Single targeting of MET in EGFR-mutated and MET-amplified non-small cell lung cancer

Abstract

Background

In EGFR-mutant and MET-amplified lung cancer resistant to EGFR inhibitors, double blockade of EGFR and MET is considered as a reasonable strategy despite increasing toxicity. This study evaluated the single MET inhibition in these specific tumours.

Methods

We investigated the efficacy of a single MET inhibitor in EGFR-mutant, MET-amplified lung cancer cells (HCC827GR) and the matched clinical cases and patient-derived cells. Acquired resistance mechanisms to single MET inhibitor were further explored.

Results

Single MET inhibitor sufficiently inhibited the EGFR downstream signalling and proliferation in the HCC827GR cells. The MET-inhibitor-sensitive clones had similar EGFR mutation allele frequency as the MET-inhibitor-resistant clones. The patients with EGFR-mutant, MET-amplified lung cancer resistant to EGFR inhibitors showed definite response to single MET inhibitor but the response duration was not durable. The MET gene copy number in their plasma circulating tumour DNA was significantly decreased during the treatment and was not re-increased after progression. In the cells resistant to single MET inhibitor, the EGFR pathway was reactivated, and gefitinib alone successfully suppressed their growth.

Conclusions

Single MET inhibition produced a short-lived response in EGFR-mutant and MET-amplified lung cancer. A further study of a novel combination therapy schedule is needed to achieve long-lasting efficacy and less toxicity.

Introduction

The epidermal growth factor receptor (EGFR) gene has been recognised as a major driver oncogene in lung cancer [1]. Small anticancer molecules blocking the activity of this gene were developed and marketed in the early 2000s. Large numbers of clinical and translational studies revealed these EGFR tyrosine kinase inhibitors (TKIs) produce dramatic and durable responses in lung cancer harbouring drug-sensitive somatic mutations on the EGFR gene, including exon 19 deletion and exon 21 L858R [2–6]. Thus, the current standard treatment for advanced non-small cell lung cancer (NSCLC) with these EGFR gene mutations is upfront treatment with an EGFR-TKI. Furthermore, the development of next-generation EGFR-TKIs continues to overcome the limitations of existing EGFR-TKIs. However, almost all lung cancer patients who have an initial excellent response to an EGFR-TKI experience acquired drug resistance and ultimately disease progression. A secondary EGFR T790M mutation in exon 20 of the EGFR gene is the most common resistance mechanism against first- or second-generation EGFR-TKIs such as gefitinib, erlotinib, or afatinib and is detected in 50-60% of EGFR-TKI-resistant tumours [7–9]. Third-generation EGFR-TKIs, which selectively and irreversibly target EGFR T790M and activating EGFR mutations, show promising efficacy in this EGFR-TKI-resistant lung cancer with EGFR T790M mutation [10]. Currently, third-generation EGFR-TKIs are the new standard treatment for chemo-naive patients with EGFR-mutant advanced NSCLC.

MET amplification is the second most common mechanism of resistance to EGFR-TKIs, regardless of the type of EGFR-TKIs [11, 12]. In a preclinical study by Engelman et al., it was first found that MET amplification leads to resistance to EGFR-TKIs through activation of the ERBB3-PI3K pathway rather than EGFR mutation [13]. Multiple translational studies determined the prevalence of MET amplification as an acquired resistance mechanism ranges from 5 to 22% in EGFR-mutant lung cancer resistant to EGFR-TKIs [12]. Recently, several clinical trials of EGFR and MET inhibitor combination therapy have been conducted in patients with EGFR-mutant lung cancer that developed MET amplification as a resistance mechanism after EGFR-TKI use [14, 15]. The TATOON Phase Ib study is the most representative study and assessed the combination of savolitinib and osimertinib in 186 patients with advanced NSCLC with EGFR mutation and MET amplification who experienced progression on an EGFR-TKI [14]. In this study, the combination therapy showed promising efficacy with a 66% response rate and thus, a few Phase 2 or 3 studies, SAVANNAH, ORCHARD and SAFFRON are ongoing to further evaluate this regimen. The majority of these clinical trials chose a combination therapy strategy involving dual inhibition of EGFR and MET based on two well-known preclinical studies which found concurrent treatment with an EGFR-TKI and a MET-TKI was more effective in inhibiting the growth of resistant cells than MET-TKI treatment alone [13, 16]. Despite its proven preclinical efficacy, treatment-related toxicity is one challenge in developing this two-drug combination approach. Concurrent administration of two different TKIs may increase the incidence of severe adverse effects because the toxicity profiles of TKIs usually overlap. For example, the frequency of diarrhoea, skin rash, fatigue, hepatitis, and pneumonitis may be increased by combined treatment with two types of TKI. Thus, only targeting MET signalling pathway has recently attracted attention due to this safety issue. Furthermore, the transactivation of EGFR signalling by MET activation can be more fundamental rationale of targeting MET in the context of EGFR [17].

This study aimed to evaluate the efficacy of single MET inhibition in EGFR-mutant lung cancer that acquired MET amplification after progression on an EGFR-TKI. This study may provide supportive evidence for the development of an innovative drug combination strategy with higher efficacy, better tolerability, and less toxicity in this patient population.

Methods

Cell line and reagents

The HCC827 cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). The cell line was maintained in RPMI-1640 supplemented with 10% foetal bovine serum (FBS). The HCC827 cell line was authenticated using short tandem repeat profiling. Gefitinib was purchased from LC Laboratories (Woburn, MA, USA), and PHA665752, AZD4547, AZD9291 and crizotinib were purchased from Selleckchem Korea (Seoul, Korea).

Gefitinib-resistant cell lines

Gefitinib-resistant HCC827GR cells were established by continuously exposing HCC827 cells to gefitinib with the stepwise escalation of drug dose from 0.01 to 1 μM. PHA665752-resistant HCC827GR_PR cells were also established using the same method. Drug resistance was confirmed with cell viability assays.

Cell viability assay

Resistant cell lines were maintained in a drug-free medium for 2 weeks. The cells were then seeded at a density of 1 × 10³ cells/well in 96-well plates. After 24 h, they were treated with different concentrations of the drug for 72 h. After that, cell viability was measured with the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Droplet digital polymerase chain reaction (PCR) for circulating tumour DNA (ctDNA) sequencing

We lysed 1 ml of plasma using MagNA Pure ctDNA Buffer Set (cat no. 0779439800, Roche, Germany) and extracted ctDNA using the automated method MagNA Pure 24 Total NA Isolation Kit (cat no. 07658036001, Roche, Germany) according to the protocol recommended by the manufacturer. Invitrogen QubitTM 1× dsDNA HS Assay Kit (cat no. 33231) was used to quantify the concentration of ctDNA according to the protocol recommended by the manufacturer. Droplet digital PCR (ddPCR) was performed using a QX200 Droplet Digital PCR system (Bio-Rad, Hercules, CA, USA). Reactions were performed according to the manufacturer’s protocol. The TaqMan Probe mix was used to detect EGFR L858R mutation and EGFR 19 deletion and to measure MET gene copy number (EGFR L858R, FAM: #10031246, HEX: #10031249; EGFR 19 deletion, FAM; #10031246, HEX; #10031249; MET CNV FAM: #10031240 HEX: #10031243). After PCR was complete, a droplet reader was used to read TaqMan Probe fluorescence in individual droplets using QuantaSoft software.

Quantitative real-time PCR (RT-PCR)

Total RNA was prepared using TRIzol (Ambion, Austin, TX, USA) and cDNA was synthesised from total RNA using Superscript II (Invitrogen, Carlsbad, CA, USA). Gene expression was investigated using RT-PCR on a Lightcycler®480 instrument (Roche, Basel, Switzerland). Experiments were run in triplicate. Reaction mixtures were prepared with Sensi FAST SYBR No-ROX mix (Bioline, London, UK) according to the manufacturer’s protocol.

Immunoblotting

Cells for immunoblot assay were collected and lysed in lysis buffer containing phosphatase inhibitor cocktail set V (#524629; MERCK, Kenilworth, NJ, USA) and protease inhibitor cocktail set III (#535140 MERCK). Cell lysates were separated using SDS-polyacrylamide gel electrophoresis, blotted onto polyvinylidene fluoride membranes (#10600030; Amersham, Little Chalfont, UK) and incubated with primary antibody overnight. Proteins were detected via incubation with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, #34095) and LAS-3000 detection system (Fujifilm, Tokyo, Japan). Antibodies for p-EGFR (Y1068_ Cat# 3777, RRID:AB_2096270), EGFR (Cat# 4267, RRID:AB_2246311), p-HER2 (Y1221/1222_ Cat# 2243, RRID:AB_490899), HER2 (Cat# 2165, RRID:AB_1069249 0), p-HER3 (Y1289_ Cat# 4791, RRID:AB_2099709), HER3 (Cat# 4561, RRID:AB _2099707), p-MET (Y1234/1235_Cat# 3077, RRID:AB_2143884), MET (Cat# 8198, RRID:AB_10858224), P-FGFR1 (Y653/654_ Cat# 3471, RRID:AB_33107 2), FGFR (Cat# 9740, RRID:AB_11178519), p-ERK (T202/Y204_ Cat# 4370, RRID:AB_231511 2), ERK (Cat# 4695, RRID:AB_390779), p-AKT (S473_ Cat# 9271, RRID:AB_329825), AKT (Cat# 9272, RRID:AB_329827), and VIMENTIN (Cat# 5741, RRID:AB_10695459) were purchased form Cell Signaling Technology (Danvers, MA, USA).

Patient-derived cells (PDCs)

PDCs were isolated from pleural effusion by density gradient centrifugation using Lymphocyte Separation Medium (LSM, #091692249 MP Biomedicals, France). After centrifugation, tumour cells were harvested and cultured in AR-5 medium (5% FBS, 1× GlutaMAX (Thermo Fisher Scientific), 1× ITS (Insulin-Transferrin-Selenium, Thermo Fisher Scientific), 1% Penicillin/streptomycin, 50 nM hydrocortisone, 1 mM Sodium Pyruvate and 1 ng/ml EGF in RPMI) at 37 °C in a 5% CO₂ atmosphere.

Receptor tyrosine kinase (RTK) array

A human phospho-RTK array kit was purchased from R&D Systems and used according to the manufacturer’s protocol (#ARY001B).

Direct sequencing

Genomic DNA was extracted from cells using a phenol-chloroform method. PCR was performed with according to the manufacturer’s protocol (SG-Bio, SG-P004EX). The primers are shown in Supplementary Table 1.

Results

Clinical efficacy of single MET inhibition

We retrospectively collected the patients who were treated with a single MET inhibitor for EGFR-mutant and MET-amplified lung cancer after progression to EGFR-TKIs between May 2020 and May 2022 in National Cancer Center Korea (South Korea, Goyang). Finally, nine patients were identified, and their clinicopathologic characteristics and treatment outcomes were summarised in Table 1. Eight patients had acquired resistance of MET amplification after the 1st or 2nd generation EGFR-TKIs treatment, while one patient had that after the 3rd generation EGFR-TKI treatment. Three single MET-targeting drugs were used; crizotinib (n = 4), REGN5093 (n = 4), and ABN-401 (n = 1). Overall, the single MET-targeting treatment showed 44.4% (4/9) of response rate and 2.8 months (95% confidence interval, 2.7–2.9) of median progression-free survival.

Table 1.

Characteristics of patients with EGFR-mutant and MET-amplified non-small cell lung cancer who received singe MET inhibitor.

Patient	Age/sex	Histology	EGFR mutation type	Prior EGFR-TKI	Single MET inhibitor				Retreatment with EGFR-TKI after MET inhibitor
					Drug	Toxicity	Response	PFS
No. 1	71/M	ADC	L858R	Afatinib	Crizotinib	No	PR (–60%)	2.0 mo	–
No. 2	57/M	ADC	19del	Gefitinib	Crizotinib	No	PR (–40%)	3.1 mo	Erlotinib (SD)
No. 3	59/M	ADC	19del	Osimertinib	Crizotinib	No	SD (–15%)	2.2 mo	Osimertinib + Crizotinib (PD)
No. 4	64/F	ADC	19del	Erlotinib	Crizotinib	Nausea	PD (+30%)	0.5 mo	Erlotinib + Crizotinib (PR)
No. 5	67/M	ADC	19del	Gefitinib	ABN-401	Skin Rash	SD (–10%)	5.2 mo	Dacomitinib (PR)
No. 6	57/M	ADC	19del	Dacomitinib	REGN5093	No	SD (–9%)	2.8 mo	Erlotinib (PD)
No. 7	78/F	ADC	L858R	Gefitinib	REGN5093	oedema	PR (–38%)	2.8 mo	–
No. 8	72/F	ADC	19del	Gefitinib	REGN5093	anorexia	SD (0%)	4.9 mo	–
No. 9	63/M	ADC	L861Q	Erlotinib	REGN5093	No	PR (–84%)	2.7 mo	–

EGFR-TKI EGFR tyrosine kinase inhibitor, PFS progression-free survival, PR partial response, SD stable disease, PD progression disease, ADC adenocarcinoma.

We further describe a patient (No. 1 in Table 1) with EGFR-mutant and MET-amplified NSCLC who showed a definite response to single treatment with the multi-targeted MET-TKI crizotinib. This was a 71-year-old male patient who was initially diagnosed with stage IV adenocarcinoma of the lung with EGFR L858R mutation and MET amplification (Fig. 1). The MET gene copy number was 6 on the tissue-based targeted next-generation sequencing (NGS). The patient initially received the second-generation EGFR-TKI, afatinib, at a dose of 40 mg daily, but his tumour rapidly progressed, with metastases to the liver and mediastinal lymph nodes within two months (Fig. 1a). At the time of progression on afatinib, the MET gene copy number in his plasma ctDNA was significantly increased whereas the EGFR L858R mutation allele frequency was decreased (Fig. 1b). This suggested that MET-amplified-resistant subclones had driven his rapid disease progression during afatinib treatment. Thus, he began treatment with crizotinib 250 mg twice daily and showed a profound tumour response with rapid symptom relief after one month of treatment. However, his disease progressed only after two months of crizotinib treatment. At the time of progression on crizotinib, the MET copy number in his plasma ctDNA was decreased but the EGFR mutation allele frequency was significantly increased. The plasma MET gene copy number was not increased again after crizotinib was discontinued (Fig. 1b). There was no MET amplification detected on fluorescence in situ hybridisation (FISH) analysis in the post-crizotinib tumour (C-MET/CEP7 ratio 1.9), in contrast to the pre-crizotinib tumour (C-MET/CEP7 ratio 2.6) (Fig. 1c). These results suggest that post-crizotinib tumour relapse was driven primarily by EGFR-mutant cells that did not carry concomitant MET gene copy number alterations.

Fig. 1. A case with response to single MET inhibition.

a Radiologic response to crizotinib in a patient with stage IV EGFR-mutant MET-amplified NSCLC resistant to afatinib. b Serial monitoring of EGFR mutation and MET gene copy number in plasma ctDNA during the treatment with an EGFR-TKI or a MET-TKI. c Dual-colour FISH was performed to detect MET amplification in the patient’s tumour tissues (CEP7 (green)/D7S522 (red).

Taken together, these clinical data suggested single MET inhibitor may induce a definite tumour response in some lung cancers with both EGFR mutation and MET amplification, even though the response duration cannot be durable.

In vitro efficacy of single MET inhibition in lung cancer cells with acquired resistance to gefitinib

We established an EGFR-mutant lung cancer cell line resistant to the selective EGFR inhibitor gefitinib (HCC827GR) which had de novo MET amplification as an acquired resistance mechanism (Fig. 2a). HCC827GR cell growth was inhibited by the selective MET inhibitor PHA665752 and completely inhibited by the combination of gefitinib and PHA665752 (Fig. 2b). To verify these findings, we performed a clonogenic cell survival assay under multiple experimental conditions. To exclude the remaining effect of previous gefitinib treatment, we cultured HCC827GR cells in a gefitinib-free media for either 3 days or 14 days before comparing their sensitivity to PHA665752. We found that the HCC827GR cells were still sensitive to single treatment with PHA665752 even after being cultured in gefitinib-free media for 14 days, with similar findings after 3 days in gefitinib-free media (Fig. 2c). In addition, PHA665752 exposure duration did not affect sensitivity to PHA665752 in the HCC827GR cells (Fig. 2d).

Fig. 2. Gefitinib-resistant cells sensitive to single MET inhibition.

a MET gene copy number variation were checked by ddPCR assay in the HCC827 and HCC827GR cells. b Cell viability of the HCC827 and HCC827GR cells against gefitinib, PHA665752 and gefitinib plus PHA665752 combination were measured after 72 h of drug treatment. The concentration of drugs was 0.01 μM, 0.01 μM, 1.0 μM, 5.0 μM and 10.0 μM. Combination treatment were performed after mixing the two drugs at the same concentration. c The HCC827GR cells were treated with PHA665752 after they had been maintained in the gefitinib-free medium for 3 days or 14 days. Crystal violet staining was done after 7 days. The bars in the histogram represent the mean of three tests. d The HCC827GR cells were exposed to PHA665752 for 3 days or 7 days and then crystal violet staining was done. The bar histograms represent the mean of three tests. e The HCC827 and HCC827GR cells were exposed to gefitinib, PHA665752 and gefitinib plus PHA665752 combination for 48 h at concentrations of 1 μM. The cell extracts were immunoblotted to detect the indicated proteins. Statistics were calculated using t tests. *P ≤ 0.05.

In the HCC827GR cells, phosphorylation of MET was upregulated while phosphorylation of EGFR was downregulated (Fig. 2e). In the parental HCC827 cells, ERK and AKT, the downstream effector molecules of EGFR, were fully activated and inhibited by an EGFR inhibitor alone, but not by a MET inhibitor alone. Moreover, an EGFR inhibitor alone suppressed both MET and p-MET expression of the parental HCC827 cells. However, in the gefitinib-resistant HCC827GR cells, the phosphorylation of ERK and AKT was sustained on a single treatment with gefitinib but suppressed by single treatment with PHA665752. This means that the HCC827GR cells depend on MET activation rather than the EGFR activation for survival and growth, unlike the HCC827 cells. We further found that combination treatment did not further inhibit phosphorylation levels of MET, ERK and AKT compared to single MET inhibition.

The EGFR gefitinib-sensitising mutation is retained in MET-amplified cells with acquired resistance to gefitinib

To investigate the mechanism underlying the efficacy of single MET inhibition in the EGFR-TKI-resistant cells, we assessed whether the EGFR-sensitive mutation was lost in those resistant cells. We separated the PHA665752-treated HCC827GR cells into live cells, which were resistant to single treatment with PHA665752, and dead cells, which were sensitive to single treatment with PHA665752, by FACS sorting with Annexin-V (Fig. 3a). Then, EGFR 19del mutation allele frequency was measured by ddPCR assay (Fig. 3b). The droplet threshold for each fluorophore used was determined based on the results of prior tests with positive controls (PC9 cells) and negative controls (lung cancer cells with no EGFR 19 deletion including A549 cells and H1975 cells) (Supplementary Fig. 1). There was no significant difference in the EGFR 19del mutation allele frequency between the live cells (Annexin-V negative) and the dead cells (Annexin-V positive) under PHA665752 treatment (Fig. 3c). This result suggests that the sensitivity to single MET inhibition in the EGFR-mutant and MET-amplified-resistant cells may not have originated from the loss of EGFR mutations.

Fig. 3. EGFR mutation status in EGFR-mutated and MET-amplified cells sensitive to single MET inhibition.

a HCC827GR cells were sorted according to annexin-V-APC and 7AAD expression levels after 48 h of treatment with DMSO or PHA665752 (0.5 µM). b The allele frequency of EGFR 19 deletion in each sorted group was measured using a ddPCR assay. The blue dots represent individual droplets having at least one copy of EGFR deletion and no copies of the EGFR wild-type gene while the green dots vice versa. MT mutation droplets, WT wild-type droplet. c Among the HCC827GR cells treated with DMSO or PHA665752, the EGFR 19 deletion allele frequencies of live cells and dead or apoptotic cells were compared. The bars in the histogram represent the mean of three tests. Statistics were calculated using paired t tests.

Reactivation of the EGFR pathway after single MET inhibition

We chronically exposed the HCC827GR cells to PHA665752 and established MET-TKI-resistant cells (HCC827GR_PR) after 25 weeks (Fig. 4a). The HCC827GR_PR cells were resistant to PHA665752 but had regained sensitivity to gefitinib (Fig. 4b). The HCC827GR_PR cells had both the spindle shape of the EGFR-TKI-sensitive parental cells (HCC827) and the branched shape of the EGFR-TKI-resistant cells (HCC827GR) (Fig. 4c). The proliferation rate of the HCC827GR_PR cells was lower than that of both the HCC827 and HCC827GR cells.

Fig. 4. Reactivation of EGFR signalling in PHA665752-resistant HCC827GR_PR cells.

a Summary of the establishment of PHA665752-resistant HCC827GR_PR cells. b Cell viability of the HCC827GR_PR cells against gefitinib, PHA665752 and gefitinib plus PHA665752 combination were measured after 72 h of drug treatment. The bars in the histogram represent the mean cell viability after treatment with 1 μM each of gefitinib, PHA665752 and gefitinib plus PHA665752 combination. c Representative photomicrograph phenotype changes and mean relative cell proliferation rates of the HCC827, HCC827CR, and HCC827GR_PR cells. d EGFR and MET mRNA expression of parental and acquired resistance cell lines were measured by RT-PCR assay. The error bars represent the mean ± SEM. Statistical analysis was performed using t test. *P ≤ 0.05; ns not significant. e The HCC827GR_PR cells were exposed to gefitinib, PHA665752 and gefitinib plus PHA665752 combination for 48 h at a concentration of 1 μM. The cell extracts were immunoblotted to detect the indicated proteins. f Radiologic response to retreatment with erlotinib following progression on crizotinib in a patient with stage IV EGFR-mutant MET-amplified NSCLC resistant to gefitinib. Patient-derived cells were established from the patient’s pleural effusion fluid before and after treatment with crizotinib. g Dual-colour FISH (CEP7 (green)/D7S522 (red)) was performed to detect MET amplification in the patient’s tumour tissues. h Drug sensitivity tests were performed for gefitinib, afatinib, osimertinib, and crizotinib using patient-derived cells which were established before and after treatment with crizotinib.

To explore the acquired resistance mechanism of the HCC827GR_PR cells, we first evaluated hot spot mutations in the EGFR and MET genes. No secondary resistance mutations in the EGFR and MET kinase domains were observed in the HCC827GR_PR cells (Supplementary Fig. 2A). We performed a RTK array and found p-EGFR was reactivated in the HCC827GR_PR cells compared to the HCC827GR cells (Supplementary Fig. 2B). RNA sequencing also revealed EGFR expression in the HCC827GR_PR cells was significantly increased compared with the HCC827GR cells, which was confirmed by RT-PCR (Fig. 4d and Supplementary Fig. 2C). In the western blotting analysis, basal p-EGFR, p-ERK, and p-AKT expression of the HCC827GR_PR cells was enhanced in the same as the HCC827 cells (Figs. 2e and 4e). Basal p-MET expression of the HCC827GR_PR cells was sustained as it was in the HCC827GR cells. Alike the HCC827 cells, single treatment with EGFR inhibitor, gefitinib, fully inhibited p-EGFR, p-ERK, and p-AKT expression and decreased total MET and p-MET expression in the HCC27GR_PR cells. On the other hand, single treatment with MET inhibitor, PHA 665752, partially inhibited p-MET expression of the HCC827GR_PR cells unlike the HCC827GR cells which p-MET expression was completely inhibited by PHA6665752. There was no significant difference in the average MET copy number between the HCC827GR_PR cells and the HCC827GR cells (6.38 vs. 5.54, t test, P = 0.85) (Supplementary Fig. 3B). Taken together, these results suggest that reactivation of the EGFR pathway drives acquired resistance to single MET inhibition in EGFR-mutant lung cancer cells with MET amplification.

We here describe one case supporting our preclinical data about the reactivation of the EGFR pathway after progression on single MET inhibition (No. 2 in Table 1). This was a 57-year-old male patient who was treated with gefitinib for stage IV adenocarcinoma of the lung with EGFR L858R mutation (Fig. 4f–h). After 23 cycles of gefitinib, the patient’s disease progressed, especially the right pleural and hepatic lesions (Fig. 4f). Genetic testing using cytological samples from pleural effusion fluid revealed de novo MET amplification without EGFR T790M mutation. Then, the patient received single treatment with crizotinib and his pleural effusion and mediastinal lymph node metastases significantly decreased. However, the patient’s response to crizotinib was not sustained and the pleural effusion and liver metastasis worsened after 3 months. In addition, the MET FISH analysis revealed there was no longer MET amplification in the post-crizotinib tumour (Fig. 4g). We performed in vitro drug sensitivity testing using his PDC samples which were serially established before and after crizotinib treatment (Fig. 4h). Interestingly, the post-crizotinib PDCs were highly sensitive to three EGFR-TKIs, including gefitinib, afatinib, and osimertinib, in contrast to the pre-crizotinib PDCs, which were resistant to all EGFR-TKIs. These results demonstrated the recovery of sensitivity to EGFR-TKIs following single treatment with crizotinib. Based on the PDC drug sensitivity testing results, the patient was treated with erlotinib and had stable disease during the treatment with that drug. A total of three patients, including this case, resumed single treatment of EGFR-TKIs after progression to MET inhibitors, and one (33.3%) patient showed partial response to the EGFR-targeting drugs (Table 1).

Intra-tumoral heterogenous resistance mechanism to single MET inhibition

In addition to EGFR, several genes were profoundly increased in the HCC827GR_PR cells, including HGF, IL6, BCL2, GAS6, NRG1, SHC2, GAB1, FGFR2 and AXL (Supplementary Fig. 2C). This result suggested an activation of alternate receptor kinases and their more immediate downstream biochemical effectors to sustain ERK and AKT signalling. Thus, we further explored the resistance mechanisms of the HCC827GR_PR single clones to verify the significance of genes upregulated in the total HCC827GR_PR cells. According to their sensitivity to gefitinib and PHA665752, we classified nine single colonies of HCC827GR_PR cells into three representative subgroups (Supplementary Fig. 3A). The first resistant subgroup (S10) constitutes the majority of the HCC827GR_PR cell population. These colonies were highly sensitive to gefitinib (IC₅₀ = 74 nM), similar to the EGFR-TKI-naive parental cells (HCC827), even though they had MET gene amplification, with copy numbers comparable to those of the HCC827GR cells (Fig. 5a, b and Supplementary Fig. 3B). The second resistant subgroup (S7) was relatively resistant to gefitinib compared to the HCC827 cells (IC₅₀ = 600 nM) and had higher copy numbers of MET gene than the HCC827GR cells (mean, 10.9 vs. 5.5, t test, P = 0.001) (Fig. 5a and Supplementary Fig. 3A). MET phosphorylation in these cells was strongly enhanced by PHA665752 in contrast to the other subgroups (Fig. 5b). It seems that increased MET amplification enhanced MET signalling, which reduced sensitivity to EGFR-TKIs. The third resistant subgroup (S6) was also relatively resistant to EGFR-TKIs compared to the HCC827 cells and had FGFR signalling activation as a bypass mechanism to activate the EGFR pathway (Fig. 5c). The combination of a FGFR inhibitor, AZD4547, and gefitinib produced a synergistic effect, inhibiting the cell growth in these colonies (Fig. 5d).

Fig. 5. Different resistance mechanisms of subclones in PHA665752-resistant HCC827GR_PR cells.

a Cell viability of S10, S7, S6 subgroups of HCC827GR cells against gefitinib, PHA665752 and gefitinib plus PHA665752 combination were measured after 72 h of drug treatment. The means of three replicates ±SE are shown. Statistics were calculated using Kruskal–Wallis test among S10, S7, and S6. *P ≤ 0.05. ns, not significant. b Three representative colonies of HCC827GR_PR cells were exposed to PHA665752 for 48 h at a concentration of 1 μM. The cell extracts were immunoblotted to detect the indicated proteins. c Immunoblots of S10, S7, S6 colonies of HCC827GR cells after treatment of gefitinib, PHA665752 and gefitinib plus PHA665752 combination indicate the status of p-FGFR1 protein activation. d Cell viability of three representative colonies of HCC827GR_PR cells against gefitinib, AZD4547 (FGFR inhibitor) and genfitinib plus AZD4547 combination were measured after 72 h of drug treatment. The concentration of drugs was 0.01 μM, 0.01 μM, 1.0 μM, 5.0 μM and 10.0 μM. Combination treatment were performed after mixing the two drugs at the same concentration. e Radiologic progression on osimertinib plus crizotinib following progression on crizotinib in a patient with stage IV EGFR-mutant MET-amplified NSCLC resistant to erlotinib. Patient-derived cells (PDC) was serially established from the patient’s pleural effusion fluid. f Dual-colour FISH (CEP7 (green)/D7S522 (red)) was performed to detect MET amplification in the patient’s tumour cells. g Serial monitoring of EGFR mutation and MET gene copy number variation in plasma ctDNA during the treatment with an EGFR-TKI and a MET-TKI. h Drug sensitivity test for osimertinib, crizotinib, pazopanib, and carbozantinib were performed using PDC samples established in the indicated period. The means of three replicates ±SE are shown. Statistics were calculated using t tests between PDC1 and PDC2 or PDC3. *P ≤ 0.05. G + P   Gefitinib and PHA 665752 Combination treatment.

We present a case related to EGFR-TKI-resistant cells with FGFR signalling activation (No. 3 in Table 1). A 59-year-old male patient received erlotinib as first line treatment for stage IV adenocarcinoma of the lung with EGFR 19 deletion (Fig. 5e–h). After 18 cycles of erlotinib, a left pleural lesion progressed and left pleurectomy was performed to evaluate acquired resistance mechanisms (Fig. 5e). Genomic analysis of the resected tumour identified MET amplification together with EGFR T790M mutation. We established PDCs using the resected tumour sample and evaluated in vitro sensitivity to osimertinib, crizotinib, and both agents in combination (Fig. 5h). The initial drug sensitivity test revealed the sensitivity to crizotinib (IC₅₀ = 0.76 μM) was similar to the sensitivity to osimertinib (IC₅₀ = 0.60 μM). The patient was started on osimertinib in accordance with practical guidelines but showed disease progression after three months. Then, single treatment with crizotinib was given. After 1 month on crizotinib, his tumour burden was decreased and his respiratory symptoms also dramatically improved. However, the response to crizotinib lasted two months. The post-crizotinib tumour showed no longer MET amplification on the FISH analysis and plasma ctDNA MET gene copy number was significantly decreased after crizotinib (Fig. 5f, g). The post-crizotinib PDCs were resistant to osimertinib and crizotinib on their own, but the two drugs in combination showed a synergistic effect (Supplementary Fig. 4A). Thus, he was treated with osimertinib 80 mg daily and crizotinib 200 mg twice daily. However, the patient’s disease continued to progress, and he passed away after two months. The other new finding of the post-crizotinib PDC analysis was that the sensitivity of the PDCs to a FGFR inhibitor was increased after progression on crizotinib (Fig. 5h). The IC₅₀ of pazopanib, an inhibitor of multiple tyrosine kinases including FGFR1, deceased by half after progression on single treatment with crizotinib. Notably, the post-crizotinib PDCs were more sensitive to cabozantinib, which targets both MET and FGFR1, than to pazopanib. Furthermore, the targeted sequencing analysis, which detected FGFR1 gene amplification in the post-crizoinib tumour, supports the results of the PDC analysis (Supplementary Fig. 4B).

Discussion

Concurrent two-drug combination therapy has been generally adopted as a treatment strategy for the management of resistance to targeted therapy in oncogene-addictive tumours. This approach can be biologically ideal but practically not feasible due to increased toxicities leading to treatment discontinuation. Single treatment with an anticancer drug targeting the resistance mechanism would be expected to be less toxic but remains under investigation because of efficacy. This study evaluated single MET inhibition in terms of the anti-tumour efficacy in patients with EGFR-mutant NSCLC that acquired MET-amplification after progression on an EGFR-TKI.

Using diverse experimental models, we demonstrated that some tumours are dominantly dependent on MET signalling and thus are sensitive to single treatment with a MET inhibitor even though they have both EGFR mutations and MET amplification. At first, we were unsure whether cancer cells with MET amplification responsive to single MET inhibition had lost their original EGFR mutations. However, further experiments using cell sorting techniques showed that the MET-TKI-sensitive cells also had EGFR mutations but were no longer dependent on EGFR signalling, unlike the parental EGFR-TKI-sensitive cells. These findings are line with the most recent study by Eser et al. [18]. The authors also identified MET-exclusive dependence even in the presence of retained mutant EGFR activation and high sensitivity to single MET inhibition in xenograft models derived from patients with EGFR-mutant, MET-amplified lung cancer resistant to EGFR-TKIs [18]. Thus, they proposed single-agent MET-TKI treatment in the patients with tumours having such biology in that study.

In this study, the median progression-free survival for single MET treatment was 2.8 months. The response duration of single MET treatment seems to be quite short compared to that of dual targeting treatment of the TATTON study which showed 48% of response rate and 7.6 months of median progression-free survival [14]. Multiple patient reports, including our study cases, showed that single MET inhibition did not induce a durable response in these specific lung cancer patients (Supplementary Table 2) [19–23]. Wang et al. reported on eight patients with EGFR-mutant NSCLC with MET-amplification who progressed on an EGFR-TKI and were treated with crizotinib alone. They showed a 50% response rate with a median progression-free survival of 6.0 months [20]. In most previous reports, single treatment with a MET-TKI resulted in rapid symptom improvement with dramatic tumour shrinkage but the response to the MET-TKI was not durable. As seen in previous studies, our study presented clinical cases with advanced NSCLC having EGFR mutation and secondary MET-amplification who showed a profound but short-lived response to single treatment with crizotinib. Interestingly, van Veggel et al. demonstrated that in post-crizotinib tumours, MET/CEP9 ratios were not high and MET gene copy numbers were lower than in pre-crizotinib tumours in the same patient population [21]. Based on these findings, the authors concluded that MET-amplified EGFR-TKI-resistant cells are subclones, and thus, the anti-tumour efficacy of single MET inhibition may be short-lived and heterogeneous. In our study, MET gene copy number in plasma ctDNA was significantly decreased after short-duration treatment with crizotinib and did not increase again even after the disease progressed on crizotinib. Furthermore, the post-crizotinib tumours did not have the MET amplification which had been observed in the pre-crizotinib tumours, similar to van Veggel et al. All these findings suggest that the tumour cells acquiring de novo MET amplification following EGFR-TKI treatment may be minor clones and transient single MET inhibition could eradicate those kinds of tumour cells. And thus, we ultimately need to identify the major other clones that drive drug resistance as well as the MET-amplified resistant minor subclones.

Our preclinical study using established resistant cell lines indicates it is possible that single MET inhibition may re-sensitise tumour cells to EGFR-TKIs in EGFR-mutant lung cancer cells resistant to EGFR-TKIs. In the first case of our study (Fig. 1), the EGFR mutation allele frequency in plasma ctDNA was significantly increased after the single treatment with crizotinib. Additionally, in the second case (Fig. 4f–h), the PDC sample from the pleural effusion fluid collected at the time of progression on crizotinib showed high sensitivity to an EGFR-TKI which was not seen in the pre-crizotinib PDC samples. In this case, retreatment with erlotinib provided the clinical benefit of disease stabilisation to the patient even though the tumour response was not as dramatic as seen in the PDC sample. However, in some resistant tumours, other resistance mechanisms may exist concurrently with reactivation of the EGFR pathway. For example, in the third case (Fig. 5e–h), FGFR pathway activation seemed to result in disease progression after single treatment with a MET-TKI, acting as a bypass resistance mechanism that activated the EGFR pathway. This was also demonstrated in a resistant subclone population (HCC827GR_PR_S6) in our preclinical study. Moreover, in the same case, the patients showed no response to the combination of osimertinib and crizotinib in contrast to the PDC sample. This could be attributed to the molecular heterogeneity of the extensively pre-treated tumour, suggesting the concomitant presence of several mechanisms of acquired drug resistance involving different receptor tyrosine kinases.

Acquired drug resistance is the most challenging issue encountered when using molecular-targeted drugs for driver oncogene-addictive tumours. Thus, recent clinical trials in patient populations with these tumours have focused on concurrent combination therapy to overcome multiple drug resistance mechanisms. However, we also have to consider whether combination therapy can increase the risk of adverse effects compared to each single therapy. A Phase I study of crizotinib plus dacomitinib demonstrated that the combination was associated with increased toxicity compared with either agent alone [24]. Even in the most recent Phase I/IIb study of capmatinib and gefitinib, 27 of the total 161 patients (17%) stopped the two drug treatments and 71 (44%) required dose adjustment or interruption due to adverse effects [25]. The proportion of patients experiencing adverse effects which led to dose reduction in this combination study was much higher than the 23% in the Phase II study with the same MET inhibitor alone [26]. Moreover, potential pharmacokinetic drug interactions can make it difficult to continuously use the effective dose of each drug. Actually, in a Phase I study with crizotinib plus erlotinib in 27 patients with advance NSCLC, crizotinib increased the area under the plasma concentration-time curve for erlotinib by 1.5-fold to 1.8-fold [27]. As a result, the maximum tolerated dose of this regimen was determined to be crizotinib 150 mg twice daily with erlotinib 100 mg once daily, which is less than the approved standard dose of each drug. Thus, we might consider an intermittent or sequential drug schedule to overcome these limitations of combined EGFR-TKI and MET-TKI therapy. Some preclinical studies have evaluated new combination strategies to obtain long-term efficacy without higher toxicity. For example, Wang et al. demonstrated that AXL-low expressing EGFR-mutated lung cancer cells are highly sensitive to osimertinib, but IGF-1R-mediated drug tolerance develops in some subclones [28]. The researchers designed a new therapeutic schedule for the long-term use of an IGF-1R inhibitor and osimertinib. Thus, they found that transient treatment with a combination of an IGF-1R inhibitor and osimertinib followed by continuous treatment with osimertinib eliminated tumours and prevented recurrence even after osimertinib cessation without significant weight loss in mice. Based on Wang et al. transient treatment with a combination of a MET inhibitor and an EGFR inhibitor followed by maintenance therapy with an EGFR inhibitor might be considered a novel combination approach for patients with EGFR-mutant MET-amplified NSCLC resistant to EGFR-TKIs.

The number of patients included in this study was too small to draw the final conclusion, even though genetic study and functional experiments using the corresponding PDCs were parallelly conducted to support the clinical findings. Similar to our findings, one randomised Phase II study has recently started to evaluate the efficacy of only savolitinib, MET inhibitor, in patients with EGFR- mutated and MET-amplified, advanced NSCLC who have progressed following treatment with osimertinib (SAVANNAH, NCT03778229). This retrospective study might be validated by that prospective clinical study in the future.

In conclusion, some EGFR-TKI-resistant lung cancers with EGFR mutation and MET amplification may be dependent on only the MET signalling pathway for resistance and thus single MET inhibition may be effective enough to inhibit the growth in those tumours. However, the anti-tumour effect of single MET inhibition is expected to be short-lived. To obtain a long-lasting response with less toxicity in these lung cancer subsets, further studies are needed to determine the optimal sequence, duration, and doses of EGFR-TKIs and MET-TKIs.

Author contributions

Y-RC: methodology, validation, formal analysis, investigation, data curation, writing—original draft, visualisation, writing—review & editing, project administration. EHK, SK, S‑YP and J-YH: resources and methodology. YL: conceptualisation, methodology, validation, formal analysis, investigation, data curation, writing—original draft, visualisation, writing—review & editing, project administration, supervision and funding acquisition.

Funding

This study was supported by a National Cancer Center grant (grant number 2210740-2).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Competing interests

Youngjoo Lee; consulting fee: Roche, Merck, Yuhan, Bayer. Ji-Youn Han; research grants: Roche, ONO, Pfizer and Takeda; consulting fee: Astra Zeneca, BMS, Eli Lilly, Merck, Novartis, Pfizer, Abion, Jints Bio; honoraria for lecture: Astra Zeneca, BMS, Merck, Takeda and Novartis. The remaining authors declare no competing interests.

Ethics approval and consent to participate

This study was performed with approval from the National Cancer Center Institutional Review Board (approval number NCC2016-0208). All patients provided written informed consent.

Consent for publication

Not applicable.

Supplementary information

The online version contains supplementary material available at 10.1038/s41416-023-02264-4.