Circumvention of Gefitinib Resistance by Repurposing Flunarizine via Histone Deacetylase Inhibition

Abstract

Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) for treating advanced nonsmall cell lung cancer (NSCLC). However, drug resistance seriously impedes the clinical efficacy of gefitinib. This study investigated the repositioning of the non-oncology drug capable of inhibiting histone deacetylases (HDACs) to overcome gefitinib resistance. A few drug candidates were identified using the in silico repurposing tool "DRUGSURV" and tested for HDAC inhibition. Flunarizine, originally indicated for migraine prophylaxis and vertigo treatment, was selected for detailed investigation in NSCLC cell lines harboring a range of different gefitinib resistance mechanisms (EGFR T790M, KRAS G12S, MET amplification, or PTEN loss). The circumvention of gefitinib resistance by flunarizine was further demonstrated in an EGFR TKI (erlotinib)-refractory patient-derived tumor xenograft (PDX) model in vivo. The acetylation level of cellular histone protein was increased by flunarizine in a concentration-and time-dependent manner. Among the NSCLC cell lines evaluated, the extent of gefitinib resistance circumvention by flunarizine was found to be the most pronounced in EGFR T790Mbearing H1975 cells. The gefitinib−flunarizine combination was shown to induce the apoptotic protein Bim but reduce the antiapoptotic protein Bcl-2, which apparently circumvented gefitinib resistance. The induction of Bim by flunarizine was accompanied by an increase in the histone acetylation and E2F1 interaction with the BIM gene promoter. Flunarizine was also found to upregulate E-cadherin but downregulate the vimentin expression, which subsequently inhibited cancer cell migration and invasion. Importantly, flunarizine was also shown to significantly potentiate the tumor growth suppressive effect of gefitinib in EGFR TKIrefractory PDX in vivo. The findings advocate for the translational application of flunarizine to circumvent gefitinib resistance in the clinic.