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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: J Thorac Oncol. 2020 Oct 7:S1556-0864(20)30765-6. doi: 10.1016/j.jtho.2020.09.019

Allele-specific role of ERBB2 in the oncogenic function of EGFR L861Q in EGFR-mutant lung cancers

Hiroki Sato 1,2, Michael Offin 3, Daisuke Kubota 1,2, Helena A Yu 3, Clare Wilhelm 3, Shinichi Toyooka 4, Romel Somwar 1,2, Mark G Kris 3, Marc Ladanyi 1,2,#
PMCID: PMC7775889  NIHMSID: NIHMS1644769  PMID: 33038514

Abstract

Introduction:

Unlike common EGFR mutations, many less common EGFR mutations remain poorly characterized in terms of oncogenic function and drug sensitivity. Herein, we characterize the subset of lung adenocarcinoma harboring EGFR L861Q through both preclinical and clinical investigations.

Experimental Design:

We reviewed clinical and genomic data from patients with EGFR-mutant lung cancer. We established cells expressing EGFR mutations and performed functional analysis of L861Q in comparison with common EGFR mutations.

Results:

Among patients with lung cancer, 3.4% (47/1367) possess an EGFR L861Q mutation. Of patients with L861Q, 23.4% (11/47) had a concurrent exon 18 mutation (typically involving G719). In vitro studies revealed that the oncogenic activity of L861Q is dependent on asymmetric dimerization. Cells expressing L861Q were less sensitive to EGFR-specific inhibitors compared to cells expressing L858R but were similarly sensitive to pan-ERBB inhibitors. In cells expressing L861Q, ERBB2 phosphorylation was significantly higher compared to cells expressing L858R and an enhanced interaction between EGFR and ERBB2 was observed in co-immunoprecipitation studies. In addition, treatment with osimertinib enhanced expression of the anti-apoptotic protein MCL1, and knockdown of ERBB2 suppressed the expression of MCL1 in L861Q, raising the possibility of differential allele-specific cross-phosphorylation of ERBB2. Moreover, compared to EGFR-specific inhibitors, pan-ERBB inhibitors exerted superior growth inhibitory effects on cells expressing compound L861Q/G719X mutations.

Conclusions:

Our results suggest that ERBB2 plays a previously unrecognized role in EGFR L861Q-driven tumorigenesis, and pan-ERBB inhibitors are likely to be more effective than selective EGFR tyrosine kinase inhibitors in this setting.

Keywords: EGFR L861Q mutation, targeted therapy, Lung adenocarcinoma, EGFR uncommon mutation, L861Q, ERBB2, pan-ERBB inhibitor

Introduction

Since the first reports in 2004, a variety of actionable somatic mutations and indels in the epidermal growth factor receptor (EGFR) have been discovered in lung adenocarcinoma.1–4 These alterations aberrantly activate receptor tyrosine kinase (RTK) signaling, leading to dysregulated cellular proliferation and oncogene-driven cancer. Molecularly directed therapy has shown significant antitumor activity against targetable mutations, representing a breakthrough in the treatment of lung adenocarcinoma.5

EGFR is a member of the ERBB family of receptor tyrosine kinase proteins, that also includes ERBB2 (a.k.a. HER2), ERBB3, and ERBB4. The ERBB family of proteins plays a key role in the regulation of cellular proliferation, differentiation, cell survival, and cell cycle progression. However, aberrant activation of ERBB protein signaling causes the development and progression of many human cancers, including lung adenocarcinoma.6 Somatic mutations in the tyrosine kinase domain of EGFR are detected in 40-50% of lung adenocarcinomas in Asians and 10-20% in Caucasians.7–9 Exon 19 deletion and exon 21 L858R point mutations, also termed ‘common mutations,’ account for approximately 80-90% of mutations in EGFR-mutant lung adenocarcinoma, and are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs).7 Uncommon mutations comprise the remaining 10-20% of EGFR mutations in lung adenocarcinoma.8,10, 11 Uncommon mutations are a highly heterogenous, with some of the better known being exon 20 insertions (approximately 6% of all EGFR mutations), the substitution mutations of G719X in exon 18 (3%), L861Q in exon 21 (1%), and S768I in exon 20 (1%).10 While the evidence for molecular targeted therapy for EGFR common mutations is well established based on several large phase III studies,1, 11–13 EGFR TKI response data for lung adenocarcinomas harboring uncommon mutations remains limited. In a post hoc analysis of the LUX-Lung trials, Yang and colleagues reported that afatinib demonstrated favorable responses in patients with tumors harboring G719X, L861Q, and S768I mutations.11 Several other studies have also suggested improved outcomes for patients with uncommon EGFR mutations upon treatment with second-generation EGFR TKIs.14–16 A phase II trial recently showed that the third-generation EGFR TKI, osimertinib was active and had favorable activity against certain uncommon mutations, although the sample sizes were small.17 In contrast, exon 20 insertions are largely resistant to clinically available EGFR TKIs, including afatinib.18, 19 Thus, responsiveness to EGFR TKIs varies among uncommon mutations, and there is no clear consensus on the optimal therapeutic strategy for patients with lung adenocarcinoma harboring uncommon mutations due to the low incidence and paucity of both experimental and clinical data.

In this study, we focused on the L861Q mutation in exon 21, one of the more prevalent uncommon mutations. Similar to G719X mutations, second-generation EGFR TKIs, including afatinib, appear efficacious for lung adenocarcinomas harboring L861Q.14–16 However, very little is known about the functional properties of this mutation and detailed mechanisms of its response to EGFR TKIs. The aim of this study was to characterize the subset of lung adenocarcinoma harboring L861Q through both preclinical and clinical investigations.

Materials and methods

Patients

This study was approved by the Memorial Sloan Kettering Cancer Center (MSK) institutional review board. We performed a retrospective analysis of all patients with non-small cell lung cancer (NSCLC) who underwent targeted next-generation sequencing at MSK from January 2014 through April 2020. MSK-IMPACT was the primary platform,20 and genomic data were available via the cBioPortal (http://www.cbioportal.org/).21, 22 The status of EGFR mutations and patient records were reviewed to collect demographic, clinical outcome, and molecular data. Results of the first MSK-IMPACT test were used to evaluate the frequency of EGFR mutations. Patients with EGFR T790M or C797X mutations were excluded from the subject of this study. To assess the efficacy of the TKIs, we evaluated time to treatment discontinuation (TTD) due to progression of disease. Patients with metastatic EGFR-mutant (L858R, Del19 and L861Q) lung cancers who underwent MSK-IMPACT between 2014 and 2018 were identified and followed through April of 2020. Most patients with EGFR L861Q were treated with a first-generation EGFR TKI, so we excluded patients who were treated with first-line osimertinib for the proper comparison among EGFR mutants. TTD was calculated from the date of metastatic diagnosis to the date of confirming progression of disease. Log-rank test and Mantel-Cox test were used to assess the differences between the groups and the Kaplan-Meier method was used to generate curves for TTD.

Plasmid construction, lentiviral generation and generation of cell lines

Wildtype EGFR was PCR amplified from HEK-293T cells and cloned into pENTR/TOPO vector. EGFR L704N, G719A, G719C, G719S, L858R, L861Q, G719A+L861Q, G719C+L861Q, G719S+L861Q, and E746_A750 deletion were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, SantaClara, CA) per the manufacturer’s protocol. The primer sequences used for mutagenesis are shown in Supplementary Table 1. Mutations were cloned into pLenti-CMV-Blast-DEST vector (Addgene, plasmid #17451) by the LR reaction using the Gateway LR Clonase II Enzyme mix (Thermo Fisher Scientific). The virus was generated by FuGENE HD (Promega, Madison, WI, USA)-mediated transfection using HEK-293T cells. Cells were subsequently infected with lentivirus followed by selection with blasticidin (10 μg/mL).

Three-dimensional structure analysis

The structure of wildtype EGFR in a complex with erlotinib was generated by SWISS-MODEL (https://swissmodel.expasy.org/) using Protein Data Bank (PDB) 4HJO (https://www.rcsb.org/) as a template. The structure of mutant EGFR was modeled using PyMOL 2.3 (https://pymol.org/2/).

Focus formation assay

Cells were seeded onto 6-well plates at a density of 100,000 cells/well. Media was changed every 3 days. Foci formation was evaluated by light microscopy on day 7.

Statistical analyses

All in vitro experiments were performed with at least triplicate determinations. Data are expressed as the mean ± standard error. Mann-Whitney U tests were applied to compare the scores between two independent groups. All data were analyzed using GraphPad Prism Ver. 7 (GraphPad Software, San Diego, CA). All statistical tests were two-sided, and a p-value less than 0.05 was considered significant.

Results

EGFR mutations in MSK clinical sequencing cohort

Among 6,024 patients with NSCLC who underwent next-generation sequencing via MSK-IMPACT,20 we identified 1,367 (22.7%) patients with somatic EGFR kinase domain mutations. A total of 1,503 mutations were detected; 136 patients had compound mutations in the EGFR kinase domain. As shown in Figure 1A, deletions in exon 19 (Del19, n=605, 40.3%) were the most frequent alterations, followed by L858R mutations (n=451, 30.0%). Among uncommon mutations, insertions in exon 20 were detected in 6.1% (n=92), followed by G719X mutation (n=71, 4.7%), L861Q mutation (n=47, 3.1%) and S768I mutation (n=40, 2.7%). Among cases with compound mutations, EGFR L858R and L861Q mutations were less frequently identified as compound mutations, compared to EGFR G719X and S768I mutations (11.3%, 51/451 for L858R; 29.8%, 14/47 for L861Q; 73.2%, 52/71 for G719X; and 95.0%, 38/40 for S768I) (Figure 1B). Of patients with compound L861Q mutation, 78.6% (11/14) had concurrent exon 18 mutations (G719X: n=9, S720F: n=2) while none had a concurrent exon 19 deletion or L858R mutation.

Figure 1. Clinical characteristics of patients with lung cancer harboring EGFR L861Q.

Figure 1.

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(A) The frequency of EGFR mutations in non-small cell lung cancer. A total of 1,503 mutations were identified among 1,367 patients. The frequency of each mutation is noted above the corresponding bar. (B) The ratio of single and compound mutations is shown in the pie chart. (C) Box plot of time to treatment discontinuation (TTD) due to progression of disease as a function of treatment and EGFR TKI inhibitor generation. Erlotinib is a first-generation inhibitor; afatinib/dacomitinib are second-generation inhibitors; and osimertinib is a third-generation inhibitor. The plus sign denotes the median. (D) Kaplan-Meier curve for TTD. TTD for first- or second-generation TKI treatment was compared between groups. (E) Kaplan-Meier curve for TTD. TTD for a first-generation TKI treatment was compared between groups.

Treatment and outcomes in patients with EGFR L861Q mutations

We then investigated the clinical course of patients with lung adenocarcinoma harboring EGFR L861Q mutation. Of 47 patients with L861Q mutation, 25 patients were metastatic during disease course, and received one or more targeted therapies sequentially before or after standard cytotoxic chemotherapy and immunotherapy (Table 1 and Supplementary table 2A). Among them, 18 patients were treated with first-or second-generation TKIs. Six patients switched to a second EGFR TKI and one switched to a third TKI. Comparing TTD among three generations of TKI including second- or third line therapy, the average of TTD for first-, second- and third-generation TKIs were 5.5, 26.1 and 9.2 months, respectively. Patients who received second-generation TKIs (afatinib or dacomitinib) had a significantly longer TTD than those who received a first- or third- generation TKI (Mann-Whitney U test, second vs first: p < 0.01, second vs third: p = 0.04) (Figure 1C). As for the three patients with compound L861Q and G719X mutations, two received afatinib and one received erlotinib as initial therapy. Afatinib also showed favorable disease control for patients with compound L861Q and G719X mutations. The detailed clinical course of these patients is summarized in Supplementary Table 2B. TTD for patients who received first- or second-generation TKIs as first line therapy was compared across 67 patients with tumors harboring EGFR L858R, 106 patients with EGFR Del19, and 17 patients with EGFR L861Q. Patients with EGFR L861Q had a significantly shorter TTD compared to patients with EGFR L858R or EGFR Del19 (comparison among three groups by log rank for trend: p < 0.001, comparison between binary cohorts by Mantel-Cox test: L861Q vs L858R: p = 0.07, L861Q vs Del19: p < 0.01) (Figure 1D and Supplementary Table 2C). Moreover, this tendency became more pronounced when the analysis was restricted to patients treated with a first-generation TKI (63 patients with EGFR L858R, 96 with EGFR Del19, and 14 with EGFR L861Q, comparison among three groups by log rank for trend: p < 0.0001, comparison between binary cohorts by Mantel-Cox test: L861Q vs L858R: p < 0.0001, L861Q vs Del19: p < 0.0001) (Figure 1E and Supplementary Table 2D). These results suggest a need for the establishment of a standard therapy alternative to a first-generation TKI for lung adenocarcinoma harboring L861Q mutation.

Table 1:

Cases with EGFR L861Q mutation

Patient Concurrent EGFR alteration Sex Age at metastasis stage Ethnicity Smoking TKI#1 TTD (months) TKI#2 TTD (months) TKI#3 TTD (months)
1 S720F F 61 IV White Former Erlotinib 7.8
2 L747F M 77 IV White Never Erlotinib 4
3 F 63 IV White Never Erlotinib 1
4 M 59 IV Asian Never Erlotinib 2
5 M 83 IIIA White Former Erlotinib 6
6 F 68 IIIB Asian Never Erlotinib 4.5
7 F 60 IIIA White Former Erlotinib 0.3
8 F 65 IV White Former Erlotinib 4
9 F 66 IV White Former Erlotinib 1.4 Afatinib 22.4 Osimertinib 12.3
10 F 72 IV White Former Erlotinib 11.1 Osimertinib 14.7
11 F 55 IIIA Asian Never Erlotinib 3.7 Osimertinib 3
12 F 54 IV White Never Erlotinib 4.2 Osimertinib 26.2
13 F 86 IV White Never Erlotinib 18.1 Osimertinib 20.5
14 G719S F 69 IV White Former Erlotinib 12.8
15 G719A M 68 IV White Former Afatinib 9.3
16 G719C F 56 IV White Former Afatinib 36.2 Erlotinib 1.4
17 F 50 IV Asian Never Afatinib N.D.
18 F 61 IV White Former Dacomitinib 36.7
19 M 70 IIIA White Former Osimertinib N.D.
20 F 66 IIIB White Former Osimertinib 2.6
21 F 65 IV White Former Osimertinib 1
22 F 83 IV White Former Osimertinib 8.4
23 F 74 IV Asian Never Osimertinib 13.1
24 F 84 IV White Former Osimertinib 1.9
25 M 79 IV White Former Osimertinib 0.9
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N.D.: not determined

ERBB2 plays an important role in cells expressing EGFR L861Q

To characterize the functional properties of the L861Q mutation, we established stable isogenic NIH-3T3, HBECp53, and Ba/F3 cells expressing EGFR wild type, L858R, E746_A750 deletion (Del19), and L861Q alterations using lentiviral plasmids harboring the respective cDNAs. Similar to Ba/F3 cells expressing L858R or Del19, Ba/F3 cells expressing L861Q can grow without IL-3, suggesting that Ba/F3 cells were transformed by the L861Q transduction (Figure 2A). There was no difference in Ba/F3 growth among these mutations. In addition, HBECp53 cells expressing L858R, Del19, or L861Q yielded EGF-independent growth (not shown). These results suggest that the induction of L861Q confers oncogenic properties as effectively as common mutations. We next examined how EGFR is activated in cells expressing L861Q. Although the formation of asymmetric dimers is indispensable for enzymatic activation and the consequent oncogenic activity of L858R, Del19 is active and oncogenic without the formation of asymmetric dimers.23, 24 We therefore introduced the dimerization-impairing L704N mutation into 3T3 cells expressing L858R, Del19, or L861Q. The introduction of a dimerization-impairing mutation abrogated phosphorylation of EGFR in cells expressing L858R or L861Q but had no effect on the phosphorylation status of EGFR in cells expressing Del19 (Figure 2B). Additionally, focus formation was also abolished by the inhibition of dimerization in cells expressing L858R or L861Q, but not Del19 (Supplementary Figure 1). These results indicated that the oncogenic activity of L861Q is dependent on asymmetric dimerization. Subsequently, to assess the effect of L861Q on the status of other ERBB family members and downstream signaling, the phosphorylation status of ERBB2, ERBB3, ERBB4, and key signaling elements were examined by western blot analysis. As shown in Figure 2C and Supplementary Figure 1B, phosphorylation of AKT and ERK1/2 was highly induced in cells expressing L858R or L861Q. Interestingly, ERBB2 and ERBB4 were more prominently activated in cells expressing L861Q, compared to cells expressing L858R. To further examine the mechanisms underlying ERBB2 and ERBB4 activation in cells expressing L861Q, we performed co-immunoprecipitation analysis to look at the association of EGFR and other ERBB family members. We found that EGFR is strongly co-immunoprecipitated with ERBB2 in cells expressing L861Q (Figure 2D). In contrast, cells expressing L861Q exhibited little co-immunoprecipitation of ERBB4 with EGFR. These results suggest the possibility that L861Q mutation enhances the interaction of EGFR with ERBB2. To gain insight into the significance of ERBB2 activation in cells expressing L861Q, we knocked down ERBB2. Depletion of ERBB2 led to suppression ERK and AKT phosphorylation in cells expressing L861Q, suggesting the importance of ERBB2 in cell growth and survival (Figure 2E). Taken together, our results indicated that through the direct interactions of EGFR and ERBB2, L861Q mutation induced the strong stimulation of ERBB2, resulting in the activation of pivotal downstream signaling pathways.

Figure 2. ERBB2 plays an important role in cells expressing EGFR L861Q.

Figure 2.

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(A) Ba/F3 cells stably expressing the indicated cDNAs were cultured without IL-3 and counted daily. Data are representative of three independent experiments conducted with duplicate determinations (mean ± SE). (B) Lysates were extracted from 3T3 cells stably expressing the indicated cDNAs and subjected to western blot analysis. (C) Cells were serum-starved for 6 h, lysed, and then probed by western blot. (D) Isogenic stable cell lines were serum-starved for 6 h, lysed, and subjected to co-immunoprecipitation followed by western blot analysis. (E) HBECp53 cells were transfected with the construct indicated and incubated for 48 h after which lysates were extracted and subjected to western blot analysis.

Pan-ERBB inhibitors impair the growth of cells expressing EGFR L861Q

To explore therapeutic strategies for lung adenocarcinomas harboring EGFR L861Q mutations, we tested its sensitivity to EGFR TKIs using established cell line models (Figure 3A, Supplementary Figure 2A, and Supplementary Table 3A). Although the induction of L861Q conferred sensitivity to all generations of EGFR TKIs (first-generation; erlotinib, second-generation; afatinib, third-generation; osimertinib), cells expressing L861Q were less sensitive to erlotinib and osimertinib compared to cells expressing L858R. However, afatinib, which can inhibit EGFR, ERBB2, and ERBB4, inhibited growth with similar potency in cells expressing L861Q or L858R. To better elucidate how these drugs affect cell growth, we examined their effect on phosphorylation of EGFR, ERBB2 and other downstream signaling proteins. As shown in Figure 3B, phosphorylation of AKT and ERK was inhibited by osimertinib in a dose-dependent fashion in cells expressing L858R but not L861Q. ERBB2 phosphorylation was also insensitive to osimertinib in L861Q-expressing cells. Moreover, compared to osimertinib, afatinib more potently inhibited phosphorylation of AKT and ERK in cells expressing L861Q. Indeed, knockdown of ERBB2 increased the sensitivity to EGFR-specific TKIs in cells expressing L861Q (Figure 3C and Supplementary Table 3B). Given the possibility that EGFR, ERBB2, and ERBB4 are highly activated in cells expressing L861Q, we hypothesized that the broad inhibition of ERBB family members may be critical for the treatment of lung adenocarcinoma harboring EGFR L861Q. Based on this hypothesis, we tested the efficacy of dacomitinib and poziotinib, irreversible pan-ERBB inhibitors. Dacomitinib and poziotinib strongly inhibited the growth of cells expressing L861Q (Figure 3D, Supplementary Figure 2B and Supplementary Table 3C). In addition, western blot analysis showed that phosphorylation of ERBB2, AKT, and ERK was effectively suppressed by dacomitinib and poziotinib (Figure 3E and Supplementary Figure 2C). To extend these results, we tested the sensitivity of a patient-derived xenograft (PDX)-derived cell line harboring EGFR L861Q, LUAD-0080 to EGFR TKIs including the pan-ERBB inhibitor, poziotinib. Poziotinib inhibited growth in this model more potently than any of the selective EGFR TKIs (Figure 3F and Supplementary Table 3D). Collectively, these results indicated that pan-ERBB inhibitors may be a better treatment option for lung adenocarcinoma harboring EGFR L861Q.

Figure 3. Pan-ERBB inhibitors inhibit the growth of cells expressing EGFR L861Q.

Figure 3.

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(A) Growth inhibition dose response curves of isogenic cell lines harboring EGFR variants were treated with each drug as indicated for 96 h. Data is representative of three independent experiments conducted with six determinations at each drug concentration (mean ± SE). (B) Isogenic cell lines harboring L858R or L861Q mutations were treated with osimertinib or afatinib at the indicated concentrations for 4 h. Cells were then lysed and subsequently subjected to western blot analysis. (C) Cells were transfected with the siRNA construct indicated. After 24 h, cells were treated with each drug as indicated for 96 h. Growth inhibition for each condition was determined. Data represent the mean ± SE of four independent experiments. (D) Isogenic cell lines, unmodified cells or cells harboring EGFR wild-type (WT) or mutant proteins, were treated with dacomitinib as indicated for 96 h. Data is representative of three independent experiments conducted with six replicate determinations (mean ± SE). (E) HBECp53 cells harboring EGFR with the L861Q mutation were treated with dacomitinib at the indicated concentration for 4 h. Then, cells were lysed and subjected to western blot analysis. (F) LUAD-0080 cells harboring the EGFR L861Q mutation were treated for 96 h with each drug as indicated. Data is representative of three independent experiments conducted with six replicate determinations (mean ± SE). EV: empty vector; WT: wild-type

Anti-apoptotic response mediated by ERBB2 contributes to the survival of cells expressing L861Q

To further interrogate the mechanisms of resistance to EGFR-specific TKIs observed in cells expressing L861Q, we assessed whether the induction of EGFR L861Q affected the apoptotic cascade. We first examined the basal expression of pro-apoptotic (BIM, BAX, and BAK) and anti-apoptotic markers (BCL2 and MCL1) at the protein level. Expression of pro-apoptotic proteins, especially BIM, was downregulated (Figure 4A). We subsequently examined how TKIs affect the expression of BIM and MCL1. As shown in Figure 4B, expression of BIM was dose-dependently enhanced by osimertinib and afatinib in both L858R- and L861Q-expressing cells, suggesting that TKI treatment promoted a pro-apoptotic response. These results are consistent with previous reports showing that the induction of BIM plays an essential role in apoptosis triggered by EGFR TKIs.25, 26 On the other hand, whereas expression of MCL1 was dose-dependently suppressed in L858R-expressing cells treated with osimertinib and afatinib, and in L861Q-expressing cells treated with only afatinib, osimertinib increased expression of MCL1 in cells expressing L861Q. Thus, this anti-apoptotic signal may dominate in cells expressing L861Q treated with osimertinib. To gain insight into this phenomenon, we performed time-course experiments to evaluate the long-term impact of osimertinib on the apoptosis cascade in cells expressing L861Q (Figure 4C). As expected, osimertinib induced the increased expression of BIM and reduced expression of MCL1 in cells expressing L858R, resulting in the cleavage of PARP. In contrast, in cells expressing L861Q, osimertinib enhanced expression of MCL1, which persisted even at 120 hours later, and cleavage of PARP was not observed. Consistent with this result, the number of living cells were increased despite treatment with osimertinib (Figure 4D). Finally, we examined whether the activation of ERBB2 played a role in the anti-apoptotic response observed in L861Q-expressing cells treated with osimertinib. ERBB2 depletion increased expression of MCL1 and BIM in L858R expressing cells. ERBB2 depletion in cells expressing L861Q increased BIM expression but did not exhibit a concurrent increase in MCL1. These results suggest that activation of ERBB2 is necessary for the anti-apoptotic response to EGFR inhibition in cells expressing L861Q. Taken together, EGFR L861Q conferred intrinsic resistance to EGFR-specific inhibition via the regulation of apoptotic proteins, which was dependent on ERBB2 activation.

Figure 4. Anti-apoptotic response mediated by ERBB2 contributes to the survival of cells expressing L861Q.

Figure 4.

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(A) Cells were serum-starved for 6 h, whole-cell lysates were then prepared and subjected to western blot analysis. (B) Isogenic cell lines were treated with osimertinib or afatinib at the indicated concentration for 4 h. Whole-cell lysates were subjected to western blot analysis. (C) HBECp53 cells harboring the L858R or L861Q mutation were treated with osimertinib (20 nM) as indicated. Then, cells were lysed and subjected to western blot analysis. (D) HBECp53 cells harboring L858R (blue) or L861Q (orange) mutations were continuously treated with osimertinib (20 nM) as indicated time. The number of living cells were calculated by trypan blue staining every 24 h. No living cells were detected in the cell line expressing the L858R mutation at day 7. Each condition was assayed with duplicate determinations and data is representative of three independent experiments (mean ± SE). (E) HBECp53 cells harboring L858R or L861Q mutations were transfected with the constructs indicated. After 48 h, cell lysates were extracted and subjected to western blot analysis.

Therapeutic options for lung adenocarcinoma harboring compound L861Q and G719 mutations

G719 point mutations were the most frequent concurrent exon 18 alteration in patients with EGFR L861Q mutations (G719A; n = 6, G719S; n = 2, G719C; n = 1), consistent with prior observations.10, 14, 27, 28 However, an optimal therapeutic strategy for compound L861Q and G719X mutations is still unclear given the paucity of mechanistic data based on in vitro studies. To address the implications of G719X mutations co-existing with L861Q, we transiently transfected EGFR L858R, L861Q, G719A, G719C, and G719S mutations into HEK-293T cells, and compared the phosphorylation status of EGFR and ERBB2 by western blot analysis. Cells expressing the L858R or L861Q mutation were used for comparison. Even though there was no difference in the expression level of total EGFR, EGFR phosphorylation was significantly reduced in cells transfected with G719X mutations, compared to cells transfected with L858R or L861Q (Figure 5A). Consistent with this result, Yun and colleagues reported that the EGFR L858R mutant has approximately 5-fold stronger kinase activity, compared to G719X mutant.29 Next, vectors expressing mutant (L861Q, L861Q+G719A, L861Q+G719C, and EGFR L861Q+G719S) EGFR were transiently transfected into HEK-293T cells, and phosphorylation of EGFR and ERBB2 was evaluated by western blot analysis. Compared to cells expressing L861Q alone, phosphorylation of EGFR was slightly decreased and ERBB2 phosphorylation was markedly reduced in cells expressing L861Q plus G719X (Figure 5B).

Figure 5. Therapeutic options for lung adenocarcinoma harboring compound L861Q plus G719 mutation.

Figure 5.

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(A and B) Forty-eight hours after transfection of HEK-293T cells with the construct expressing single or dual mutation EGFR mutations, lysates were extracted and subjected to western blot analysis. (C) Positional relationship between representative EGFR mutation sites and the ATP-binding pocket of the EGFR kinase domain in the modeled EGFR-erlotinib complex structure (PDB id: 4HJO). Residues at the mutation site of the EGFR kinase domain (G719, S768, L858, and L861) are shown in orange. Yellow: Erlotinib (shown as a stick), Blue: P-loop, Red: C-helix, Green: A-loop. (D) In-silico protein structure modeling of wildtype (WT) and mutant EGFR proteins are shown in surface. E872-K875 is omitted in models of compound mutation to improve visualization of the ATP-binding pocket. Cyan: Erlotinib (shown as a stick), Blue: P-loop, Red: C-helix, Green: A-loop, Orange: L861, Yellow: G719. (E) Isogenic Ba/F3 cells were treated with each drug at the indicated concentration for 96 h. Each condition was assayed in six replicate determinations and data is representative of three independent experiments (mean ± SE).

To assess whether G719X mutations have an impact on the structure of ATP-binding pocket in L861Q-mutated EGFR, we performed in-silico protein structure modeling. The positional relationships between the ATP-binding pocket and representative EGFR point mutations are shown in Figure 5C. Both EGFR L858 and L861 lie in the activation loop (A-loop) of the C-lobe where substitutions have little effect on the structure of ATP-binding pocket. By contrast, G719 lies in the ATP-binding loop (P-loop) of the N-lobe. We then modeled the structure of the EGFR protein with concurrent L861Q and G719X mutations. In-silico protein structure modeling showed that substitution from glycine to alanine, cysteine, or serine at G719 causes spatial restriction in the ATP-binding pocket and steric hinderance to TKI binding (Figure 5D).

Finally, we established Ba/F3 cells expressing L861Q plus G719A and examined their sensitivity to EGFR TKIs. Viability assays revealed that cells expressing L861Q plus G719A showed decreased sensitivity (8 – 58-fold reduction) to erlotinib and osimertinib compared to cells expressing L861Q alone (Figure 5E, Supplementary figure 3 and Supplementary Table 3). On the other hand, sensitivity to pan-ERBB inhibitors was relatively retained in cells expressing L861Q plus G719 although a slight decrease was observed compared to cells expressing L861Q alone. These results suggest that pan-ERBB family inhibitors may be promising treatments for lung adenocarcinomas harboring EGFR L861Q alone, and with L861Q plus G719X.

Discussion

In this large, prospectively collected, clinical cancer genomics dataset, we found that L861Q made up 3.1% of all EGFR mutations in patients with NSCLC, and that approximately 20% of L861Q mutations exist as compound mutations with G719X. Our in vitro studies demonstrated that the EGFR L861Q mutation enhanced the interaction between EGFR and ERBB2, resulting in stronger phosphorylation of ERBB2 and ERBB4 in comparison with cells expressing EGFR L858R. Whereas EGFR-specific inhibitors (erlotinib and osimertinib) could not sufficiently inhibit the activation of ERBB2 in cells expressing L861Q, pan-ERBB inhibitors (afatinib, dacomitinib and poziotinib) successfully inhibited the activation of both EGFR and ERBB2, which led to a strong inhibitory effect on the growth of cells expressing L861Q. Additionally, pan-ERBB inhibitors also exerted superior growth inhibitory effects on cells expressing compound L861Q plus G719X mutations, compared to EGFR-specific TKIs. Our results provide preclinical evidence for the effectiveness of pan-ERBB family inhibition for patients with EGFR L861Q mutation.

Similar to the L858R mutation, the L861Q mutation increases the heterodimerization affinity of EGFR through the suppression of its intrinsically disordered state, and stabilizes the active conformation of the C-helix, resulting in aberrant activation of EGFR.30, 31 Such mutations have reduced affinity for ATP and increased affinity for inhibitors, such as EGFR TKIs.29, 30 In addition, our in-silico protein modeling showed that L861Q, like L858R, has no effect on the structure of the ATP-binding pocket. The question remains as to what causes the difference in sensitivity of L858R and L861Q to first- or third-generation EGFR TKIs. Our results provide one possible explanation, namely that the higher activation of ERBB2 observed in cells expressing L861Q may play an important role in drug efficacy from the viewpoint of both the mitogenic signaling cascade and the apoptotic response. Genetic depletion of ERBB2 increased expression of BIM in both L858R- and L861Q-expressing cells. However, while the expression of MCL1 was increased by ERBB2 knockdown in cells expressing L858R, suggesting the compensatory activation of MCL1 to neutralize increased BIM, the increased expression of MCL1 was not observed in cells expressing L861Q. MCL1 is regulated by EGF signaling via EGFR or ERBB2 in several cancer types and its expression is correlated with EGFR and ERBB2 expression.32–35 Henson and colleagues found that inhibition of ERBB2 signaling reduced the expression of MCL1 in ERBB2-overexpressing breast cancer, which rendered cells sensitive to chemotherapeutic drugs.36 Taken together with our results, this suggests that targeting ERBB2 in ERBB2-dependent cancer exerts a pro-apoptotic effect via suppression of MCL1. Indeed, knockdown of ERBB2 restored sensitivity to osimertinib and erlotinib, confirming that inhibition of ERBB2 may be particularly important for the treatment of lung adenocarcinoma harboring EGFR L861Q. Considering that amplification of ERBB2 is an important mechanism of resistance to osimertinib,37, 38 selective EGFR inhibition in lung adenocarcinoma harboring L861Q, in which ERBB2 is potentially activated, might cause increased dependency on ERBB2, resulting in rapid acquisition of resistance. Therefore, pan-ERBB TKIs should be considered instead.

In our study, 23.4% of EGFR L861Q cases co-existed with mutations in exon 18, with G719X as the most common partner. There have been reports of patients harboring compound L861Q plus G719X mutations showing a better overall response rate to first-generation EGFR TKIs than those harboring a single L861Q mutation.10, 39 In contrast, our results indicated that cells expressing L861Q plus G719A showed decreased sensitivity to first, second, and third-generation EGFR TKIs compared to cells expressing L861Q alone. Additionally, we demonstrated that concurrent mutation of G719X and L861Q results in spatial restriction of the ATP-binding pocket. Yun and colleagues have previously noted that the EGFR G719S mutation can disrupt the binding between the P-loop and inhibitors in the ATP-binding pocket.29 Yang and colleagues also reported that the EGFR G719A mutation causes steric hindrance of the drug-binding pocket, resulting in resistance to osimertinib.40 These results indicate there are two different factors that can alter drug sensitivity in lung adenocarcinoma harboring L861Q plus G719X (i.e., the activation of ERBB2 by L861Q and physical hindrance of inhibitor binding by G719X). Given these two potential mechanisms, it is not surprising that TKI sensitivity was reduced in cells co-expressing L861Q and G719A in comparison to cells expressing L861Q alone. Of note, our results indicated that pan-ERBB inhibitors are superior to EGFR-specific inhibitors at inhibiting the growth of PDX-derived cells harboring EGFR L861Q and cells expressing L861Q plus G719X. Considering that concentrations of afatinib required to exhibit clinical activity are relatively high, which can lead to dose-limiting toxicity,41,42 other ERBB inhibitors, such as dacomitinib or poziotinib, might be better therapeutic options for lung adenocarcinomas harboring EGFR L861Q mutation.

In conclusion, our results indicate that the uncommon EGFR L861Q mutation, unlike common mutations in EGFR, causes prominent activation of ERBB2, resulting in decreased efficacy of EGFR-specific inhibitors. Pan-ERBB inhibitors exhibit favorable growth inhibitory effects on both single EGFR L861Q and compound L861Q plus G719X mutations. Our study provides a pre-clinical rationale for clinical trials evaluating pan-ERBB inhibitors in this molecularly distinct subset of EGFR-mutated lung adenocarcinoma.

Supplementary Material

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Acknowledgments

Financial Support:

This work was supported by the National Institutes of Health/NCI Cancer Center Support Grant P30 CA008748 and Lung Cancer Program Project grant P01 CA129243. The sponsors had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.

Conflict of interest disclosure statements:

Hiroki Sato, Daisuke Kubota, and Clare Wilhelm report no conflicts of interest to disclose. Dr. Offin reports personal fees from PharmaMar, personal fees from Novartis, personal fees from Targeted Oncology, personal fees from Bristol-Myers Squibb, personal fees from Merck Sharp & Dohme, outside the submitted work. Dr. Yu has consulted for AstraZeneca and has research funding to her institution from AstraZeneca, Lilly, Pfizer, Novartis, and Daiichi. Dr. Mark G. Kris reports personal fees from AstraZeneca, personal fees from Pfizer, personal fees from Regeneron, and personal fees from Daiichi-Sankyo outside the submitted work; and he has received honoraria for participation in educational programs from WebMD, OncLive, Physicians Education Resources, Prime Oncology, Intellisphere, Creative Educational Concepts, Peerview, i3 Health, Paradigm Medical Communications, AXIS, Carvive Systems, AstraZeneca, and Research to Practice. Funds for travel and lodging, and food and beverage have been provided by AstraZeneca, Pfizer, Regeneron, and Genentech. Dr. Kris is an employee of Memorial Sloan Kettering. Memorial Sloan Kettering has received research funding from The National Cancer Institute (USA), The Lung Cancer Research Foundation, Genentech Roche, and PUMA Biotechnology for research conducted by Dr. Kris. MSK has licensed testing for EGFR T790M to MolecularMD. Romel Somwar has received research funding from Helsinn Healthcare, Loxo Oncology and 14NER Oncology. Shinichi Toyooka had honoraria from Chugai pharmaceutical CO.LTD., AstraZeneca, Boehringer Ingelheim. Marc Ladanyi has received advisory board compensation from Boehringer Ingelheim, AstraZeneca, Bristol-Myers Squibb, Takeda, and Bayer, and research support from LOXO Oncology, Helsinn Healthcare and 14NER Oncology.

Footnotes

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Supplementary Materials

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NIHMS1644769-supplement-1.docx (21.1KB, docx)
2
NIHMS1644769-supplement-2.docx (30KB, docx)
3
NIHMS1644769-supplement-3.pptx (1.8MB, pptx)

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