NRF2-mediated changes in Glutathione Metabolism mediate the resistance of EGFR-T790M Mutant Lung Cancer Cells to Erlotinib
Olivier Pardo,
Team Leader,
Imperial College London
EGFR tyrosine-kinase inhibitors (TKI) such as erlotinib are novel agents in the treatment of lung cancer. However, their efficacy is impaired by the development of drug-resistance through secondary receptor mutations, such as the T790M substitution. Although increased affinity of the mutants for ATP that competes out the inhibitors was considered responsible for this, we show that additional factors are at play. Following metabonomics profiling of erlotinib-sensitive/resistant cell pairs we found that the levels of glutathione (GSH) are considerably reduced in erlotinib-resistant (ER) cells. Using RNA interference and pharmacological inhibitors of GSH pathway enzymes, we demonstrate that increasing GSH levels in ER cells sensitises these to erlotinib. Conversely, reducing GSH levels renders sensitive cells resistant to the drug. We show that the reduction in GSH levels in ER cells is associated with the decreased transcription of the GSH synthesising enzymes, GCLC and GSS, a phenomenon also observed in clinical samples from patients with T790M-EGFR. This correlates with inhibition of NRF2, through increased levels of its inhibitor KEAP1 and/or decreased expression of the co-activators SQSTM1 and PALB2. We demonstrate these changes to be directly linked to the T790M mutation, as introducing the T790M-EGFR in HEK293 cells reduces GSH levels and decreases expression of SQSTM1 and PALB2. Finally, administration of ethacrynic acid, a clinically used diuretic that is also a GST inhibitor, increases intratumoral GSH levels and re-sensitises resistant tumours to erlotininb in a xenograft mouse model. Our data identify a new resistance mechanism to EGFR TKIs and propose a novel therapeutic strategy to tackle this problem in the clinic.
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