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Acquired resistance mechanisms to capmatinib, a MET inhibitor in MET-amplified non-small cell lung cancer cells

ABSTRACT: Purpose: MET is a receptor tyrosine kinase (RTK) that has been considered a druggable target in non-small cell lung cancer (NSCLC). To understand the mechanisms of resistance to MET-TKIs and establish therapeutic strategies, we developed an in vitro model using capmatinib-resistant cell lines (EBC-CR1, CR2, and CR3) derived from the MET-amplified NSCLC cell line EBC-1. Methods: We established capmatinib-resistant NSCLC cell lines from the MET-amplified NSCLC cell line EBC-1 and identified alternative signaling pathways using 3’mRNA sequencing and human phospho-RTK arrays. Copy number alterations were evaluated by quantitative PCR and cell proliferation assay; activation of RTKs and downstream effectors were compared between the parental cell line EBC-1 and the EBC-CR1, -CR2, and -CR3 resistant cell lines. Results: We found that epidermal growth factor (EGFR) mRNA expression and protein activation were increased in EBC-CR1–3 cells compared to EBC-1 cells. EBC-CR1 cells showed EGFR-dependent growth and sensitivity to afatinib, an irreversible EGFR TKI. EBC-CR2 cells, which overexpressed the EGFR-MET heterodimer, responded dramatically to the combination of capmatinib and the phosphoinositide-3 kinase catalytic subunit α (PIK3CA) inhibitor afatinib. In addition, EBC-CR3 cells, which had activated EGFR along with amplified PIK3CA, were sensitive to the combination of afatinib and the PI3Kα inhibitor. Conclusions: 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 an effective therapeutic strategy in MET-TKI-resistant NSCLC patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE94405 | GEO | 2017/05/01

SECONDARY ACCESSION(S): PRJNA369611

REPOSITORIES: GEO

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Project description:EGFR tyrosine kinase inhibitors (TKIs) have demonstrated tremendous clinical benefits in non-small cell lung cancer (NSCLC) patients. However, resistance emerges rapidly due to a variety of mechanisms including a secondary mutation of T790M in EGFR that abrogates the binding of the drugs. It has been postulated that EGFR TKIs, such as afatinib (BIBW2992), with activity against the T790M mutant EGFR kinase might overcome the drug resistance problem or, when used as the first-line treatment, delay or suppress the emergence of resistance in EGFR. In this study, we generated BIBW2992-resistant cells, HCC827-BR1 and HCC827-BR2, from the parental HCC827 cells. In HCC827-BR cells, EGFR, MET, and Erb2 were down-regulated and no secondary mutation was found to be present in the coding region of EGFR. Gene set enrichment analysis (GSEA) revealed an obvious signature of epithelial to mesenchymal transition (EMT) in the drug resistant cells. Subsequently, the HCC827-BR cells were shown to be more invasive. Most importantly, we strived to seek if alternative medicine might be applied alone or in combination to treat the BIBW2992-resistant cells or to, under BIBW2992 treatment, diminish the emergence of resistant cells. Compared to the parental cells, the HCC827-BR cells were more sensitive to dasatinib, an FDA-approved kinase inhibitor. Furthermore, as revealed in the clonogenicity assay, the reduction of tumor-colony-forming cells after exposure to BIBW2992 was substantially potentiated by low concentration of dasatinib. Thus, prospective clinical investigations may be needed to evaluate if dasatinib can be beneficial to patients receiving second-generation EGFR TKIs for the treatment of NSCLC. Over a period of 3-4 months, BIBW2992-resistant cells were isolated in cell culture by maintenance of HCC827 cells in the presence of escalating concentrations of BIBW2992 up to 2 Î¼M. Two cell lines, HCC827-BR1 and HCC827-BR2, were established based on individual clones. Total RNA of these HCC827, HCC827-BR1 and HCC827-BR2, were extracted for gene expression microarray analysis using Illumina HumanHT12 v3 BeadChip.

Project description:Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC. Examination of mRNA levels in DMSO and gefitinib-resistant cultures of HCC4006 and HCC827. Each group has two replicates.

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Acquired resistance mechanisms to capmatinib, a MET inhibitor in MET-amplified non-small cell lung cancer cells

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