EML4-ALK G1202R and EML4-ALK L1196M mutations induce crizotinib resistance in non-small cell lung cancer cells through activating epithelial-mesenchymal transition mediated by MDM2/MEK/ERK signal axis

Cell Biol Int. 2025 Jan;49(1):55-67. doi: 10.1002/cbin.12249. Epub 2024 Sep 24.

Abstract

Crizotinib, as the first-generation of anaplastic lymphoma kinase (ALK) inhibitor, effectively improves the survival time of ALK-positive non-small cell lung cancer (NSCLC) patients. However, its efficacy is severely limited by drug resistance caused by secondary mutations. G1202R and L1196M are classical mutation sites located in ALK kinase domain. They may hinder the binding of ALK inhibitors to the target kinase domain, resulting in drug resistance in patients. However, the exact mechanism of drug resistance mediated by these mutations remains unclear. In this study, we aimed to evaluate how G1202R and L1196M mutations mediate crizotinib resistance. To explore the resistance mechanism, we constructed EML4-ALK G1202R and L1196M mutant cell lines with A549 cells. The results showed that the mutant cells exhibited significant epithelial-mesenchymal transition (EMT) and metastasis compared to control (A549-vector) or wild type (A549-EML4-ALK) cells. Subsequently, it was found that the occurrence of EMT was correlated to the high expression of murine double minute 2 (MDM2) protein and the activation of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in mutant cells. Down-regulation of MDM2 inhibited the activation of MEK/ERK pathway, thus reversed the EMT process and markedly increased the inhibitory effect of crizotinib on the growth of mutant cells. Collectively, resistance of ALK-positive NSCLC cells to crizotinib is induced by G1202R and L1196M mutations through activation of the MDM2/MEK/ERK signalling axis, promoting EMT process and metastasis. These findings suggest that the combination of MDM2 inhibitors and crizotinib could be a potential therapeutic strategy.

Keywords: EML4‐ALK G1202R mutation; EML4‐ALK L1196M mutation; EMT; MDM2; crizotinib resistance.

MeSH terms

  • A549 Cells
  • Animals
  • Carcinoma, Non-Small-Cell Lung* / drug therapy
  • Carcinoma, Non-Small-Cell Lung* / genetics
  • Carcinoma, Non-Small-Cell Lung* / metabolism
  • Carcinoma, Non-Small-Cell Lung* / pathology
  • Cell Line, Tumor
  • Crizotinib* / pharmacology
  • Drug Resistance, Neoplasm* / drug effects
  • Drug Resistance, Neoplasm* / genetics
  • Epithelial-Mesenchymal Transition* / drug effects
  • Humans
  • Lung Neoplasms* / drug therapy
  • Lung Neoplasms* / genetics
  • Lung Neoplasms* / metabolism
  • Lung Neoplasms* / pathology
  • MAP Kinase Signaling System* / drug effects
  • Mice
  • Mice, Nude
  • Mutation*
  • Oncogene Proteins, Fusion* / genetics
  • Oncogene Proteins, Fusion* / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-mdm2* / genetics
  • Proto-Oncogene Proteins c-mdm2* / metabolism

Substances

  • Crizotinib
  • Proto-Oncogene Proteins c-mdm2
  • EML4-ALK fusion protein, human
  • Oncogene Proteins, Fusion
  • MDM2 protein, human
  • Protein Kinase Inhibitors