Cancer Res Treat. 2019;51(3):951-962.

Acquired Resistance of MET-Amplified Non-small Cell Lung Cancer Cells to the MET Inhibitor Capmatinib.

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제1저자 김슬기, 김태민 · 교신저자 김동완

PMID 30309221 PMC6639226 DOI 10.4143/crt.2018.052 IF 4.2 혈액종양내과

소속 (2)

Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea.

Abstract

PURPOSE Amplified mesenchymal-epithelial transition factor, MET, is a receptor tyrosine kinase (RTK) that has been considered a druggable target in non-small cell lung cancer (NSCLC). Although multiple MET tyrosine kinase inhibitors (TKIs) are being actively developed for MET-driven NSCLC, the mechanisms of acquired resistance to MET-TKIs have not been well elucidated. To understand the mechanisms of resistance and establish therapeutic strategies, we developed an in vitro model using the MET-amplified NSCLC cell line EBC-1.

MATERIALS AND METHODS We established capmatinib-resistant NSCLC cell lines and identified alternative signaling pathways using 3' mRNA sequencing and human phospho-RTK arrays. Copy number alterations were evaluated by quantitative polymerase chain reaction and cell proliferation assay; activation of RTKs and downstream effectors were compared between the parental cell line EBC-1 and the resistant cell lines.

RESULTS We found that EBC-CR1 showed an epidermal growth factor receptor (EGFR)‒dependent growth and sensitivity to afatinib, an irreversible EGFR TKI. EBC-CR2 cells that had overexpression of EGFR-MET heterodimer dramatically responded to combined capmatinib with afatinib. In addition, EBC-CR3 cells derived from EBC-CR1 cells that activated EGFR with amplified phosphoinositide-3 kinase catalytic subunit α (PIK3CA) were sensitive to combined afatinib with BYL719, a phosphoinositide 3-kinase α (PI3Kα) inhibitor.

CONCLUSION Our in vitro studies suggested that activation of EGFR signaling and/or genetic alteration of downstream effectors like PIK3CA were alternative resistance mechanisms used by capmatinib-resistant NSCLC cell lines. In addition, combined treatments with MET, EGFR, and PI3Kα inhibitors may be effective therapeutic strategies in capmatinib-resistant NSCLC patients.

Keywords

Acquired resistanceCapmatinibMET amplificationMET tyrosine kinase inhibitorNon-small cell lung carcinoma

Figures

**Fig. 1.** Molecular characterization of capmatinib-resistant cell lines. (A) Capmatinib-resistant cell lines (EBC-CR1, EBC-CR2, and EBC-CR3) were derived from EBC-1, which is a non-small cell lung cancer cell line that harbors mesenchymal-epithelial transition factor (*MET*) amplification. Cell lines were treated with capmatinib and crizotinib for 72 hours and growth inhibition was determined by cell viability assay (Ez-cytox). Tests were performed as three independent experiments. The EBC-CR1, -CR2, and -CR3 cell lines showed resistance to capmatinib and cross-resistance to crizotinib. (B) The resistant cell lines showed significant *MET* copy number loss after long-term treatment with capmatinib (^*p < 0.05). *MET* copy number was confirmed by quantitative polymerase chain reaction. hgDNA, human genomic DNA (C) Capmatinib-resistant cells had persistent expression of phosphorylated ERK1/2 and AKT in the presence of capmatinib. EBC-1 and the resistant cell lines were treated with capmatinib at 1 μmol/L for 2 hours then analyzed by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control for the Western blot. (D) Hierarchical clustering analysis of the resistant cell lines revealed a cluster containing seven receptor tyrosine kinases (RTKs) that was increased in all resistant cell lines. The values for clustering analysis fulfilled the requirements of normalized read counts (log2) > 6 and p < 0.05. The fold changes in expression of these RTKs are indicated in Table 1. Each color represents relative gene expression with the highest expression as red, lowest expression as green, and median expression as black.

**Fig. 2.** Shift from mesenchymal-epithelial transition factor (MET) to epidermal growth factor receptor (EGFR) kinase pathway in EBC-CR1. (A) EBC-1 and resistant cell lines were treated with afatinib for 72 hours. The 50% inhibitory concentrations (IC₅₀) were calculated using Sigma Plot 12.0; results are indicated as mean±standard deviation. (B) Heparin-binding epidermal growth factor‒like growth factor (HBEGF) expression was measured by quantitative reverse transcription polymerase chain reaction in three independent experiments. HBEGF expression was increased in the EBC-CR1 cell line compared to the parental and other capmatinib-resistant cell lines (^**p < 0.01). (C) For Western blot, EBC-1 and EBC-CR1 cells were treated with serial 10-fold dilutions ranging from 0.1 to 1 μmol/L of capmatinib with or without afatinib at 1 μmol/L for 24 hours. EBC-CR1 showed almost complete dependency on downstream signaling due to afatinib treatment with or without capmatinib. Phosphorylation of AKT and ERK1/2 was efficiently inhibited by capmatinib in EBC-1 cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

**Fig. 3.** Combined treatment with capmatinib and afatinib effectively inhibited phosphorylation of downstream signaling and proliferation in EBC-CR2 cells. (A) For Western blot, EBC-CR2 cells were treated with increasing concentrations of capmatinib with or without afatinib at 1 μmol/L for 24 hours. Capmatinib significantly increased epidermal growth factor receptor (EGFR) phosphorylation in a dose-dependent manner. The combination of capmatinib with afatinib at 1 μmol/L effectively inhibited phosphorylation of AKT and ERK at a low concentration of capmatinib, 100 nmol/L. (B) The heterodimerization of receptor tyrosine kinases was analyzed by co-immunoprecipitation. Although immunoprecipitation (IP) with mesenchymal-epithelial transition factor (MET) showed EGFR-MET interactions in EBC-CR1, 2, and 3 cells, IP with EGFR showed an interaction between EGFR and MET only in EBC-CR2 cells. Therefore, EBC-CR2 cells had more dominant EGFR-MET heterodimers than the other resistant cell lines. (C) EBC-CR2 cells treated with capmatinib, afatinib, and capmatinib with afatinib at 0.1 μmol/L, respectively, for 72 hours. Treatment with the combination of capmatinib with afatinib synergistically inhibited EBC-CR2 cell proliferation.

**Fig. 4.** *PIK3CA* amplification in EBC-CR3, resulting to afatinib-resistance and schematic model of resistant mechanisms to capmatinib in *MET*-amplified non-small cell lung cancer (NSCLC) cell lines. (A) Treatment of EBC-CR3 cells with afatinib, BYL719, and BYL719 with afatinib at 0.1 μmol/L, respectively, for 72 hours. BYL719 with afatinib synergistically inhibited proliferation of EBC-CR3 cells. (B) *PIK3CA* copy numbers in the cell lines were measured by quantitative polymerase chain reaction. Compared to the other cell lines and the human genomic DNA control, *PIK3CA* was amplified in the EBC-CR3 cell line (^**p < 0.01). (C) Treatment of EBC-CR3 cells with afatinib 500 nmol/L, BYL719 1 μmol/L, or BYL719 with afatinib at 500 nmol/L, respectively, for 24 hours. The combination of BYL719 with afatinib completely inhibited AKT phosphorylation. (D) In a drug-sensitive cell line harboring *MET* amplification, cell proliferation and survival signals are highly dependent on constitutively activated MET kinase without ligand binding and/or homodimerization. In addition, MET activates the epidermal growth factor receptor (EGFR) signal pathway via multiple mechanisms. In contrast, the MET pathway cannot act as a survival signal and alternative pathways must be activated in capmatinib-resistant cells. We confirmed three different mechanisms of resistance to capmatinib in MET-dependent NSCLC cell lines. (1) heterodimerization between MET and EGFR, (2) increased EGFR and EGF-like growth factor (HBEGF) expression; and (3) *PIK3CA* amplification via activated EGFR-dependent PIK3CA stimulation, resulting in afatinib resistance. Each mechanism has different molecular characteristics and therapeutic strategies. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PI3K, phosphoinositide-3-kinase; mTOR, mammalian target of rapamycin.

그림 출처: PubMed Central 공개 논문 (PMC6639226)

Conflict of interest statement

Conflict of interest relevant to this article was not reported.

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