Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

Introduction

Lung cancer ranks first among the incidences of various cancers globally. and is the leading cause of cancer-related deaths worldwide. In China, the number of new cases and deaths of lung cancer in 2020 was still the highest among all cancers (1). Non-small cell lung cancer (NSCLC) is the most common type, accounting for about 85%, and adenocarcinoma is more common (2). Since the early symptoms are mostly atypical symptoms such as cough and shortness of breath, patients are easy to ignore those symptoms, leading to delays in diagnosis. Most NSCLC patients are diagnosed with lung cancer at an advanced stage and have missed the best time for surgical resection (3-5). For such patients, the traditional treatment method is chemotherapy. However, it is difficult to make a breakthrough in curative effect, the adverse reactions are severe, and the benefit to patients is limited (6).

In recent years, with the development of tumor molecular biology and genomics, the concept of “precision medicine” has been deepened, and the clinical treatment of patients with advanced NSCLC has gradually begun to be individualized (7). Molecular targeted therapy has been the focus of NSCLC research in recent years. One of the hotspots, compared with traditional chemotherapeutic drugs, it has the characteristics of solid targeting and less toxic side effects. It is a new type of anti-tumor drug with multiple mechanisms of action. Targeted drugs represented by epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs) have effectively treated lung cancer with mutated targets (8).

First-generation EGFR-TKIs, including gefitinib, enhance the treatment outcomes of patients with EGFR mutation-positive advanced NSCLC, prolonging the median progression-free survival (mPFS) to approximately a half year (9,10). For example, mPFS was 11.0 months with gefitinib in treatment-naïve patients, compared with 5.6 months with chemotherapy. Phase III studies of first- and second-generation EGFR-TKIs have shown that EGFR-TKIs can significantly improve the overall survival rate of EGFR-positive patients and show strong efficacy in the first-line treatment of locally advanced or metastatic NSCLC patients with sensitive EGFR mutations (11,12). However, despite the remarkable effectiveness, most patients still develop resistance to EGFR-TKIs about 1 year after treatment, leading to disease progression. Therefore, the essential treatment for the EGFR T790M mutation is using third-generation EGFR-TKI drugs to prevent disease progression or intolerable adverse events.

Osimertinib is the first third-generation EGFR-TKI, which can play a role in both EGFR-sensitive gene mutations and EGFR T790M drug-resistant gene mutations, and is used for the treatment of advanced NSCLC that is positive for T790M mutations after receiving EGFR-TKI therapy (13,14). The results of the FLAURA study showed that compared with the first-generation EGFR-TKI treatment, the use of osimertinib treatment could reduce the risk of disease progression or death in patients by 54%. However, adverse reactions such as rash and diarrhea remain prominent. Therefore, it is necessary to optimize the third-generation EGFR inhibitors to provide effective first-line treatment of EGFR-mutant NSCLC with good tolerance. In March 2020, aumolertinib was approved for marketing by the National Medical Products Administration, becoming the second third-generation EGFR-TKI approved for EGFR T790M-positive advanced NSCLC patients, and was approved in China as a first-line treatment for adults with untreated advanced NSCLC and EGFR ex19del or L858R mutations (15,16). Therefore, it is necessary to investigate the mechanism of aumolertinib to optimize EGFR-TKI treatment outcomes and develop adjuvant strategies for prolonging patient survival.

This study employed next-generation sequencing technology and in vitro/in vivo experiments to obtain data on aumolertinib’s efficacy in treating NSCLC. The research analyzed and validated the potential molecular mechanisms underlying aumolertinib’s therapeutic effects, providing valuable insights for enhancing its clinical outcomes. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-aw-2071/rc).

Methods

Cell culture

PC-9 cell line was purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were inoculated in RPMI 1640 medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum, penicillin 1×10⁵ U/L, and streptomycin 100 mg/L, at 37 ℃, 5% CO₂ saturated humidity cultured in a constant temperature incubator. All experiments used cells in a logarithmic growth phase.

Cell viability assay

Collect PC-9 cells stably growing in the logarithmic phase, count them using a cell counting plate, adjust the cell density to 5×10⁴/mL, and seed them in a 96-well culture plate with 100 µL of cell suspension added to each well. Place the plate in a 37 ℃, 5% CO₂ constant-temperature incubator. After cell adhesion, replace the medium in each well with different concentrations (1, 2, 4, 8, 16, and 60 µmol/L) of aumolertinib (MedChemExpress, Monmouth Junction, NJ, USA). The cells are then cultured for 24 and 48 hours for detection, with three replicate wells set for each concentration. At the 24 and 48 hours time points, discard the old medium, add 10 µL of Cell Counting Kit-8 (CCK-8) solution and 100 µL of serum-free medium to each well, and incubate in the dark at 37 ℃ for 2 h. Finally, measure the absorbance at 450 nm wavelength using a microplate reader (Bio-Rad, Hercules, CA, USA).

Wound healing assay

The cells of each group were inoculated in six-well plates at 2×10⁵ cells/well, and the cells of each group were treated according to grouping and cultured routinely. Vertical lines were drawn perpendicular to the six-well plate, and the six-well plate was slowly washed with phosphate-buffered saline (PBS) to remove the cells treated with the line. Routinely cultivated for 24 hours, and observed the cell migration at the scratch under an optical microscope.

Flow cytometry detection

Collect the cells of each group in the flow tube, wash the cells with pre-cooled PBS, centrifuge at 1,000 r/min for 8 min, discard the supernatant, add 200 µL 1× Annexin V binding solution to resuspend the cells, add 3 µL Annexin V-fluorescein isothiocyanate (FITC), incubated on ice for 15 min, then added 5 µL propidium iodide (PI) staining solution, mixed gently, protected from light, set on ice for 10 min; added 200 µL 1× Annexin V binding solution, mixed well, and the sample was dissolved within 1 hour detected by flow cytometry.

Cells in the logarithmic growth phase were harvested, digested with trypsin to make cell suspension, and transferred to 15 mL centrifuge tubes, with about (2–3)×10⁶ cells in each centrifuge tube, washed with PBS, and centrifuged at 1,000 r/min at room temperature. 70% pre-cooled ethanol was added and placed at 4 ℃ overnight. Take it out from the refrigerator, centrifuge at room temperature for 10 min, wash the centrifuged cell pellet with PBS, resuspend it in PBS, and count the total number of cells to 1×10⁶, centrifuge at room temperature for 10 min. Add 500 µL PI, and stain at room temperature for 20 min in the dark. The suspension was filtered through a 40 µm cell strainer and tested on a flow cytometer.

Public data analysis

To analyze the mechanism of lung cancer EGFR-TKI treatment, we collected transcriptome sequencing data of PC-9 cells treated with osimertinib and gefitinib from the Gene Expression Omnibus (GEO) database, GSE193258 and GSE178975, respectively. After downloading the expression data, we analyzed both datasets’ differential expression and functional enrichment. After obtaining the differentially expressed genes (DEGs), we compared the differential expression of genes in the two public datasets with that of aumolertinib-treated cells. Finally, we analyzed the relationship between the transcriptome changes caused by the three-drug treatments. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

RNA sequencing (RNA-seq) analysis

Collect PC-9 cells stably growing in the logarithmic phase, seed them into 10 cm culture dishes, add aumolertinib (2 µM, experimental group) to the cell culture medium, and use solvent-only as the control group. After 48 hours of culture, collect samples for transcriptome sequencing. The RNA samples were treated with the RiboMinus Eukaryote Kit (Qiagen, Valencia, CA, USA) to remove ribosomal RNA (rRNA) before constructing the RNA-seq libraries. Following the manufacturer’s instructions, assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Qubit 4.0) were used to assessing the library’s concentration. Agilent Bioanalyzer, Agilent D1000 Reagents, and Agilent D1000 Reagents (Agilent, Santa Clara, CA, USA) were used to determine the library fragments and sequenced by Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). After loading, the data was split, and quality control was performed according to Illumina standard procedures.

We used htseq-count software to calculate the read count information for each gene. Then, we analyzed the DEGs between aumolertinib-treated and control cells based on the read count data using the DESeq2 R package. After calculating the DEGs, we used the clusterProfiler R package to analyze the enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional pathways in the DEGs between the two groups of samples. We also generated the heatmap of the expression levels of DEGs across all samples.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) assay

To investigate the role of the ETS variant transcription factor 4 (ETV4), loss-of-function experiments were conducted using RNA interference technology. Shanghai GenePharma Co., Ltd. (Shanghai, China) synthesized human-specific ETV4 small interfering RNA (siRNA). The ETV4 siRNA (siETV4) sequences were as follows: siETV4-1: GAGCAACGGAAUUUCCUGATT; siETV4-2: CCAGCCAUGAAUUACGACATT; siETV4-3: GUGAGCGUUACGUGUACAATT.

PC-9 cells were cultured in 1640 complete medium and maintained in a 37 ℃, 5% CO₂ incubator. Before transfection, the cells were seeded in six-well plates at a density of 2.5×10⁵ cells per well and cultured overnight until the cell confluence reached 70–80%. Transfection was performed according to the instructions of Lipofectamine^® 2000 (Thermo Fisher Scientific): the experimental group was transfected with 50 nM siETV4, while the control group was transfected with an equal concentration of control reagent. Forty-eight hours after transfection, the transfection efficiency was detected by qRT-PCR and Western blot analysis.

Total RNA was extracted using TRIzol™ reagent (Thermo Fisher Scientific). RNA concentration and purity were determined by measuring absorbance at 260 and 280 nm. One microgram of total RNA was reverse-transcribed into complementary DNA (cDNA) using PrimeScript RT Master Mix (Takara Bio Inc., Kusatsu, Japan). qRT-PCR was performed using SYBR Premix Ex Taq™ (Takara Bio Inc.) on an insert instrument name, e.g., Applied Biosystems 7500 Real-Time PCR System. The primer sequences were as follows:

ETV4 forward (F):
5'-CAGTGCCTTTACTCCAGTGCC-3';
ETV4 reverse (R):
5'-CTCAGGAAATTCCGTTGCTCT-3';
18S F: 5'-cagccacccgagattgagca-3';
18S R: 5'-tagtagcgacgggcggtgtg-3'.

Using 18S rRNA as the internal reference gene, the relative expression level of ETV4 messenger RNA (mRNA) was calculated using the 2^−ΔΔCt method, with the control siRNA group serving as the calibrator.

Protein extraction and western blotting assay

Cells were lysed using cold lysis buffer (Beyotime, Nantong, China), and quantification of protein concentration was performed with a BCA protein assay kit (Beyotime Institute of Biotechnology). Then, equal amounts (20 µg) of proteins from each group were purified with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 ℃. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, 5174, Cell Signaling Technology, Danvers, MA, USA) was used as an internal control. Next, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000, 7074, Cell Signaling Technology) for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) reagent (Millipore). ImageJ was used to analyze the grey level of the bands.

Mouse and xenograft tumor models

All experimental animals were housed in a designated pathogen-free environment for a period of 1 week prior to the experiment, and all experimental animals in this study had free access to food and water. Five-week-old female BALB/C nude mice were used for the subcutaneous injection experiment. PC-9 cells in the logarithmic growth phase were digested with trypsin, and the supernatant was removed by centrifugation. The cells were then resuspended in serum-free medium, and the cell concentration was adjusted to 5×10⁷ cells/mL. Twenty nude mice were randomly divided into four groups (n=5 per group). 5×10⁶ cells (100 µL) was injected under the skin of the right forelimb of each mouse. Once the subcutaneous tumor size reached 100 mm³, treatment was administered. The following drug treatments were applied: Normal saline group (control): administered once daily by oral gavage, n=5. siETV4 tail vein injection group: 30 µg per mouse, injected once every 3 days, n=5. Oral aumolertinib group: 15 µg per mouse, administered once daily, n=5. Combination treatment group: siETV4 (30 µg/animal every 3 days) + aumolertinib (15 mg/kg daily), n=5. Tumors were measured twice weekly, and the tumor volumes were determined from caliper measurements of the tumor length (L) and width (W) according to the formula: (L × W²)/2. Since no mice died, CO₂ euthanasia was performed on the 20 mice, followed by cervical dislocation to confirm death. All animal experiments were performed under a project license (No. 2025-HDYY-043) granted by the Laboratory Animal Ethics and Welfare Committee of Fudan University, in compliance with national guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. The unpaired two-tailed t-test was used to compare the two groups, and P<0.05 indicated statistical significance.

Results

In this study, we first explored the inhibitory effect of EGFR-TKI aumolertinib on the activity of PC-9 cells. The results of the CCK-8 experiment showed that with the increase of drug concentration and the extension of time, the inhibitory effect of the aumolertinib on PC-9 cells was gradually enhanced, which was time and dose-dependent (Figure 1A). Then we verified the effects of aumolertinib on lung cancer cell proliferation and tumorigenesis were investigated. As shown in Figure 1B, CCK-8 assay demonstrated that aumolertinib administration (2 µM) led to inhibition of cell proliferation. Meanwhile, the wound healing assay demonstrated that aumolertinib administration inhibited migration abilities (Figure 1C,1D). Flow cytometry with Annexin V/PI staining (Figure 1E,1F) demonstrated that aumolertinib administration led to enhancement of apoptosis. Meanwhile, aumolertinib administration was significantly induced cell cycle arrest (Figure 1G,1H). The above results show that aumolertinib has a good effect on inhibiting lung cancer cell proliferation and tumorigenesis.

Figure 1 Aumolertinib inhibits proliferation, migration, and induces apoptosis in PC-9 cells. (A) CCK-8 assay was used to evaluate the inhibitory effects of aumolertinib on cell viability of PC-9 cells. (B) CCK-8 assay was used to evaluate the inhibitory effects of aumolertinib on proliferation of PC-9 cells (P=0.003). (C,D) In vitro wound healing assay was performed to assess the effects of aumolertinib on migration ability of PC-9 cells (P=0.02), magnification 100×. (E,F) Flow cytometry with Annexin V/PI staining was used to determine the effects of aumolertinib on apoptosis of PC-9 cells (P<0.001). (G,H) Flow cytometric analysis of the cell cycle in PC-9 cells treated with aumolertinib (P=0.049). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ETV4, ETS variant transcription factor 4; FITC, fluorescein isothiocyanate; PI, propidium iodide; siETV4, ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Next, we used RNA-seq data to analyze the effects of EGFR-TKIs on gene differential expression after the treatment of PC-9 cells. The principal component analysis (PCA) results showed that the features of PC-9 samples treated with three aumolertinib and control samples were significantly different (Figure 2A). Heatmap results showed that the DEGs in the aumolertinib treatment group and the control group were significantly different (Figure 2B). Differential expression analysis was conducted in the aumolertinib treatment group and the control group. Then, 389 up-regulated and 547 down-regulated DEGs were obtained with P<0.05 (Figure 2C). The following GO analysis found that most of these DEGs were enriched in biological process (BP), cell component (CC), and molecular function (MF), such as response to cytoplasmic translation, cytosolic ribosome, and structural constituent of ribosome (Figure 2D). KEGG analysis found that DEGs were enriched in the ribosome, efferocytosis, and hippo signaling pathway (Figure 2E). As shown in Figure S1, heatmap results showed that the DEGs were in the gefitinib (GSE178975) treatment group and the osimertinib (GSE193258) treatment group. Venn analysis was used to find the DEGs of three types of EGFR-TKI after treatment, and found that ETV4 and DUSP6 responded to aumolertinib, osimertinib, and gefitinib after treatment (Figure 2F).

Figure 2 Transcriptomic analysis reveals aumolertinib-induced differential gene expression and pathway modulation in PC-9 cells. (A) PCA of grouped samples from the control and aumolertinib administration cohorts. (B) Heatmap showing the DEGs in control and aumolertinib administration cohorts. (C) Volcano plot showing the DEGs in control and aumolertinib administration cohorts. (D) GO analysis revealed that DEGs were mainly focused on BPs in aumolertinib administration cohorts. (E) KEGG analysis indicated that DEGs in aumolertinib administration cohorts. (F) Venn plot illustrating the DEGs among our RNA-seq data, GSE178975 dataset (gefitinib), and GSE193258 dataset (osimertinib). Alm, aumolertinib administration; BP, biological process; CC, cell component; DEG, differentially expressed gene; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; NC, negative control; PC, principal component; PCA, principal component analysis; RNA-seq, RNA sequencing.

Previous studies have elucidated the regulatory role of DUSP6 in the treatment of NSCLC (17-19). In contrast, little research has been done on the regulatory function of ETV4 in EGFR-TKI treatment of NSCLC. Next, we conducted further experiments to reveal the molecular mechanism by which ETV4 affects proliferation and tumorigenesis during the treatment of lung cancer cells with aumolertinib. We knocked down the expression of ETV4 by siRNA, detected the mRNA expression level of ETV4 in the knocked down expression cell line by qRT-PCR (Figure 3A), and verified the expression of ETV4 protein by Western blot (Figure 3B,3C). It was found that siETV4-3 significantly reduced ETV4 mRNA and protein expression levels in PC-9 cells. Subsequently, we explored the differences in the therapeutic effect of aumolertinib on lung cancer cells after the knockdown of ETV4. Firstly, the proliferation of cells within 72 hours was observed via CCK-8 detection. It was found that, in comparison with the control group, the inhibitory effect of aumolertinib on cell proliferation was significantly enhanced in the ETV4 knockdown group (Figure 3D). Wound healing experiments showed that ETV4 knockdown enhanced the ability of aumolertinib to inhibit the migration of lung cancer cells (Figure 4A,4B). Annexin V-FITC/PI staining was used to detect the effect of knockdown ETV4 on the apoptosis of lung cancer cells induced by aumolertinib. The results showed that the low expression of ETV4 significantly promoted the ability of aumolertinib to induce apoptosis of lung cancer cells (Figure 4C,4D). As shown in Figure 4E,4F, PI staining assays were performed to detect the apoptosis-inducing ability of aumolertinib on PC-9 cells with ETV4 knockdown. The proportion of the G2/M phase in ETV4 knockdown groups was markedly increased compared to the control group. The above results indicate that low ETV4 expression enhances the ability of aumolertinib to induce apoptosis in PC-9 cells, while also improving its effect on inhibiting PC-9 cell migration.

Figure 3 Silencing ETV4 potentiates aumolertinib’s growth inhibition in PC-9 cells. (A) PC-9 cells were transfected with siETV4 (siETV4-1, siETV4-2, and siETV4-3) or siNC. Knockdown efficiency using siETV4 in PC-9 cells was analyzed by qRT-PCR (P<0.001). (B,C) Knockdown efficiency using siETV4 in PC-9 cells was analyzed by immunoblotting. GAPDH was used as the control (P<0.001). (D) CCK-8 assay was used to evaluate the effects of siETV4 on proliferation of PC-9 cells that treated with aumolertinib (P=0.001). **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ETV4, ETS variant transcription factor 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; siETV4, ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Figure 4 ETV4 knockdown enhances aumolertinib’s anti-tumor effects in PC-9 cells. (A,B) Wound healing assay was performed to evaluate the effect of ETV4 knockdown on migration ability of PC-9 cells that treated with aumolertinib (P=0.03), magnification 100×. (C,D) Flow cytometry with Annexin V/PI staining was used to assess the influence of ETV4 knockdown on apoptosis of PC-9 cells that treated with aumolertinib (P<0.001). (E,F) Flow cytometric analysis was used to assess the influence of ETV4 knockdown on cell cycle of PC-9 cells that treated with aumolertinib. *, P<0.05; **, P<0.01; ***, P<0.001. ETV4, ETS variant transcription factor 4; FITC, fluorescein isothiocyanate; PI, propidium iodide; siETV4, ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

To further investigate the therapeutic role of ETV4 in aumolertinib-treated lung cancer, this study employed a subcutaneous xenograft model in nude mice to evaluate the efficacy of ETV4 knockdown combined with aumolertinib. Twenty nude mice were randomly divided into four groups (n=5 per group). The treatment regimens included: (I) normal saline, (II) siETV4, (III) aumolertinib, and (IV) a combination of siETV4 and aumolertinib (Figure 5A). As shown in Figure 5B, tumors in the control group grew the fastest, reaching 1 cm³ by day 21. The aumolertinib-alone and siETV4-alone groups exhibited similar tumor growth rates, both significantly slower than the control group. Notably, the combination therapy group (aumolertinib + siETV4) showed the most pronounced suppression of tumor growth. Figure 5C,5D presents the analysis of tumor weights across groups. The control group had the heaviest tumors, while the aumolertinib and siETV4 monotherapy groups displayed comparable and significantly lower tumor weights. The combination therapy group achieved the greatest reduction in tumor mass. These results demonstrate that both aumolertinib and siETV4 monotherapies effectively inhibit tumor growth, but the combination therapy yields superior efficacy compared to either treatment alone.

Figure 5 ETV4 enhances the inhibitory effects of aumolertinib on tumor growth in vivo. (A) Photographs of mice models treated with ETV4 inhibitors and/or aumolertinib. (B) Tumor growth curves of mice models treated with ETV4 inhibitors and/or aumolertinib (P<0.001). Tumor volume (mm³) was measured every 3 days. (C,D) Comparison of tumor weights (g) treated with ETV4 inhibitors and/or aumolertinib (P<0.001). *, P<0.05; ***, P<0.001. ALM, aumolertinib administration; ETV4, ETS variant transcription factor 4; NC, negative control; siETV4, ETV4 siRNA; siRNA, small interfering RNA.

Discussion

Among various types of lung cancer, NSCLC has the highest proportion. Most patients have metastasized at initial diagnosis or relapsed after surgery or radiotherapy. Therefore, for NSCLC patients, effective inhibition of tumor migration and invasion will be the primary method to improve the prognosis of patients (20). Targeted therapy has become one of the most attractive and promising treatments because of its good curative effect and minor adverse reactions. Small molecule EGFR-TKIs, such as gefitinib and erlotinib, have become the treatment of choice for patients with EGFR-mutant NSCLC (21). However, after using the first and second-generation EGFR-TKIs, patients will inevitably develop resistance to them, the most representative of which is the T790M mutation.

Aumolertinib, as the third-generation EGFR-TKI after osimertinib, not only successfully solved the problem of T790M drug resistance but also has better safety and tolerance and more substantial blood-brain barrier penetration (22). In addition, it has a definite curative effect on patients with brain metastases, and clinical studies have shown that it has an excellent therapeutic effect on lung cancer. The AENEAS study is a multicenter, randomized, double-blind, controlled phase III clinical trial, which compared the efficacy and safety of aumolertinib and gefitinib in the first-line treatment of EGFR mutation-positive advanced NSCLC. Four hundred and twenty-nine patients were enrolled in 53 centers, 214 in the aumolertinib group and 215 in the gefitinib group. All patients had locally advanced or metastatic NSCLC, had not received other regimens before, and had 19del or L858R mutation. The study’s preliminary results showed that the mPFS of first-line treatment patients with aumolertinib was 19.3 months, which was significantly higher than the mPFS (9.9 months) of first-line treatment patients with gefitinib. In terms of durable remission, the median duration of response (mDOR) of the aumolertinib group reached 18.1 months, while the mDOR of the gefitinib group was only 8.3 months. Among all 19del patients, the mPFS was 20.8 months and 12.3 months in the aumolertinib and gefitinib groups, respectively; among all L858R mutation patients, the mPFS was 13.4 months in the aumolertinib and gefitinib groups, respectively. Among all patients with central nervous system metastases, the mPFS of the aumolertinib and the gefitinib groups were 15.3 and 8.2 months, respectively. In patients without central nervous system metastases, the mPFS in the two groups were 19.3 and 12.6 months, respectively, and the objective response rate and disease control rate were similar in the aumolertinib group and the gefitinib group (15). Compared with the global multi-center phase III clinical study of Osimertinib, the mPFS of aumolertinib was similar to the global data of osimertinib in terms of value (19.3 vs. 18.9) but significantly higher than that of osimertinib in China (19.3 vs. 17.8) (23). At the same time, the risk of disease progression [hazard ratio (HR) =0.38; 95% confidence interval (CI): 0.24–0.60] in patients with brain metastases who received first-line treatment with aumolertinib was significantly lower than the global data of osimertinib (HR =0.48; 95% CI: 0.26–0.86) and China subgroup data (HR =0.66; 95% CI: 0.30–1.38), suggesting that aumolertinib is more suitable for Chinese patients and has a better effect on brain metastases (24).

Although EGFR-TKIs have excellent disease control effects, acquired drug resistance still inevitably occurs, which is also a complex problem for targeted therapy. A variety of acquired resistance mechanisms of EGFR-TKI have been discovered, the most important of which is the second point mutation, which is caused by the amino acid substitution at position 790 (T790M) of exon 20, resulting in ATP-competitive TKI decreased binding capacity and drug activity (25,26). Other mechanisms include MET, HER2 amplification, PIK3CA mutation, BRAF mutation, small cell lung cancer, and epithelial-mesenchymal transition (27). Although the results of the AURA3 study proved that the third-generation EGFR-TKI (osimertinib) had shown great clinical benefits in patients with T790M-positive advanced NSCLC after resistance to the first and second-generation EGFR-TKIs, these patients’ drug resistance also occurs after receiving third-generation EGFR-TKI treatment (28). Therefore, almost all patients receiving EGFR-TKI treatment, no matter their EGFR-TKI, will eventually develop acquired drug resistance. Consequently, it is urgent to deeply explore the mechanism of action of aumolertinib, delay the occurrence of EGFR-TKI resistance, and adjuvant treatment strategies to prolong the survival time of patients.

Previous research has demonstrated that the high expression of ETV4 is correlated with tumor invasion, metastasis, and prognosis (29). For instance, there is a significant difference in the expression of ETV4 mRNA between normal breast tissue and tumor tissue, which may play a crucial role in the onset of breast cancer. Baker et al. utilized the MMTV/Wnt1 transgenic mouse model and discovered that silencing the expression of the ETV4 gene could inhibit the growth of mouse breast cancer cells (30). Zhu et al. also verified in the study of primary breast cancer cells that ETV4 is associated with the pathological grade and prognosis of breast cancer (31). Moreover, ETV4 mRNA and protein are also overexpressed in colorectal cancer, and ETV4 overexpression is associated with short survival (32,33). A previous study on ETV4 in lung cancer has shown that by activating ETV4-mediated upregulation of MMP24, it regulates the migration and invasion ability of tumor cells, and elevated levels of ETV4 and MMP24 are biomarkers of tumor progression and poor prognosis (34). In this study, through transcriptome sequencing of cell samples treated with aumolertinib, abnormal expression of ETV4 was found in the GEO database, and ETV4 was inferred to be a potential target affecting the efficacy of aumolertinib.

Previous studies have shown that in human cancers, the PI3K/AKT and MAPK/ERK signaling pathways are frequently activated. They promote malignant transformation of cells and tumor growth by enhancing cell growth and proliferation while inhibiting apoptosis (35). In lung cancer, mutations in upstream receptors (such as EGFR) are frequent (36). The persistent activation of MAPK/ERK signaling is associated with poor prognosis and is therefore regarded as a negative prognostic indicator and a therapeutic target in cancer (37). BCL2 family proteins are key components of the intrinsic pathway. Overexpression of certain anti-apoptotic family members, such as BCL2, inhibits apoptosis in tumor cells (38). Studies using specific inhibitors of the PI3K/AKT and ERK pathways in EGFR-mutant cells indicate that the ERK pathway primarily regulates the status of BIM downstream of EGFR. This suggests that targeting the intrinsic pathway may represent a therapeutic strategy to further improve clinical outcomes for patients with EGFR-mutant lung cancer (39).

Recent studies have indicated that the ETV4 gene exhibits abnormal expression in diverse human malignancies, including gastric carcinoma and esophageal carcinoma. These studies disclose that ETV4 assumes a crucial function in facilitating tumor cell proliferation, migration, and invasion. Specifically, in the research on gastric carcinoma, scientists have ascertained that ETV4 targets the KDM5D gene and diminishes its expression level, thereby expediting the metastasis process of gastric carcinoma cells. Moreover, ETV4 can augment the expression of KIF2A, promoting cell proliferation and inhibiting apoptosis through the activation of the AKT signaling pathway. This finding offers a novel research perspective for the treatment of gastric carcinoma (40). In the study on esophageal squamous cell carcinoma, researchers have discovered that neuropilin 2 activates the MAPK/ERK and ETV4 signaling pathways, thereby inducing tumorigenesis and metastasis (41). In the research on esophageal adenocarcinoma, ETV4 promotes cell proliferation and invasion by mediating the MMP1 gene associated with the MAPK/ERK pathway, thus driving the progression of esophageal adenocarcinoma (42). It is worth noting that this study found that ETV4 knockout significantly enhanced the migration inhibition mediated by aumolertinib. While existing research has established that ETV4 promotes tumor cell migration and invasion through MMP1 upregulation, cell migration remains a complex process involving dynamic regulation of MMPs, integrins, and cytoskeletal components. Thus, EGFR-TKIs may also regulate cell migration through ETV4-independent pathways, such as the PI3K-AKT and JAK/STAT signaling pathways, mediating PI3K-AKT, JAK/STAT, and other signaling pathways to control cell migration (43,44).

This study, through integrating transcriptomic datasets of EGFR-TKIs (aumolertinib, osimertinib, and gefitinib) and in vivo/in vitro model experiments, confirms that ETV4 knockdown significantly enhances lung cancer cells’ sensitivity to aumolertinib treatment, revealing its novel role as a therapeutic target. However, there are certain limitations in this study. First, there is insufficient investigation into the regulatory relationships of ETV4 within upstream and downstream signaling pathways, as well as the specific molecular mechanisms through which ETV4 enhances the therapeutic efficacy of aumolertinib in non-small cell lung cancer. Second, although the study verified the role of ETV4 in enhancing the sensitivity of lung cancer cells to aumolertinib through an in vivo nude mouse model, the in vitro investigations were solely carried out using the PC-9 cell line. Elucidating the regulatory network of ETV4 signaling pathways and its underlying molecular mechanisms will offer a more robust basis for the development of subsequent therapeutic targets.

Conclusions

In conclusion, our study demonstrates that the third-generation EGFR-TKI Aumolertinib exerts potent antitumor effects in NSCLC, including inhibition of cell proliferation, induction of G2/M arrest, promotion of apoptosis, and suppression of migration, as revealed by comprehensive in vitro and in vivo studies. These findings not only expand our understanding of EGFR-TKI mechanisms but also provide a compelling rationale for developing ETV4 as both a predictive biomarker and therapeutic target in NSCLC. Further investigation into the ETV4 regulatory network and validation in clinical samples will be crucial for translating these findings into improved treatment strategies for EGFR-mutant lung cancer patients.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-aw-2071/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-aw-2071/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-aw-2071/prf

Funding: This work was supported by the Lian Yun Gang Shi Hui Lan Public Foundation (No. HL-HS2020-70).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-aw-2071/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were performed under a project license (No. 2025-HDYY-043) granted by the Laboratory Animal Ethics and Welfare Committee of Fudan University, in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Chen P, Liu Y, Wen Y, et al. Non-small cell lung cancer in China. Cancer Commun (Lond) 2022;42:937-70. [Crossref] [PubMed]
2. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. [Crossref] [PubMed]
3. Thai AA, Solomon BJ, Sequist LV, et al. Lung cancer. Lancet 2021;398:535-54. [Crossref] [PubMed]
4. Jovanoski N, Abogunrin S, Di Maio D, et al. Systematic Literature Review to Identify Cost and Resource Use Data in Patients with Early-Stage Non-small Cell Lung Cancer (NSCLC). Pharmacoeconomics 2023;41:1437-52. [Crossref] [PubMed]
5. Wang C, Chen B, Liang S, et al. China Protocol for early screening, precise diagnosis, and individualized treatment of lung cancer. Signal Transduct Target Ther 2025;10:175. [Crossref] [PubMed]
6. Gerber DE, Schiller JH. Maintenance chemotherapy for advanced non-small-cell lung cancer: new life for an old idea. J Clin Oncol 2013;31:1009-20. [Crossref] [PubMed]
7. Park K, Tan EH, O’Byrne K, et al. Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial. Lancet Oncol 2016;17:577-89. [Crossref] [PubMed]
8. Deng S, Sun C, Liu D, et al. Antibody-drug conjugates: A new twist to overcome EGFR-TKIs resistance in non-small cell lung cancer. Pharmacol Res 2026;223:108066. [Crossref] [PubMed]
9. Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 2010;362:2380-8. [Crossref] [PubMed]
10. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 2012;13:239-46. [Crossref] [PubMed]
11. Zhou C, Wu YL, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol 2011;12:735-42. [Crossref] [PubMed]
12. Wu YL, Zhou C, Hu CP, et al. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Lancet Oncol 2014;15:213-22. [Crossref] [PubMed]
13. Ramalingam SS, Vansteenkiste J, Planchard D, et al. Overall Survival with Osimertinib in Untreated, EGFR-Mutated Advanced NSCLC. N Engl J Med 2020;382:41-50. [Crossref] [PubMed]
14. Remon J, Steuer CE, Ramalingam SS, et al. Osimertinib and other third-generation EGFR TKI in EGFR-mutant NSCLC patients. Ann Oncol 2018;29:i20-7. [Crossref] [PubMed]
15. Lu S, Dong X, Jian H, et al. AENEAS: A Randomized Phase III Trial of Aumolertinib Versus Gefitinib as First-Line Therapy for Locally Advanced or MetastaticNon-Small-Cell Lung Cancer With EGFR Exon 19 Deletion or L858R Mutations. J Clin Oncol 2022;40:3162-71. [Crossref] [PubMed]
16. Wang J, Wu L. An evaluation of aumolertinib for the treatment of EGFR T790M mutation-positive non-small cell lung cancer. Expert Opin Pharmacother 2022;23:647-52. [Crossref] [PubMed]
17. Zhang Z, Kobayashi S, Borczuk AC, et al. Dual specificity phosphatase 6 (DUSP6) is an ETS-regulated negative feedback mediator of oncogenic ERK signaling in lung cancer cells. Carcinogenesis 2010;31:577-86. [Crossref] [PubMed]
18. Moncho-Amor V, Pintado-Berninches L, Ibañez de Cáceres I, et al. Role of Dusp6 Phosphatase as a Tumor Suppressor in Non-Small Cell Lung Cancer. Int J Mol Sci 2019;20:2036. [Crossref] [PubMed]
19. Phuchareon J, McCormick F, Eisele DW, et al. EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function. Proc Natl Acad Sci U S A 2015;112:E3855-63. [Crossref] [PubMed]
20. Thyagarajan A, Gajjar V, Sahu RP. Are metformin-based combination approaches beneficial for non-small cell lung cancer: evidence from experimental and clinical studies. Mil Med Res 2025;12:61. [Crossref] [PubMed]
21. Rolfo C, Giovannetti E, Hong DS, et al. Novel therapeutic strategies for patients with NSCLC that do not respond to treatment with EGFR inhibitors. Cancer Treat Rev 2014;40:990-1004. [Crossref] [PubMed]
22. Lu S, Wang Q, Zhang G, et al. Efficacy of Aumolertinib (HS-10296) in Patients With Advanced EGFR T790M+ NSCLC: Updated Post-National Medical Products Administration Approval Results From the APOLLO Registrational Trial. J Thorac Oncol 2022;17:411-22. [Crossref] [PubMed]
23. Lamb YN. Osimertinib: A Review in Previously Untreated, EGFR Mutation-Positive, Advanced NSCLC. Target Oncol 2021;16:687-95. [Crossref] [PubMed]
24. Cheng Y, He Y, Li W, et al. Osimertinib Versus Comparator EGFR TKI as First-Line Treatment for EGFR-Mutated Advanced NSCLC: FLAURA China, A Randomized Study. Target Oncol 2021;16:165-76. [Crossref] [PubMed]
25. Jänne PA, Yang JC, Kim DW, et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. N Engl J Med 2015;372:1689-99. [Crossref] [PubMed]
26. Hata AN, Niederst MJ, Archibald HL, et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat Med 2016;22:262-9. [Crossref] [PubMed]
27. Wu SG, Shih JY. Management of acquired resistance to EGFR TKI-targeted therapy in advanced non-small cell lung cancer. Mol Cancer 2018;17:38. [Crossref] [PubMed]
28. Mok TS, Wu Y-L, Ahn M-J, et al. Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer. N Engl J Med 2017;376:629-40. [Crossref] [PubMed]
29. Cosi I, Pellecchia A, De Lorenzo E, et al. ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21. J Hematol Oncol 2020;13:112. [Crossref] [PubMed]
30. Baker R, Kent CV, Silbermann RA, et al. Pea3 transcription factors and wnt1-induced mouse mammary neoplasia. PLoS One 2010;5:e8854. [Crossref] [PubMed]
31. Zhu T, Zheng J, Zhuo W, et al. ETV4 promotes breast cancer cell stemness by activating glycolysis and CXCR4-mediated sonic Hedgehog signaling. Cell Death Discov 2021;7:126. [Crossref] [PubMed]
32. Lee JS, Kim E, Lee J, et al. Capicua suppresses colorectal cancer progression via repression of ETV4 expression. Cancer Cell Int 2020;20:42. [Crossref] [PubMed]
33. Fonseca AS, Ramão A, Bürger MC, et al. ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer. BMC Cancer 2021;21:207. [Crossref] [PubMed]
34. Okimoto RA, Breitenbuecher F, Olivas VR, et al. Inactivation of Capicua drives cancer metastasis. Nat Genet 2017;49:87-96. [Crossref] [PubMed]
35. Poulikakos PI, Zhang C, Bollag G, et al. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 2010;464:427-30. [Crossref] [PubMed]
36. Tu HY, Ke EE, Yang JJ, et al. A comprehensive review of uncommon EGFR mutations in patients with non-small cell lung cancer. Lung Cancer 2017;114:96-102. [Crossref] [PubMed]
37. Bartholomeusz C, Gonzalez-Angulo AM, Liu P, et al. High ERK protein expression levels correlate with shorter survival in triple-negative breast cancer patients. Oncologist 2012;17:766-74. [Crossref] [PubMed]
38. McGill GG, Horstmann M, Widlund HR, et al. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 2002;109:707-18. [Crossref] [PubMed]
39. Lum JJ, Bauer DE, Kong M, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 2005;120:237-48. [Crossref] [PubMed]
40. Cai LS, Chen QX, Fang SY, et al. ETV4 promotes the progression of gastric cancer through regulating KDM5D. Eur Rev Med Pharmacol Sci 2020;24:2442-51. [Crossref] [PubMed]
41. Fung TM, Ng KY, Tong M, et al. Neuropilin-2 promotes tumourigenicity and metastasis in oesophageal squamous cell carcinoma through ERK-MAPK-ETV4-MMP-E-cadherin deregulation. J Pathol 2016;239:309-19. [Crossref] [PubMed]
42. Keld R, Guo B, Downey P, et al. The ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma. Mol Cancer 2010;9:313. [Crossref] [PubMed]
43. Baumgartner U, Berger F, Hashemi Gheinani A, et al. miR-19b enhances proliferation and apoptosis resistance via the EGFR signaling pathway by targeting PP2A and BIM in non-small cell lung cancer. Mol Cancer 2018;17:44. [Crossref] [PubMed]
44. Liu X, Pu W, He H, et al. Novel ROR1 inhibitor ARI-1 suppresses the development of non-small cell lung cancer. Cancer Lett 2019;458:76-85. [Crossref] [PubMed]