–negative or -positive lung cancers resistant to EGFR-TKIs are still dependent on the ErbB signaling axis for survival. To improve the prognosis of patients with EGFR mutation–positive EGFR-TKI–refractory lung cancer, the development of treatments resulting in enhanced inhibition of EGFR-mediated signals is needed. In human clinical trials of afatinib or dacomitinib, severe adverse events, such as skin rash, diarrhea, or stomatitis, were observed in a significant proportion of patients with NSCLC (13, 14). By taking advantage of the synergistic effects of afatinib/dacomitinib and cetuximab combination therapy, more effective and less toxic EGFR-targeted therapy may be possible. To this end, dose-adjusted human clinical trials will be mandatory. In addition, we evaluated the efficacy of this combination therapy in previously established osimertinib-resistant lung cancer cell lines PC9-AZDR and PC9-COR (29). We found that the combination therapy was effective in PC9-COR cells (Supplementary Fig. S11), indicating that the combination treatment might be useful for some patients with EGFR-mutated lung cancer after experiencing osimertinib failure in clinic.

Here, we present a preclinical rationale for the use of afatinib or dacomitinib and cetuximab combination therapy for refractory EGFR-mutated lung cancers. In summary, we describe a novel and clinically relevant characteristic of EGFR-TKIs, that of monomer preference. These findings provide insights relevant to the treatment strategy for lung cancers refractory to treatment with EGFR-TKIs.

K. Soejima reports receiving a commercial research grant from Taiho Pharmaceutical, other commercial research support from Nippon Boehringer Ingelheim Co., Ltd and AstraZeneca K.K., and has received speakers bureau honoraria from Chugai Pharmaceutical, Ono Pharmaceutical, AstraZeneca K.K., Bristol-Myers Squibb Japan, and MSD Oncology. No potential conflicts of interest were disclosed by the other authors.

Conception and design: A. Oashi, H. Yasuda, S. Ikemura, I. Kawada, K. Soejima

Development of methodology: A. Oashi, H. Yasuda, T. Tani, J. Hamamoto, S. Ikemura, I. Kawada

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Oashi, H. Yasuda, K. Kobayashi, T. Tani, J. Hamamoto, K. Masuzawa, T. Manabe

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Oashi, H. Yasuda, K. Kobayashi, T. Tani, J. Hamamoto, K. Masuzawa, T. Manabe, H. Terai, I. Kawada, K. Naoki

Writing, review, and/or revision of the manuscript: A. Oashi, H. Yasuda, K. Kobayashi, J. Hamamoto, H. Terai, I. Kawada, K. Naoki, K. Soejima

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Oashi, H. Yasuda, K. Kobayashi, K. Naoki

Study supervision: H. Yasuda, S. Ikemura, I. Kawada, K. Naoki, K. Soejima

We would like to thank Professor Tomoko Betsuyaku for financial assistance and technical advice. We are grateful to Ms. Mikiko Shibuya and Ms. Chinatsu Yonekawa for their excellent technical assistance. We are also grateful to the Collaborative Research Resources at the Keio University School of Medicine (Tokyo, Japan) for assistance with cell sorting. This work was supported, in part, by Japan Society for the Promotion of Science (JSPS; 15K09229, to K. Soejima; 17K09667, to H. Yasuda; and 18K08184, to H. Terai). This work was supported, in part, by Takeda Science Foundation (to H. Yasuda and H. Terai).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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