Dong Kwon Kim; Jin Woo Park; Hyeon Bin Cho; Su Jin Choi; Sung Sook Lee; YeongMun Kim; Jii Bum Lee; Sun Min Lim; Mi Ra Yu; Byoung Chul Cho

doi:10.4143/crt.2025.1399

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Original Article Synergistic Antitumor Activity of Combination Therapy with a MET TKI Vabametkib and a Third-Generation EGFR TKI Lazertinib in MET-Amplified EGFR-Mutant Non–Small Cell Lung Cancer: Dong Kwon Kim¹, Jin Woo Park¹, Hyeon Bin Cho², Su Jin Choi¹, Sung Sook Lee³, YeongMun Kim⁴, Jii Bum Lee⁵, Sun Min Lim⁵, Mi Ra Yu⁶, Byoung Chul Cho^5,6; DOI: https://doi.org/10.4143/crt.2025.1399
Published online: March 31, 2026

¹Yonsei Biomedical Research Institute, Yonsei University College of Medicine, Seoul, Korea

²Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA

³Department of Hematology-Oncology, Inje University Haeundae Paik Hospital, Busan, Korea

⁴R&D Center, ABION Inc., Seoul, Korea

⁵Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea

⁶Yonsei New Il Han Institute for Integrative Lung Cancer Research, Yonsei University College of Medicine, Seoul, Korea

Correspondence: Sun Min Lim, Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
Tel: 82-2-2228-0869 E-mail: limlove2008@yuhs.ac

Co-correspondence: Mi Ra Yu, Yonsei New Il Han Institute for Integrative Lung Cancer Research, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
Tel: 82-2-2228-0870 E-mail: mira0802@yuhs.ac

^*Dong Kwon Kim and Jin Woo Park contributed equally to this work.

• Received: December 23, 2025 • Accepted: March 18, 2026

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract

Purpose
Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) improved outcomes in EGFR-mutant non–small cell lung cancer (NSCLC); however, the subsequent development of resistance emphasizes the necessity of overcoming this therapeutic limitation. MET amplification is one of the major resistance mechanisms in EGFR-mutant NSCLC, bypassing EGFR inhibition by activating cell survival, proliferation, and metastasis. Combining MET and EGFR TKIs is thus emerging as a promising therapeutic strategy to overcome resistance to EGFR TKIs.
Materials and Methods
This study investigated the combination of the selective MET TKI vabametkib and a third-generation EGFR TKI lazertinib in MET-amplified EGFR TKI resistance models. Inhibition of downstream signaling and cell proliferation by vabametkib plus lazertinib was evaluated in osimertinib-resistant NSCLC cell lines (HCC827-AR) and patient-derived organoid (YUO-010) by western blot and CellTiter-Glo assay.
Results
In vitro studies demonstrated that vabametkib plus lazertinib synergistically inhibited EGFR/MET phosphorylation, leading to markedly enhanced anti-proliferative effects through downstream phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathway blockade. To investigate the antitumor effects in vivo, we employed two patient-derived xenograft models (YHIM-1035(1) and YHIM-1053) harboring MET amplification, as characterized by whole-exome sequencing or droplet digital polymerase chain reaction. Consistent with the in vitro findings, treatment with vabametkib plus lazertinib produced pronounced suppression of tumor growth in both models through a synergistic mechanism.
Conclusion
These findings establish vabametkib plus lazertinib as a promising strategy for MET-amplified NSCLC, currently under evaluation in an ongoing phase II clinical trial (NCT05541822).
Key words: EGFR, c-MET, Tyrosine kinase inhibitors, Non-small-cell lung carcinoma

Introduction

Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) selectively target both activating EGFR mutations and the T790M resistance mutation, offering a therapeutic advantage over first- and second-generation agents [1,2]. The EGFR T790M mutation accounts for roughly half of acquired resistance to first- and second-generation EGFR TKIs and can be effectively suppressed by third-generation agents such as lazertinib and osimertinib [3,4]. This enhanced target specificity improves clinical outcomes in patients with EGFR-mutant non–small cell lung cancer (NSCLC), leading to prolonged progression-free survival (PFS) and overall survival (OS) compared to earlier-generation EGFR TKIs [5]. Despite their initial efficacy, the therapeutic benefit of these agents is ultimately limited by the emergence of subsequent acquired resistance in the majority of patients [6,7]. Subsequent acquired resistance is frequently mediated by the activation of alternative oncogenic signaling pathways that allow tumor cells to bypass EGFR dependency [8,9]. Therefore, these limitations underscore the continued need for novel therapeutic strategies to overcome or delay resistance.

The MET proto-oncogene encodes a transmembrane receptor tyrosine kinase known as c-MET, which is implicated in the development and progression of various cancers, including NSCLC [10-12]. Upon binding to its ligand, hepatocyte growth factor (HGF), c-MET undergoes dimerization and autophosphorylation at specific intracellular tyrosine residues, thereby initiating a series of intracellular signaling cascades [13]. These cascades—most notably the Ras/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways—play critical roles in regulating cellular processes such as proliferation, survival, and motility [14]. Aberrant c-MET signaling has been implicated in tumor initiation, progression, metastasis, and therapeutic resistance across multiple cancer types [15,16]. In the context of NSCLC, aberrant MET signaling—often emerging as a mechanism of acquired resistance to EGFR TKIs—has been reported in approximately 5%-22% of patients, frequently in association with secondary EGFR mutations or bypass pathway activation [17-19].

MET amplification, a representative form of aberrant MET signaling, has been extensively characterized as a key mechanism of bypass resistance to EGFR TKIs across multiple therapeutic generations in patients with EGFR-mutant NSCLC [20-22]. This genetic alteration enables tumor cells to circumvent EGFR dependency by activating alternative downstream signaling pathways, thereby sustaining proliferation and survival despite EGFR inhibition [23]. Accordingly, MET amplification has been recognized as one of the most clinically significant mechanisms of acquired resistance, particularly in the setting of treatment with third-generation EGFR TKIs such as osimertinib and lazertinib [24].

Although tumors harboring EGFR mutations and concurrent MET amplification may initially respond to either MET inhibitors or EGFR TKIs, monotherapy with either agent often leads to suboptimal and transient clinical responses. Over time, selective pressure induced by single-agent treatment frequently results in the emergence of additional resistance mechanisms, ultimately diminishing the therapeutic efficacy [8,25]. These findings have prompted increasing interest in dual-pathway inhibition as a rational therapeutic strategy to overcome resistance and enhance clinical outcomes. In this context, the combination of tepotinib—a selective type Ib MET inhibitor—and gefitinib, a first-generation EGFR TKI, has demonstrated encouraging antitumor activity and manageable safety profiles in patients with EGFR-mutant NSCLC who developed MET amplification-mediated resistance [26,27]. Despite encouraging clinical data supporting the efficacy of dual EGFR and MET inhibition, the limited activity observed with current approaches highlights the need for more advanced combination strategies. Consequently, in EGFR-mutant NSCLC patients who develop acquired MET amplification following progression on third-generation EGFR TKI therapy, it is imperative to evaluate the clinical utility of combining third-generation EGFR TKIs with MET inhibitors as a more effective approach to overcoming resistance.

Vabametkib is a highly selective MET kinase inhibitor that has demonstrated a favorable safety profile in clinical studies and exhibits potent antitumor activity against MET-altered tumor cells. This study aims to provide insights into emerging dual-TKI therapeutic strategies by investigating the combination of vabametkib with lazertinib, a third-generation EGFR tyrosine kinase inhibitor, in a preclinical model of EGFR TKI–resistant NSCLC harboring acquired MET amplification.

Materials and Methods

1. Chemicals

Vabametkib was kindly provided by ABION Corporation, and lazertinib was provided by Yuhan Corporation. Osimertinib (Cat. S7297) and savolitinib (Cat. S7674) were purchased from Selleck Chemical. All compounds were prepared and used in accordance with the manufacturer’s instructions.

2. Generation of TKI-resistant cells

HCC827 cells were purchased from the ATCC (American Type Culture Collection). To generate osimertinib (AZD9291, AR)–acquired resistant cells, we followed previously described protocols [28]. Resistant cells (HCC827-AR) were established by gradually escalating osimertinib concentrations up to 1 μM over 6 months, and subsequently derived from the surviving cell population. Cells were maintained in RPMI-1640 medium with 10% fetal bovine serum and 1% antibiotics at 37℃ with 5% CO₂.

3. Patient samples

All patient samples were obtained from EGFR-mutant NSCLC patients with acquired MET amplification after progression on EGFR TKI treatment at Yonsei University Severance Hospital (Seoul, Republic of Korea).

4. Establishment of patient-derived preclinical models

Patient-derived organoid (PDO: YUO-010) cells were established from malignant effusions of patients as previously described [29,30]. For PDO, cells were suspended in cold Matrigel (Corning #256231) and then seeded in 24-well culture plates (Corning). After 15-minute incubation at 37°C, the complete organoid medium (Dulbecco’s modified Eagle’s medium/F12 medium [Invitrogen] supplemented with 20% conditioned R-spondin1 medium, 10 nmol/L HEPES [Invitrogen], 1× GlutaMax [Invitrogen], and 1× antibiotic-antimycotic [Invitrogen]) was added to the solidified Matrigel containing cells. Tumor purity of the organoids was confirmed by positive staining for thyroid transcription factor 1 (clone EP1584Y, Abcam), calretinin (clone DAK-Calret-1, Agilent Technologies), and p53 (Leica Biosystems) using immunohistochemistry (IHC). To determine whether patient-derived preclinical models maintained patient characteristics, whole-exome sequencing (WES) was performed.

5. Establishment of patient-derived xenograft models

Patient-derived xenograft (PDX) models (YHIM-1035(1) and YHIM-1053) were established as previously described [31]. Tissues were implanted into the flanks of 6-week-old female nu/nu mice. When tumor size reached approximately 200 mm³, the mice were randomized into groups and assigned to the following treatment groups: vehicle, vabametkib (orally, once daily, 30 mg/kg), lazertinib (orally, once daily, 10 mg/kg), combination of vabametkib with lazertinib (orally, once daily). Tumor volumes were measured with an electronic caliper. Tumor volume changes from baseline (day 0) to the end of treatment (EOT) were determined using two formulas, as previously reported [32]. Tumor growth inhibition was calculated at the EOT by comparing tumor volumes in each treatment group with those in the vehicle group. Body weight kinetics of tumor bearing mice were monitored every other day throughout the study. All mice were euthanized via CO₂ inhalation at the end of the experiment.

6. WES analysis

Genomic DNA purity and concentration were assessed using the PicoGreen dsDNA assay (RRID:SCR_008410, Invitrogen) and agarose gel electrophoresis method. Genomic fragment library was constructed using the SureSelect kit (RRID:SCR_013575, Agilent Technologies) and sequenced on the Illumina HiSeq 2500 (RRID:SCR_016383) and NovaSeq 6000 platform (RRID:SCR_016387). The sequenced reads were aligned to the human genome reference (hg19) using Burrows-Wheeler Alignment (BWA, RRID:SCR_010910). Then they were analyzed with genome analysis toolkit. Somatic mutations were called using MuTect2 implemented in GATK ver. 4.1.4.1 (RRID:SCR_001876; Broad Institute, RRID: SCR_007073) within a Docker environment and were annotated using Oncotator (RRID:SCR_005183). Copy number variation analysis was performed using etal/cnvkit ver. 0.9.5 (RRID:SCR_021917) on the Docker system, with copy number (CN) ≥ 3 indicating amplification and CN ≤ 1 indicating deletion. Nine major oncogenes were selected and graphed using the OncoPrint function of the ComplexHeatmap package (RRID:SCR_017270) in R.

7. Cell proliferation assay

Approximately 3,000 cells per well were plated in 96-well plates, allowed to adhere overnight, and subsequently exposed to compounds for 72 hours. Cell viability was then assessed using the CellTiter-Glo assay (Promega) according to the manufacturer’s instructions.

8. Synergy effect analysis

Drug combination effects were evaluated using the Bliss independence model. Cell viability data from single-agent and combination treatments were used to calculate expected combination responses, assuming independent action. The expected combined effect of drugs A and B under the Bliss independence model is calculated as: E(AB)=E(A)+E(B)−E(A)×E(B), where E(A) and E(B) represent the fractional inhibition (or effect) of drug A and drug B when used alone. The observed combination effect was then compared to the Bliss expectation, and synergy was defined as observed effects significantly exceeding the predicted additive effect. Analyses were performed using GraphPad Prism (GraphPad Software Inc.), and synergy scores were interpreted according to established criteria.

9. Immunoblot analysis

Immunoblots were performed to determine relative phosphorylation of kinases and total protein levels of interest. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane for immunoblotting using the corresponding antibodies: p-MET (Tyr1234/Tyr1235), t-MET, p-EGFR (Tyr1068), t-EGFR, p-AKT (Ser473), t-AKT, p-ERK (Thr202/Tyr204), t-ERK, p-S6, t-S6, and β-actin. They were all purchased from Cell Signaling Technologies.

10. Colony formation assays

To evaluate anti-proliferative effects in a 2D culture system, we established YUOC-010 which is PDO-cell derived from PDO. Cells were seeded in 6-well plates for 24 hours and then treated with (1) 0.1% dimethyl sulfoxide, (2) vabametkib, (3) lazertinib, and (4) vabametkib plus lazertinib. After 7 to 14 days, the cells were stained with 0.5% crystal violet (Sigma-Aldrich) in 25% methanol. Colony formation was quantified using an ImageJ program.

11. Immunohistochemistry

IHC was performed in 4-μm-thick formalin-fixed paraffin-embedded (FFPE) tissue sections. The slides were baked, deparaffinized in xylene, rehydrated through a graded alcohol series, and then the antigen was recovered in 1 mmol/L EDTA, pH 8.0, for 30 seconds at 125°C. Endogenous peroxidase activity was quenched using Peroxidase Block (Dako) for 5 minutes, followed by rinsing with 50 mmol/L Tris-Cl (pH 7.4). Slides were blocked using normal goat serum (Dako) and subsequently incubated for 1 hour with anti-Ki67 antibody (No. 9027, Cell Signaling Technology). Then the slides were washed with 50 mmol/L Tris-Cl, pH 7.4, and the Signal stain boost IHC detection reagent (horseradish peroxidase, rabbit, No. 8114, Cell Signaling Technology) for 30 minutes. After additional washes, immunoperoxidase staining was developed using a 3,3′-diaminobenzidine chromogen (Dako) for 5 minutes. Finally, slides were counterstained with hematoxylin, dehydrated through graded alcohol and xylene, mounted, and coverslipped. All pathologic analyses were conducted by an independent pathologist who was blinded to the clinical information.

12. Bulk RNA sequencing

RNA sequencing data were obtained by requesting tumor FFPE samples from MACROGEN (SEL). Total RNA was extracted and used to construct mRNA sequencing libraries with the SureSelectXT RNA Direct Library Preparation Kit (Illumina) according to the manufacturer’s protocol. Libraries were sequenced on an Illumina platform using paired-end 100 bp reads. Raw image files were converted to sequence data by base-calling and quality-checked with FastQC (v0.11.7). Paired-end reads were trimmed to remove PCR and sequencing adapters using Trimmomatic (v0.39). The trimmed reads were aligned to the human reference genome using HISAT2 (v2.2.1) with Bowtie2 integration. Gene-level read counts were quantified with StringTie (v3.0.0). Differential gene expression analysis was performed at the gene level using the DESeq2 package (v1.49.3). Gene set enrichment analysis was conducted with GSEA (v4.4.0) using the Molecular Signatures Database (MSigDB, v7.0) and visualized with Cytoscape. The data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE307503. Multiple hypothesis testing correction was applied, and a significance threshold of p < 0.05 was used.

13. Code availability

All original code used for the analyses has been deposited on GitHub and is publicly available at https://github.com/DongKwonKIM/ABN401.git as of the date of publication.

14. Statistical analysis

All data are expressed as means±standard deviation or ±standard error for independent replications. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. Although the interaction between time and treatment was statistically significant, post hoc comparisons were performed with appropriate consideration of the main treatment effects.

Results

1. The combination therapy of vabametkib and lazertinib potently inhibits EGFR/MET activity and cell proliferation in in vitro models of MET-amplified EGFR-mutant NSCLC

We previously established a stable osimertinib-resistant cell line (HCC827-AR) through long-term osimertinib exposure in the HCC827 cell line and developed a PDO (YUO-010) from an EGFR TKI–resistant patient with MET-amplified EGFR-mutant NSCLC (Fig. 1A and B). These models were confirmed to have acquired MET amplification through WES and droplet digital polymerase chain reaction analyses. To investigate the potential of the combination therapy of vabametkib and lazertinib in MET-amplified, EGFR-mutant NSCLC, we evaluated in vitro cell proliferation and signal transduction using the HCC827-AR and YUO-010 cell lines. Using CellTiter-Glo assays, the combination of EGFR/MET inhibitors demonstrated potent suppression of cell viability in both HCC827-AR and YUO-010 models (Fig. 1C-F). A third-generation EGFR TKI showed limited activity as a monotherapy in these resistant cells, highlighting the critical role of MET amplification in resistance (IC₅₀ > 1 μmol/L) (S1 Fig.). Based on cell viability assay results from two cell line models, synergy scores were calculated using the Bliss independence model. The combination of vabametkib and lazertinib exhibited enhanced effects across a range of concentration combinations, including low-dose conditions (Fig. 1G and H). To evaluate MET activation status, phosphorylation of MET at tyrosine residues 1234 and 1235 was assessed using a phospho-specific antibody. These residues are located within the kinase domain and serve as key autophosphorylation sites essential for MET activation. The combination of vabametkib and lazertinib effectively inhibited EGFR and MET signaling pathways in vitro. Western blot analysis revealed significant reductions in the phosphorylation of key downstream effectors, including phospho-AKT (pAKT), phospho-ERK (pERK), and phospho-S6 (pS6), upon combination treatment. In contrast, treatment with lazertinib alone for 6 hours showed no significant changes in phosphorylation levels, underscoring the necessity of dual inhibition to disrupt resistance-associated signaling (Fig. 2A and B). Consistent with the growth inhibition observed in short-term assays, long-term clonogenic assays demonstrated that combined treatment with vabametkib and lazertinib markedly suppressed the colony-forming capacity of both HCC827-AR and YUOC-010 (Fig. 2C). In HCC827-AR cells, monotherapy with either lazertinib or vabametkib produced only a modest reduction in colony coverage compared with vehicle controls, and osimertinib monotherapy showed minimal impact, consistent with the established resistance phenotype (Fig. 2D). Similarly, in YUOC-010, neither lazertinib nor vabametkib alone achieved statistically significant inhibition, with colony coverage remaining comparable to that in control wells. By contrast, the combination of vabametkib and lazertinib resulted in a pronounced decrease in both colony size and density in both cell lines, leading to a significant reduction in quantified well coverage. These findings indicate that concurrent inhibition of MET and EGFR more effectively overcomes MET amplification–mediated resistance than either agent alone. Normal cell toxicity was evaluated using human umbilical vein endothelial cell (HUVEC) (S1A Fig.). Neither vabametkib monotherapy nor its combination with lazertinib impaired the proliferation of normal cells, demonstrating a safety profile for the combination therapy.

2. The combination of vabametkib and lazertinib shows enhanced in vivo antitumor activity in PDX models

The efficacy of vabametkib plus lazertinib was further assessed in vivo using PDX models (YHIM-1035(1) and YHIM-1053) established from MET-amplified EGFR-mutant NSCLC patients with acquired resistance to third-generation EGFR TKIs. Consistent with in vitro results, the combination of vabametkib and lazertinib induced marked tumor regression in both models. In the YHIM-1035(1) model, vabametkib monotherapy at 30 mg/kg effectively controlled tumor progression, further highlighting its potency against MET-driven resistance mechanisms (Figs. 3 and 4). Notably, no significant body weight changes were observed in any treatment group. IHC analysis was performed on tumor tissues collected 2 hours after the final administration of the drugs. In the combination group, phosphorylated EGFR and MET were markedly reduced, indicating effective inhibition of both targets. Furthermore, Ki-67 staining revealed a significant decrease in proliferating tumor cells following combination treatment compared to the vehicle or monotherapy groups. Across all PDX models tested, dual inhibition consistently suppressed both receptor activation and tumor cell proliferation (Figs. 3F and 4F). These findings underscore the therapeutic potential of the combination strategy in overcoming MET-mediated acquired resistance in EGFR-mutant tumors.

3. Divergent MET and EGFR dependency in EGFR TKI– resistant models highlights the potential of MET TKI combination strategies

Bulk RNA-seq and WES were performed to investigate the molecular basis underlying the distinct drug response profiles observed between the EGFR TKI–resistant YHIM-1035(1) and YHIM-1053 models. The WES revealed that YHIM-1035(1) harbored MET amplifications without secondary EGFR kinase domain mutations, whereas YHIM-1053 exhibited MET amplification together with an EGFR T790M missense mutation (Fig. 5A). To examine canonical pathways associated with cell proliferation and TKI resistance, we first assessed well-established signaling programs, including EGFR tyrosine kinase inhibitor resistance, MAPK signaling, and PI3K–AKT–mammalian target of rapamycin signaling. Transcriptomic analysis further demonstrated a marked upregulation of MET and its ligand HGF in YHIM-1035(1), while YHIM-1053 showed relatively greater enrichment of canonical EGFR signaling and cell cycle–related programs (Fig. 5B and C).

As both models represent resistance to EGFR TKIs, these transcriptomic findings highlight their relative molecular characteristics. Compared with YHIM-1053, YHIM-1035(1) showed significant down-regulation of extracellular matrix organization and epithelial cell differentiation, suggesting reduced reliance on canonical EGFR signaling and a concomitant shift toward microenvironment- and MET-associated survival pathways (Fig. 5D). Collectively, these findings suggest that, relative to YHIM-1053, YHIM-1035(1) displays heightened MET dependency and a shift away from canonical EGFR signaling, which may explain its favorable response to MET TKI plus EGFR TKI combination therapy. Accordingly, although both models represent EGFR TKI–resistant tumors, their distinct pathway activation and transcriptomic features imply that EGFR TKIs alone may be insufficient in certain contexts, whereas combined MET and EGFR blockade offers a rational strategy to overcome such resistance.

4. Dual inhibition of EGFR and MET overcomes acquired resistance to EGFR TKIs by suppressing compensatory signaling and enhancing antitumor efficacy in resistant tumor cells

The schematic model presented in Fig. 6 provides a conceptual overview of the proposed mechanism by which dual inhibition of EGFR and MET can overcome acquired resistance to EGFR TKIs. Specifically, this model illustrates how co-targeting these two pathways effectively suppresses compensatory signaling driven by MET amplification or reactivation, which often arises as a resistance mechanism following treatment with third-generation EGFR TKIs. By concurrently blocking both EGFR and MET signaling, the combination strategy is proposed to inhibit downstream survival and proliferation pathways more effectively than monotherapy, thereby restoring sensitivity and enhancing antitumor efficacy in resistant tumor cells.

Discussion

In NSCLC, MET amplification represents a well-characterized mechanism of acquired resistance to EGFR TKIs, facilitating oncogenic escape through activation of bypass signaling pathways [23,33,34]. While multiple targeted therapeutic strategies have been pursued to overcome this resistance, their clinical efficacy has often been constrained by the molecular heterogeneity of tumors, the complexity of dual-pathway inhibition, and limitations in biomarker-driven patient stratification [35,36]. Accumulating preclinical evidence supports the rationale for concomitant inhibition of EGFR and MET as a means to suppress MET-mediated resistance and restore antitumor sensitivity to EGFR TKIs. More recently, a combination blockade strategy targeting EGFR and MET was approved in the treatment of advanced, metastatic NSCLC. Amivantamab, which is an EGFR/MET bispecific antibody, plus lazertinib prolonged PFS and OS vs. osimertinib [37,38], highlighting the importance of EGFR and MET co-blockade.

Vabametkib is a highly selective MET kinase inhibitor with promising activity in patients with metastatic NSCLC harboring MET alterations, currently being evaluated in a Phase 2 clinical trial (NCT04052971) for solid tumors [39]. This study is part of a series investigating the efficacy and safety of combining a dual-targeted TKI with a MET inhibitor to overcome MET-mediated resistance driven by diverse oncogenic alterations in NSCLC. Specifically, we evaluated whether the combination of vabametkib and lazertinib exerts synergistic antitumor effects in preclinical models of EGFR-mutant NSCLC with MET amplification. Compared with earlier MET-EGFR combinations such as tepotinib plus gefitinib, the vabametkib–lazertinib regimen may provide a more effective strategy to overcome both T790M-mediated acquired resistance and MET-driven bypass signaling. Unlike antibody-based approaches such as amivantamabcontaining regimens, the use of two low–molecular-weight small-molecule inhibitors may allow deeper tumor penetration and more uniform intratumoral drug distribution. In addition, although MET TKI–EGFR TKI combinations such as savolitinib plus osimertinib have shown clinical activity, treatment-related toxicities including elevated liver enzymes and gastrointestinal adverse events have been reported [27,40].

Using both patient-derived models and EGFR TKI resistance cell lines, we successfully characterized genetic and phenotypic profiles of resistance models. Our study provides several novel insights. In vitro analyses indicate that concurrent inhibition of MET and EGFR signaling pathways significantly attenuates tumor cell proliferation in models harboring the corresponding oncogenic alterations. Synergy between the two agents was also observed based on the Bliss independence model, and no antagonism was detected upon combination treatment. These results suggest that the combination of the two agents enhances therapeutic efficacy through complementary mechanisms, without evidence of antagonism. The combination therapy of vabametkib and lazertinib not only effectively suppressed tumor cell proliferation, but also more comprehensively inhibited oncogenic signaling pathways compared to monotherapies. The use of p-MET (Y1234/1235) as a readout of MET activity enabled precise assessment of the pharmacodynamic effects of the treatment. Given that Y1234 and Y1235 are critical autophosphorylation sites required for full activation of the MET kinase domain, their inhibition strongly supports the conclusion that the combination therapy effectively suppresses MET-driven signaling pathways [41,42]. In particular, the dual treatment significantly inhibited not only MET and EGFR signaling, but also key downstream effectors, including phosphorylated AKT, ERK, and S6. Given that these pathways play critical roles in driving cell proliferation, survival, and drug resistance in EGFR-mutant cancers, their concurrent inhibition likely contributes to the enhanced antiproliferative effects observed in vitro. These findings provide a strong rationale for dual inhibition strategies that target both upstream receptors and downstream effectors within the EGFR signaling axis, and highlight the therapeutic potential of this combination approach in overcoming resistance and improving treatment efficacy. Colony formation in both HCC827-AR and YUOC-010 cells was markedly suppressed by the combination treatment, indicating a sustained antiproliferative effect beyond acute cytotoxicity. Since colony formation assays assess the long-term survival and clonal expansion capacity of individual tumor cells, this result suggests that the dual therapy effectively limits the ability of tumor cells to regrow after drug exposure, which is essential for durable tumor control and reduced risk of recurrence. Additionally, to evaluate the specificity of MET-targeted agents, HUVECs were employed as a representative normal cell line with physiological levels of MET expression. As primary human endothelial cells, HUVECs naturally express functional MET and are responsive to HGF, thereby serving as an appropriate control for assessing the selectivity and cytotoxicity of MET inhibitors compared to tumor cells harboring MET amplification [43,44]. In the toxicity assessment using HUVEC cells, both vabametkib monotherapy and the combination therapy with lazertinib exhibited a favorable safety profile, as they did not impair the proliferation of normal endothelial cells. These results indicate that the combination therapy selectively targets tumor cells without affecting normal cells, supporting its tumor-specific activity.

In the YHIM-1035(1) and YHIM-1053 PDX models, the combination of vabametkib and lazertinib led to marked tumor regression, consistent with the antiproliferative effects observed in vitro. Notably, monotherapy with vabametkib at a dose of 30 mg/kg effectively inhibited tumor progression in the YHIM-1035(1) model. This finding suggests that MET-driven oncogenic signaling may represent a primary dependency in this model. Differential responses observed in the two PDX models highlight the importance of molecular context in determining therapeutic sensitivity. The YHIM- 1035(1) model, characterized by EGFR exon 19 deletion and MET amplification, responded to both monotherapies and combination treatment, suggesting that either EGFR or MET signaling may be sufficient to sustain tumor growth in this context, and inhibition of either axis can yield antitumor activity. In contrast, the YHIM-1053 model, which harbors EGFR exon 19 deletion, the T790M resistance mutation, and MET amplification, exhibited sensitivity only to the combination therapy, but not to either agent alone. This finding suggests that concurrent activation of EGFR T790M–mediated signaling and MET-driven bypass pathways confers a higher level of resistance, requiring dual inhibition to sufficiently suppress downstream survival signaling. One limitation of this study is that therapeutic efficacy was not comprehensively evaluated in models harboring EGFR exon 21 L858R mutations, which may limit the generalizability of our findings to the broader EGFR-mutant NSCLC population. In our preliminary evaluation, only a modest therapeutic effect was observed in models harboring the EGFR exon 21 L858R mutation (data not shown). Further studies using additional EGFR mutation models are warranted.

Ongoing clinical trials (NCT05541822) continue to evaluate the safety and efficacy of the combination of vabametkib and lazertinib, with the goal of improving outcomes for patients with this challenging subtype of lung cancer. Future studies should explore biomarkers that predict responsiveness to MET/EGFR combination therapies and evaluate the long-term safety and efficacy of this strategy in diverse patient populations. Despite the efficacy of dual MET-EGFR inhibition, additional resistance mechanisms may emerge, including secondary EGFR mutations such as C797S, MET kinase domain mutations (e.g., D1228 or Y1230), as well as MAPK pathway reactivation through alternative bypass signaling [45,46]. Therefore, further studies investigating these potential escape pathways are warranted to better understand mechanisms of acquired resistance. Integrating these findings into clinical trials could pave the way for more personalized treatment strategies for resistant EGFR-mutant NSCLC.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

crt-2025-1399_S1_Fig.pdf

NOTES

Ethical Statement

Animal procedures were approved by the Institutional Animal Care and Use Committee and Animal Research Committee at Yonsei University College of Medicine. The study protocol has been approved by the Institutional Review Board of Severance Hospital (IRB No. 4-2016-0001) and all patients have provided written informed consent.

Author Contributions

Conceived and designed the analysis: Lim SM, Yu MR, Cho BC.

Collected the data: Kim DK, Park JW, Lee SS, Kim Y, Lim SM, Yu MR.

Contributed data or analysis tools: Cho HB, Choi SJ, Lee JB, Lim SM, Yu MR, Cho BC.

Performed the analysis: Kim DK, Park JW, Lim SM, Yu MR, Cho BC.

Wrote the paper: Kim DK, Park JW, Lim SM, Yu MR, Cho BC.

Conflicts of Interest

Conflict of interest relevant to this article was not reported.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(RS-2025-00557272). This research was supported by a grant (RS-2025-02215737) from Ministry of Food and Drug Safety in 2025. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(RS-2025-00555190).

Fig. 1.

Inhibition of cell proliferation by epidermal growth factor receptor (EGFR)/MET combination therapy in MET-amplified EGFR tyrosine kinase inhibitor–resistant cells. (A) Establishment of an osimertinib-resistant cell line. Parental HCC827 cells were chronically exposed to increasing concentrations of osimertinib (~1 μM) for 6 months to generate the resistant derivative HCC827-AR. Dose–response curves demonstrate a marked rightward shift in IC₅₀ for HCC827-AR compared with parental HCC827 cells (IC₅₀=4.5 nmol/L vs. > 1,000 nmol/L, respectively). (B) Generation of a patient-derived organoid (PDO). Tumor tissue from a patient with non–small cell lung cancer was dissociated and embedded in Matrigel supplemented with niche factors including R-spondin-1, Noggin, and additional growth factors to establish the PDO YUO-010. (C-F) ATCC cell lines (HCC827-AR) and PDO (YUO-010) were treated with increasing doses of vabametkib and lazertinib, and osimertinib for 72 hours, and cell viability was assessed using the CellTiter-Glo assay. At least two biological replicates and two technical replicates were performed. The curved graphs show the cell viability results for a single drug, and the bar graphs show the cell viability for the combination of two drugs. Control data in Fig. 1C and E are derived from the same experiment, as are those in Fig. 1D and F. (G-H) Bliss independence scores for the combination of vabametkib and lazertinib. Synergy was assessed based on deviations from expected additive effects calculated using the Bliss model.

Fig. 2.

Effect of vabametkib and lazertinib combination therapy on MET- and epidermal growth factor receptor (EGFR)–mediated downstream signaling in MET-amplified EGFR tyrosine kinase inhibitor–resistant cells. (A, B) Immunoblotting of HCC827-AR and YUO-010 cell lines after treatment with indicated vabametkib and lazertinib doses for 6 hours. Western blot analyses were independently repeated at least twice. (C) Cells were treated with the indicated concentrations of vabametkib, lazertinib, or their combination for 7-14 days. Drugcontaining media were replaced every 3 days. In vitro efficacy was assessed by a colony formation assay. Shown are representative images of crystal violet–stained colonies. (D) Quantification of colony area coverage (% of well area) from three independent replicates. Data represent mean±standard error of mean; statistical significance was determined by Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3.

Antitumor effects of the combination with vabametkib and lazertinib in YHIM-1053 patient-derived xenograft (PDX) model. In vivo antitumor activity of vabametkib plus lazertinib in the YHIM-1053 PDX model. Tumor fragments were subcutaneously implanted into the flanks of 6-week-old female BALB/c nude mice. Mice were randomized when tumor volumes reached approximately 200 mm³ and subsequently treated with the indicated drugs once daily. (A) Treatment history of YHIM-1053. (B) Tumor growth curve of YHIM-1053 PDX model in response to vabametkib/lazertinib combination therapy. Vehicle (n=12), lazertinib (n=12), osimertinib (n=4), vabametkib (n=12), combination (n=12). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant; Two-way repeated measures ANOVA with multiple comparisons. (C) Tumor volume dynamics from baseline to day 17. Individual waterfall plots depict percentage change in tumor volume relative to baseline in xenograft-bearing mice treated with vehicle, osimertinib (25 mg/kg), lazertinib (10 mg/kg), vabametkib (30 mg/kg), or vabametkib plus lazertinib for 17 days. Combination treatment achieved the strongest tumor regression compared with monotherapies. (D) Quantification of tumor growth inhibition (TGI) at day 17. Bar graphs show TGI values for each tumor in the respective treatment groups. Vabametkib plus lazertinib produced near-complete growth suppression compared with single-agent osimertinib, lazertinib, or vabametkib. (E) Body weight of the mice during the experiment. (F) Representative images of H&E staining (top) and Ki-67 immunohistochemistry (bottom) of tumor sections.

Fig. 4.

Antitumor effects of the combination with vabametkib and lazertinib in YHIM-1035(1) patient-derived xenograft (PDX) model. (A) Treatment history of YHIM-1035(1). (B) Tumor growth curve of YHIM-1035(1) PDX model in response to vabametkib/lazertinib combination therapy. Vehicle (n=10), lazertinib (n=10), vabametkib (n=10), combination (n=10). ***p < 0.001; Two-way repeated measures ANOVA with multiple comparisons. (C) Tumor volume dynamics from baseline to day 29. Individual waterfall plots depict percentage change in tumor volume relative to baseline in xenograft-bearing mice treated with vehicle, lazertinib (10 mg/kg), vabametkib (30 mg/kg), or vabametkib plus lazertinib for 29 days. Combination treatment achieved the most pronounced tumor regression compared with monotherapies. (D) Quantification of tumor growth inhibition (TGI) at day 29. Bar graphs show TGI values for each tumor in the respective treatment groups. Vabametkib plus lazertinib produced near-complete growth suppression compared with single-agent lazertinib or vabametkib. (E) Body weight of the mice during the experiment. (F) Representative images of H&E staining (top) and Ki-67 immunohistochemistry (bottom) of tumor sections.

Fig. 5.

Molecular and transcriptomic profiling of patient-derived models reveals distinct oncogenic programs. (A) The genomic characterization of the patient-derived models used in this study is expressed in the form of an oncoprint plot. Key oncogenic alterations, including epidermal growth factor receptor (EGFR), KRAS, and TP53 mutations, are annotated across HCC827, HCC827-AR, and patient-derived xenograft (PDX)–derived organoid and cell line models. (B) Volcano plot comparing transcriptomic profiles between YHIM-1035(1) and YHIM-1053. Selected differentially expressed genes (EGFR, AXL, MET, KRAS, and hepatocyte growth factor [HGF]) are highlighted. (C) Gene set enrichment analysis showing pathway-level differences between the two models. YHIM-1053 exhibits enrichment in EGFR tyrosine kinase inhibitor resistance, mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR) signaling pathways. (D) Top enriched biological processes and cellular functions, including epithelial differentiation, extracellular matrix remodeling, and cell-cell adhesion, as identified by Gene Ontology (GO)/PMID-linked gene set enrichment. Each column represents an individual tumor sample derived from independent mice (mouse 1 and mouse 2) for each PDX model.

Fig. 6.

Schematic illustration of MET-driven bypass signaling in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)–resistant non–small cell lung cancer (NSCLC) cells. Combined inhibition of EGFR and MET effectively suppresses downstream proliferative and survival signaling, thereby restoring sensitivity in MET-amplified EGFR TKI–resistant NSCLC models. EGF, epidermal growth factor; HGF, hepatocyte growth factor; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase.

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Synergistic Antitumor Activity of Combination Therapy with a MET TKI Vabametkib and a Third-Generation EGFR TKI Lazertinib in MET-Amplified EGFR-Mutant Non–Small Cell Lung Cancer

DOI: https://doi.org/10.4143/crt.2025.1399

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Synergistic Antitumor Activity of Combination Therapy with a MET TKI Vabametkib and a Third-Generation EGFR TKI Lazertinib in MET-Amplified EGFR-Mutant Non–Small Cell Lung Cancer

Fig. 1. Inhibition of cell proliferation by epidermal growth factor receptor (EGFR)/MET combination therapy in MET-amplified EGFR tyrosine kinase inhibitor–resistant cells. (A) Establishment of an osimertinib-resistant cell line. Parental HCC827 cells were chronically exposed to increasing concentrations of osimertinib (~1 μM) for 6 months to generate the resistant derivative HCC827-AR. Dose–response curves demonstrate a marked rightward shift in IC50 for HCC827-AR compared with parental HCC827 cells (IC50=4.5 nmol/L vs. > 1,000 nmol/L, respectively). (B) Generation of a patient-derived organoid (PDO). Tumor tissue from a patient with non–small cell lung cancer was dissociated and embedded in Matrigel supplemented with niche factors including R-spondin-1, Noggin, and additional growth factors to establish the PDO YUO-010. (C-F) ATCC cell lines (HCC827-AR) and PDO (YUO-010) were treated with increasing doses of vabametkib and lazertinib, and osimertinib for 72 hours, and cell viability was assessed using the CellTiter-Glo assay. At least two biological replicates and two technical replicates were performed. The curved graphs show the cell viability results for a single drug, and the bar graphs show the cell viability for the combination of two drugs. Control data in Fig. 1C and E are derived from the same experiment, as are those in Fig. 1D and F. (G-H) Bliss independence scores for the combination of vabametkib and lazertinib. Synergy was assessed based on deviations from expected additive effects calculated using the Bliss model.

Fig. 2. Effect of vabametkib and lazertinib combination therapy on MET- and epidermal growth factor receptor (EGFR)–mediated downstream signaling in MET-amplified EGFR tyrosine kinase inhibitor–resistant cells. (A, B) Immunoblotting of HCC827-AR and YUO-010 cell lines after treatment with indicated vabametkib and lazertinib doses for 6 hours. Western blot analyses were independently repeated at least twice. (C) Cells were treated with the indicated concentrations of vabametkib, lazertinib, or their combination for 7-14 days. Drugcontaining media were replaced every 3 days. In vitro efficacy was assessed by a colony formation assay. Shown are representative images of crystal violet–stained colonies. (D) Quantification of colony area coverage (% of well area) from three independent replicates. Data represent mean±standard error of mean; statistical significance was determined by Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3. Antitumor effects of the combination with vabametkib and lazertinib in YHIM-1053 patient-derived xenograft (PDX) model. In vivo antitumor activity of vabametkib plus lazertinib in the YHIM-1053 PDX model. Tumor fragments were subcutaneously implanted into the flanks of 6-week-old female BALB/c nude mice. Mice were randomized when tumor volumes reached approximately 200 mm³ and subsequently treated with the indicated drugs once daily. (A) Treatment history of YHIM-1053. (B) Tumor growth curve of YHIM-1053 PDX model in response to vabametkib/lazertinib combination therapy. Vehicle (n=12), lazertinib (n=12), osimertinib (n=4), vabametkib (n=12), combination (n=12). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant; Two-way repeated measures ANOVA with multiple comparisons. (C) Tumor volume dynamics from baseline to day 17. Individual waterfall plots depict percentage change in tumor volume relative to baseline in xenograft-bearing mice treated with vehicle, osimertinib (25 mg/kg), lazertinib (10 mg/kg), vabametkib (30 mg/kg), or vabametkib plus lazertinib for 17 days. Combination treatment achieved the strongest tumor regression compared with monotherapies. (D) Quantification of tumor growth inhibition (TGI) at day 17. Bar graphs show TGI values for each tumor in the respective treatment groups. Vabametkib plus lazertinib produced near-complete growth suppression compared with single-agent osimertinib, lazertinib, or vabametkib. (E) Body weight of the mice during the experiment. (F) Representative images of H&E staining (top) and Ki-67 immunohistochemistry (bottom) of tumor sections.

Fig. 4. Antitumor effects of the combination with vabametkib and lazertinib in YHIM-1035(1) patient-derived xenograft (PDX) model. (A) Treatment history of YHIM-1035(1). (B) Tumor growth curve of YHIM-1035(1) PDX model in response to vabametkib/lazertinib combination therapy. Vehicle (n=10), lazertinib (n=10), vabametkib (n=10), combination (n=10). ***p < 0.001; Two-way repeated measures ANOVA with multiple comparisons. (C) Tumor volume dynamics from baseline to day 29. Individual waterfall plots depict percentage change in tumor volume relative to baseline in xenograft-bearing mice treated with vehicle, lazertinib (10 mg/kg), vabametkib (30 mg/kg), or vabametkib plus lazertinib for 29 days. Combination treatment achieved the most pronounced tumor regression compared with monotherapies. (D) Quantification of tumor growth inhibition (TGI) at day 29. Bar graphs show TGI values for each tumor in the respective treatment groups. Vabametkib plus lazertinib produced near-complete growth suppression compared with single-agent lazertinib or vabametkib. (E) Body weight of the mice during the experiment. (F) Representative images of H&E staining (top) and Ki-67 immunohistochemistry (bottom) of tumor sections.

Fig. 5. Molecular and transcriptomic profiling of patient-derived models reveals distinct oncogenic programs. (A) The genomic characterization of the patient-derived models used in this study is expressed in the form of an oncoprint plot. Key oncogenic alterations, including epidermal growth factor receptor (EGFR), KRAS, and TP53 mutations, are annotated across HCC827, HCC827-AR, and patient-derived xenograft (PDX)–derived organoid and cell line models. (B) Volcano plot comparing transcriptomic profiles between YHIM-1035(1) and YHIM-1053. Selected differentially expressed genes (EGFR, AXL, MET, KRAS, and hepatocyte growth factor [HGF]) are highlighted. (C) Gene set enrichment analysis showing pathway-level differences between the two models. YHIM-1053 exhibits enrichment in EGFR tyrosine kinase inhibitor resistance, mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR) signaling pathways. (D) Top enriched biological processes and cellular functions, including epithelial differentiation, extracellular matrix remodeling, and cell-cell adhesion, as identified by Gene Ontology (GO)/PMID-linked gene set enrichment. Each column represents an individual tumor sample derived from independent mice (mouse 1 and mouse 2) for each PDX model.

Fig. 6. Schematic illustration of MET-driven bypass signaling in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)–resistant non–small cell lung cancer (NSCLC) cells. Combined inhibition of EGFR and MET effectively suppresses downstream proliferative and survival signaling, thereby restoring sensitivity in MET-amplified EGFR TKI–resistant NSCLC models. EGF, epidermal growth factor; HGF, hepatocyte growth factor; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase.

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Synergistic Antitumor Activity of Combination Therapy with a MET TKI Vabametkib and a Third-Generation EGFR TKI Lazertinib in MET-Amplified EGFR-Mutant Non–Small Cell Lung Cancer