Abstract
Introduction
EGFR tyrosine kinase inhibitors are standard therapeutic agents for patients with advanced NSCLC harboring EGFR mutations. Nevertheless, some patients exhibit primary resistance to EGFR tyrosine kinase inhibitors in the first-line treatment setting. AXL, a member of the TYRO3, AXL, and MERTK family of receptor tyrosine kinases, is involved in primary resistance to EGFR tyrosine kinase inhibitors in EGFR-mutated NSCLC.
Methods
We investigated spatial tumor heterogeneity using autopsy specimens and a patient-derived cell line from a patient with EGFR-mutated NSCLC having primary resistance to erlotinib plus ramucirumab.
Results
Quantitative polymerase chain reaction analysis revealed that AXL mRNA expression differed at each metastatic site. In addition, AXL expression levels were likely to be negatively correlated with the effectiveness of erlotinib plus ramucirumab therapy. Analysis of a patient-derived cell line established from the left pleural effusion before initiation of treatment revealed that the combination of EGFR tyrosine kinase inhibitors and an AXL inhibitor remarkably inhibited cell viability and increased cell apoptosis in comparison with EGFR tyrosine kinase inhibitor monotherapy or combination therapy of these inhibitors with ramucirumab.
Conclusions
Our observations suggest that AXL expression may play a critical role in the progression of spatial tumor heterogeneity and primary resistance to EGFR tyrosine kinase inhibitors in patients with EGFR-mutated NSCLC.
Keywords: AXL, Drug resistance, EGFR tyrosine kinase inhibitor, Heterogeneity, Non–small cell lung cancer
Introduction
Lung cancer is the leading cause of cancer-related death worldwide.1 EGFR mutations are major driver gene mutations in NSCLC. EGFR tyrosine kinase inhibitor (TKI) monotherapy is highly effective in patients with EGFR-mutated NSCLC. Nevertheless, approximately 20% of patients with EGFR-mutated NSCLC do not achieve clinical response to such first-line treatments.2, 3, 4 To overcome these issues, combined therapeutic strategies, including EGFR TKIs plus antiangiogenesis agents or chemotherapy, have been recently approved in several countries for the first-line treatment of patients with EGFR-mutated NSCLC.3,5
Various underlying mechanisms, including hepatocyte growth factor overexpression and deletion polymorphisms of BCL2-like 11, have been reported to cause primary resistance to EGFR TKIs.6,7 Our previous studies reported that AXL, a member of the TYRO3, AXL, and MERTK family of receptor tyrosine kinases, maintained a survival signal as a bypass pathway when EGFR-mutated NSCLC cell lines with high AXL expression were exposed to EGFR TKIs, and that a combination of EGFR TKIs and AXL inhibitors was effective for these cell lines.8,9 Our recent prospective observational study also revealed that in patients with EGFR-mutated NSCLC, high tumor AXL levels were significantly associated with a shorter progression-free survival after third-generation EGFR TKI osimertinib therapy in comparison with low AXL levels.10 On the basis of these studies, a clinical trial is ongoing to evaluate the safety and efficacy of the combination of osimertinib and ONO-7475, an AXL inhibitor, in patients with untreated advanced EGFR-mutated NSCLC in Japan (jRCT2051210045).
Intratumor heterogeneity is associated with the effectiveness of targeted therapies for various types of cancer.11, 12, 13 Therefore, understanding the involvement of spatial tumor heterogeneity as a primary resistance mechanism against molecular-targeted therapy in patients with NSCLC is important. In this study, to reveal the clinical impact of spatial tumor heterogeneity in patients with EGFR-mutated NSCLC, we evaluated the relationship between the clinical response and AXL expression levels in autopsy tumor samples from a patient with advanced EGFR-mutated NSCLC who had primary resistance to erlotinib plus ramucirumab. Furthermore, we performed cell line-based analysis to evaluate the efficacy of the combination of EGFR TKIs and an AXL inhibitor by using a patient-derived cell line established from left pleural effusion before initiation of the treatment.
Material and Methods
The Patient
The patient was a 67-year-old man who underwent left upper lobectomy for localized NSCLC (pT2aN0M0) in January 2021. During postoperative adjuvant chemotherapy with S-1, he had recurrence in July 2021. Because the EGFR-L858R mutation was detected from a recurrent site, combination therapy with the EGFR TKI erlotinib and the anti–vascular epithelial growth factor receptor 2 antibody ramucirumab was initiated. The patient died owing to cancer progression 15 days after initiation of the treatment. Postmortem autopsy was performed, and samples were obtained from various metastatic sites by macroscopically cutting out part of the tumor.
Real-Time Polymerase Chain Reaction
Autopsy samples were homogenized using a homogenizer. Total RNA was purified using the NucleoSpin RNA Plus (Takara Bio). Complementary DNA was synthesized using PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio). Real-time polymerase chain reaction (PCR) was performed using a QuantStudio 3 Real-Time PCR system (Applied Biosystems) and THUNDERBIRD Next SYBR quantitative PCR Mix (TOYOBO). Gene expression levels were calculated using the 2−ΔΔCT method.14 Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. The primer sequences were as follows: EGFR L858R (F: 5′-GCATTTGCCAAGTCCTACAGA-3′, R: 5′-CTATCAATGCAAGCCACGG-3′), AXL (F: 5′-GGTGGCTGTGAAGACGATGA-3′, R: 5′-CTCAGATACTCCATGCCACT-3′), and glyceraldehyde 3-phosphate dehydrogenase (F: 5′-GCCAAATATGATGACATCAAGAAGG-3′, R: 5′-GGTGTCGCTGTTGAAGTCAGAG-3′).
Patient-Derived Cell Line Establishment
Tumor cells were collected by centrifugation from the left pleural effusion of the patient before initiation of erlotinib plus ramucirumab therapy. We established the patient-derived cell line KPP-03 from tumor cells, as described previously.15 EGFR-L858R mutation was confirmed using the PNA-LNA PCR clamp method (LSI Medience Corporation).
Cell Culture and Reagents
HCC827 and PC-9 cells were purchased from the American Type Culture Collection and RIKEN Cell Bank, respectively. H1975 cells containing EGFR-L858R and T790M double mutation were provided by Dr. Yoshitaka Sekido (Aichi Cancer Center Research Institute) and Dr. John D. Minna. The cells were maintained in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37°C. All cells were passaged for less than 3 months and tested for mycoplasma using a MycoAlert Mycoplasma Detection Kit (Lonza). Erlotinib (first-generation EGFR TKI), osimertinib (third-generation EGFR TKI), ONO-7475 (AXL inhibitor), and ramucirumab (human anti–vascular epithelial growth factor receptor 2 antibody) were obtained from Selleck Chemicals.
Western Blotting
Western blotting was performed as described previously8 using primary antibodies for p-AXL (Tyr702), t-AXL, p-EGFR (Tyr1086), β-actin (13E5), p-Akt (Ser473), t-Akt, p-Erk1/2 (Thr202/Tyr204), t-ERK1/2 (1:1000; Cell Signaling Technology), and t-EGFR (1:1000; R&D Systems).
Cell Viability Assay
The cells (3 × 103 cells/well) were cultured with the indicated drugs for 72 hours. Cell viability was determined using the MTT assay (Sigma-Aldrich). Absorbance was measured using a microplate reader. The cells were also treated for 9 days, with the drugs replenished every 72 hours. The cells were stained with crystal violet and examined visually.
Apoptotic Cell Analysis
KPP-03 cells (3 × 105) were treated with the indicated drugs for 48 hours. Then, the cells were collected and incubated with Annexin V-FITC and propidium iodide for 15 minutes, after which they were analyzed using a BD Accuri C6 Plus Flow Cytometer (Becton, Dickinson & Company). Data were analyzed using FlowJo software (FlowJo LLC), as reported previously.16
siRNA Transfection
Cells were transfected with a Silencer Select small interfering (si)RNA for AXL (s1845; Invitrogen) using Lipofectamine RNAi-MAX (Invitrogen) according to the manufacturer’s instructions. The Silencer Select siRNA for negative control number 1 (Invitrogen) was the scrambled control. AXL knockdown was confirmed using Western blotting. Each sample was independently analyzed at least three times.
Statistical Analysis
Data from the MTT assay and flow cytometry were expressed as mean ± SD. Significant differences were analyzed using unpaired t tests using Prism 9.0 (GraphPad Software), with two-sided p values of less than 0.05 considered significant.
Ethical Consideration
This study was approved by the institutional review board of Kyoto Prefectural University of Medicine (No. ERB-C-977). Written informed consent was obtained from the patient and his family for the establishment of the cell line, use of the tumor specimens, and publication of the medical records and research results.
Results
Case Report
The patient was a 67-year-old man who underwent left upper lobectomy for localized NSCLC (pT2aN0M0) in January 2021. Postoperative recurrence was diagnosed using computed tomography (CT)-guided lung biopsy for a lung nodule during postoperative adjuvant chemotherapy with S-1 (80 mg/m2, d 0–13) in July 2021. The EGFR-L858R mutation was detected using the Oncomine Dx Target Test (Thermo Fisher Scientific) with a CT-guided biopsy specimen. Although combination therapy with erlotinib (150 mg/d) and ramucirumab (10 mg/kg) was initiated, the patient died owing to cancer progression 15 days after initiation of the treatment (Fig. 1A). In a comparison of CT scan results taken 9 days after initiation with those taken before initiation of the treatment, tumors in the right and left lower lobes had no remarkable changes, whereas a tumor in the right upper lobe increased rapidly, and left pleural effusion increased (Fig. 1B). These different observations may have been influenced by the heterogeneity among the metastatic tumors.
Figure 1.
AXL expression levels are negatively correlated with the efficacy of EGFR TKI treatment: clinical case. (A) A 67-year-old man underwent left upper lobectomy for localized NSCLC (pT2aN0M0) in January 2021. Postoperative recurrence was diagnosed in July 2021 during postoperative adjuvant chemotherapy with S-1. A CT scan and left thoracentesis were performed on the day before initiation of erlotinib plus ramucirumab. A CT scan was performed 9 days after initiation of treatment, and the treatment was discontinued. The patient died 15 days after initiation of the treatment. (B) CT scans taken before initiation of the treatment and those taken 9 days after initiation of the treatment. (C) The AXL mRNA expression levels of autopsy specimens measured by qPCR. (D) The EGFR-L858R–mutated mRNA expression levels of autopsy specimens measured by qPCR. (E) The AXL mRNA expression levels divided by the EGFR-L858R–mutated mRNA expression levels. CT, computed tomography; qPCR, quantitative polymerase chain reaction; TKI, tyrosine kinase inhibitor.
AXL Expression Levels Are Negatively Correlated With the Effectiveness of Treatment Regimens Including EGFR TKIs
To investigate the spatial tumor heterogeneity in relation to AXL expression, autopsy specimens from multiple metastatic sites were obtained, and AXL mRNA expression levels were compared using quantitative PCR. AXL mRNA expression levels of tumors in the right upper lobe and left pleura, which revealed tumor progression, were higher than those of tumors in the right and left lower lobes, which revealed stable disease (Fig. 1C). In comparison with AXL mRNA expression, EGFR-L858R–mutated mRNA expression revealed no remarkable association with the clinical response to treatment (Fig. 1D). To quantify the fraction of the tumor cells, AXL mRNA expression levels were divided by EGFR-L858R–mutated mRNA expression levels. The number of refractory tumors having disease progression was higher than that of the other tumors (Fig. 1E). Taken together, these findings suggest that AXL expression in the tumors may be negatively related to the effectiveness of EGFR TKIs and may lead to spatial tumor heterogeneity.
Patient-Derived Cell Lines With High AXL Expression Were Insensitive to Standard Treatments With EGFR TKIs
We next established the patient-derived cell line KPP-03 from the left pleural effusion of this patient before initiation of erlotinib plus ramucirumab therapy. The EGFR-L858R mutation was detected in the KPP-03 cells, which had higher AXL expression than HCC-827 cells with low AXL levels and PC-9 cells with high AXL levels8,9 (Fig. 2A and B). The growth inhibitory assay revealed that the KPP-03 and PC-9 cells had lower sensitivity to erlotinib plus ramucirumab than the HCC-827 cells. Similar results were observed in osimertinib treatment (Fig. 2C). We evaluated the knockdown effect of AXL using a specific siRNA to observe the dependency of cell viability on AXL signaling during EGFR TKI treatment. Treatment with AXL-specific siRNA enhanced the inhibitory effects of high AXL-expressed KPP-03 and PC-9 cells on EGFR TKIs than that of low AXL-expressed HCC827 cells, suggesting that AXL signaling might reduce drug tolerance to EGFR TKIs in high AXL-expressed cells (Fig. 2D and Supplementary Fig. 1).
Figure 2.
The patient-derived cell line KPP-03 with high AXL expression was insensitive to EGFR TKIs. (A) The AXL mRNA expression levels of KPP-03, PC-9, and HCC827 cells measured by the qPCR assay. (B) The indicated proteins of KPP-03, PC-9, and HCC827 cells detected by the Western blotting assay. (C) KPP-03, PC-9, and HCC827 cells were incubated with erlotinib plus ramucirumab (150 ng/μL) or osimertinib for 72 hours. Cell viability was determined using the MTT assay. (D) KPP-03, PC-9, and HCC827 cells treated with nonspecific control or AXL-specific siRNA were incubated with or without erlotinib (100 nmol/L) or osimertinib (100 nmol/L) for 72 hours, and cell viability was detected using MTT assays. p-, phosphorylated-; qPCR, quantitative polymerase chain reaction; si, small interfering.
The Combination of EGFR TKIs and an AXL Inhibitor Overcame Primary Resistance to EGFR TKIs in the Patient-Derived Cell Line
We performed in vitro experiments using KPP-03, PC-9, and H1975 cells to evaluate whether combining EGFR TKIs and an AXL inhibitor could inhibit the proliferation of cells expressing high AXL levels (Supplementary Fig. 2). Combining erlotinib or osimertinib with the AXL inhibitor—ONO-7475—considerably inhibited cell proliferation more than EGFR TKI monotherapy and the combination with ramucirumab in KPP-03, PC-9, and H1975 cells, except for erlotinib treatment for H1975 cells (Fig. 3A). In addition, the continuous co-treatment of KPP-03 cells with EGFR TKIs and ONO-7475 for 9 days reduced cell viability than EGFR TKI monotherapy or the combination with ramucirumab (Fig. 3B). Western blotting analysis indicated that the combination of EGFR TKIs and ONO-7475 suppressed the phosphorylation of ERK and AKT in comparison with EGFR TKI monotherapy and the combination with ramucirumab in KPP-03 cells (Fig. 3C). Apoptosis analysis revealed that the combination of EGFR TKIs and ONO-7475 considerably increased cell apoptosis in comparison with EGFR TKI monotherapy and the combination with ramucirumab in KPP-03 cells (Fig. 3D). These results indicated that AXL activation induced primary resistance to EGFR TKIs and that an initial combination of EGFR TKIs and an AXL inhibitor could overcome the insensitivity to EGFR TKI monotherapy and the combination with ramucirumab in AXL-overexpressing cells.
Figure 3.
Combination of EGFR TKIs and an AXL inhibitor overcomes primary resistance caused by AXL signal activation. (A) KPP-03, PC-9, and H1975 cells were treated with EGFR TKIs alone (erlotinib 100 nmol/liter or osimertinib 100 nmol/liter), ONO-7475 (100 nmol/L) alone, ramucirumab (150 ng/μL) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab for 72 hours. Cell viability was assessed using the MTT assay. (B) KPP-03 cells were treated with EGFR TKIs alone (erlotinib 100 nmol/L or osimertinib 100 nmol/L), ONO-7475 (100 nmol/L) alone, ramucirumab (150 ng/μL) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab for 9 days. Cells were stained with crystal violet. (C) KPP-03 cells were incubated with EGFR TKIs alone (erlotinib 100 nmol/L or osimertinib 100 nmol/L), ONO-7475 (100 nmol/L) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab (150 ng/μL) for 4 hours, and the indicated proteins were then detected by Western blotting assay. (D) KPP-03 cells were treated with EGFR TKIs alone (erlotinib 100 nmol/L or osimertinib 100 nmol/L), ONO-7475 (100 nmol/L) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab (150 ng/μL) for 48 hours, and apoptotic cells were then detected by flow cytometry. p-, phosphorylated-; TKI, tyrosine kinase inhibitor.
Discussion
Treatment with EGFR TKIs is the standard strategy for patients with untreated advanced EGFR-mutated NSCLC.17, 18, 19 Nevertheless, some patients have primary resistance to EGFR TKI treatment.2, 3, 4 High AXL protein levels in tumors are related to primary resistance and poor clinical response to osimertinib in patients with advanced EGFR-mutated NSCLC.10 On the basis of these clinical issues, elucidation of the mechanism of primary resistance to EGFR TKIs and development of novel therapies to overcome such primary resistance in patients with EGFR-mutated NSCLC are essential. Intratumor heterogeneity has been reported to be an important factor in primary resistance to EGFR TKIs.20 Nevertheless, intratumor heterogeneity has not yet been evaluated in relation to AXL expression in patients with EGFR-mutated NSCLC. In this study, we investigated the effect of AXL activation on spatial tumor heterogeneity by using autopsy specimens and a patient-derived cell line from a patient with EGFR-mutated NSCLC who had primary resistance to erlotinib plus ramucirumab.
Our observations revealed that AXL mRNA expression levels in autopsy samples were likely correlated with the clinical response to erlotinib plus ramucirumab. In addition, the patient-derived cell line KPP-03, which had high AXL expression, was less sensitive to erlotinib plus ramucirumab than HCC827 cells expressing lower levels of AXL, and the combination of EGFR TKIs and an AXL inhibitor overcame the resistance to current standard treatments. These findings suggest that AXL expression levels in tumors could predict the effective lesions for initial combined inhibition of AXL and EGFR in EGFR-mutated NSCLC tumors.
The tumor microenvironment (TME) has been reported to promote spatial tumor heterogeneity in lung cancer.21 The hypoxic environment in tumors is a critical factor in the TME. A preclinical study reported that hypoxia-inducible factors directly activated AXL expression in renal cell cancer.22 In this study, tumors with high AXL expression levels in the right upper lobe were larger than those with low AXL expression in the right and left lower lobes. These findings suggest that enrichment of AXL expression may be induced by the hypoxic environment in large tumors. Nevertheless, this is a limited analysis; therefore, further large-scale investigations are required to confirm our observations.
We revealed that the combination of EGFR TKIs and an AXL inhibitor ONO-7475 effectively suppressed phosphorylation of ERK and AKT in KPP-03 cells, indicating that KPP-03 cells depend on the AXL cascade for resistance to EGFR TKIs. The cancer immune environment is one of the major factors involved in the regulation of tumorigenicity. AXL has been reported to be involved in the immune environment and is currently being identified as a novel therapeutic target.23,24 Thus, these observations suggest that in vivo models and clinical practice might increase the dependency of AXL signaling on resistance to EGFR TKIs through its effects on the TME.25,26
This study revealed a fourfold difference in AXL expression among the metastatic lesions, suggesting that analysis by a single biopsy may underestimate the evaluation of AXL expression levels in whole tumors. Nevertheless, multiple biopsies from multiple metastatic sites are difficult to perform; therefore, evaluation by liquid biopsy when administering treatment might be useful.
This study had some limitations. First, we observed a single case. Therefore, we could not conclude the relationship between AXL expression in tumors and the effectiveness of EGFR TKIs. Nevertheless, a previous study of BRAF-mutant melanoma revealed that heterogeneity of AXL expression could affect the efficacy of MAPK pathway inhibitors, which suggested that AXL expression heterogeneity might affect the efficacy of molecular-targeted therapy, including EGFR TKIs.27 Second, the patient died shortly after initiating the treatment. Therefore, we could not evaluate the treatment efficacy after a sufficient period, which might lead to underestimating the changes in small lesions. Third, we did not establish patient-derived cell lines from multiple lesions in the present study; therefore, we could not compare the results of drug sensitivity in multiple lesions in vitro. Additional studies on the clinical efficacy of concomitant AXL inhibitors on the basis of AXL expression levels are required. Fourth, other primary resistance mechanisms were not evaluated. Nevertheless, the combination of EGFR TKIs and an AXL inhibitor remarkably suppressed the viability of KPP-03 cells, suggesting that high AXL expression might be the major resistance mechanism of EGFR TKIs.
In conclusion, AXL expression in tumors may play a critical role in the progression of spatial tumor heterogeneity and primary resistance to EGFR TKIs in patients with EGFR-mutated NSCLC. In such cases, an initial combination of EGFR TKIs and AXL inhibitors may be a promising therapeutic option for patients with EGFR-mutated NSCLC.
CRediT Authorship Contribution Statement
Ryota Nakamura: Conceptualization, Data curation, Formal analysis, Methodology, Visualization, Writing—original draft preparation.
Hiroyuki Fujii: Conceptualization, Resources.
Tadaaki Yamada: Conceptualization, Funding acquisition, Project administration, Methodology, Writing—original draft preparation.
Yohei Matsui, Takeshi Yaoi, Mizuki Honda, Noriyuki Tanaka, Aya Miyagawa-Hayashino: Resources.
Akihiro Yoshimura, Kenji Morimoto, Masahiro Iwasaku, Shinsaku Tokuda, Young Hak Kim: Writing—review and editing.
Eiichi Konishi, Kyoko Itoh, Koichi Takayama: Supervision.
Acknowledgments
This work was supported by the Takeda Science Foundation and the Princess Takamatsu Cancer Research Fund. The authors thank Editage (www.editage.jp) for help with the English-language editing.
Footnotes
Disclosure: Dr. Yamada received commercial research grants from Pfizer, Ono Pharmaceutical, Janssen Pharmaceutical K.K., AstraZeneca, and Takeda Pharmaceutical Company Limited and speaking honoraria from Eli Lilly. Dr. Takayama received research grants from Chugai-Roche and Ono Pharmaceutical and personal fees from AstraZeneca, Chugai-Roche, Merck Sharp & Dohme-Merck, Eli Lilly, Boehringer Ingelheim, and Daiichi Sankyo. The remaining authors declare no conflict of interest.
Cite this article as: Nakamura R, Fujii H, Yamada T, et al. Analysis of tumor heterogeneity through AXL activation in primary resistance to EGFR tyrosine kinase inhibitors. JTO Clin Res Rep. 2023;4:100525.
Note: To access the supplementary material accompanying this article, visit the online version of the JTO Clinical and Research Reports at www.jtocrr.org and at https://doi.org/10.1016/j.jtocrr.2023.100525.
Supplementary Data
References
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