Author of

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  MINI 09 - Drug Resistance (ID 107)

  • Event: WCLC 2015
  • Type: Mini Oral
  • Track: Biology, Pathology, and Molecular Testing
  • Presentations: 1
  • Moderators:L. Villaruz, J. Minna
  • Coordinates: 9/07/2015, 16:45 - 18:15, 205+207

  • 14179

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    MINI09.03 - Characterization of Afatinib or EGFR T790M Specific Inhibitor (WZ8040 or AZD9291) Resistant Lung Cancer Cells (PC9) (ID 1065)

    16:55 - 17:00  |  Author(s): A.M. Lin
    • Abstract
    • Presentation
    • Slides

    Background:
    Non-small cell lung cancer patients harboring epidermal growth factor receptor (EGFR) mutation respond well to EGFR tyrosine kinase inhibitors (TKI). However, all patients develop resistance to EGFR TKI after long term use. EGFR T790M mutation can be found in about half of the resistant re-biopsy tumors. Afatinib is an irreversible EGFR TKI with in vitro activity against resistant T790M mutation. However, afatinib has little activity in EGFR TKI resistant patients whose tumors developed T790M mutation. A novel alinino-pyrimidine based WZ8040 has been developed to specifically inhibit phosphorylation of EGFR with T790M mutation and not on wild type EGFR. Similar compounds such as CO1686 or AZD9291 has demonstrated high activity against T790M mutations. We plan to develop afatinib or AZD9291, WZ8040 resistant PC9 cells to study afatinib or AZD9291 resistance.
    
    Methods:
    PC9 cells were grown in culture media containing escalating concentrations of afatinib, WZ8040 or AZD9291. When cells can grow in high concentrations of drugs, cells were cloned, expanded and grew in drug-free media for more than two weeks to obtain stable afatinib, WZ8040 or AZD9291 resistant clones. Gefitinib, afatinib, WZ8040, AZD9291. Cells viability were determined by surforodamine bromide method. PC9 parental and EGFR TKI resistant cells were treated with gefitnib, afatinib, WZ8040 or AZD9291 for one hour and EGFR, AKT, ERK phosphorylation were determined by Western blot. DNA repair capacity were compared between sensitive and resistant cells after exposure of cells to ultraviolet light and measured by pGL3-luciferase plasmid transfection methods. Epithelial mesenchyma transition of these cells were tested by snail, slug, vimentin and E-cadherin western blot. Autophagy was measured by LC3-II levels by Western blot. EGFR exon 18-21 sequence of each clones were determined by Sanger’s direct sequencing.
    
    Results:
    We developed afatinib resistant PC9 cells, PC9/AFAb2, PC9/AFAc3 and WZ8040 resistant PC9/WZd7, PC9/WZf6. PC9/AFA cells were more than 100-fold resistant to afatinib and PC9/WZ cells were more than 50-fold resistant to WZ8040. 10nM of afatinib treatment inhibits EGF-induced EGFR, AKT and EKR phosphorylation in PC9 cells, but phosphorylation of these kinases were only partially inhibited in PC9/AFA cells. Phosphorylation was completely blocked at 100nM afatinib. MEK inhibitor plus afatinib did not reverse resistance to afatinib in PC9/AFA cells. On the other hand, WZ8040 or AZD9291 alone completely reversed resistance in PC9/AFA cells. EGFR, AKT and ERK phosphorylation can be blocked by 100nM WZ8040 in PC9 and PC9/WZd7 cells. However, it is curious that phosphorylation of these proteins can be inhibited by 100nM gefitinib as well. EGFR T790M mutation was only detected in PC9/AFA cells and not in PC9/gef, PC9/WZ cells. None of the PC9/WZ cells have EGFR C797S mutation. We did not detect any other EGFR resistance mechanism in PC9/AFA cells. Other of comparing EMT, autophagy and DNA repair capacity of PC9 and their resistant cells are ongoing.
    
    Conclusion:
    We developed multiple gefitinib, afatinib, WZ8040, AZD9291 resistant PC9 cells. Only afatinib resistant cells develop EGFR T790M. We demonstrated that EGFR T790M was the predominant resistant mechanism in PC9/AFA cells. The characteristics of PC9/WZ and PC9/AZD9291 are still under investigation.
    
    

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