Altered splicing of ATG16-L1 mediates acquired resistance to tyrosine kinase inhibitors of EGFR by blocking autophagy in non-small cell lung cancer.

Despite the initial efficacy of using tyrosine kinase inhibitors of epidermal growth factor receptors (EGFR-TKIs) for treating patients with non-small cell lung cancer (NSCLC), resistance inevitably develops. Recent studies highlight a link between alternative splicing and cancer drug response. Therefore, we aimed to identify deregulated splicing events that play a role in resistance to EGFR-TKI. By using RNA sequencing, reverse-transcription PCR (RT-PCR), and RNA interference, we showed that overexpression of a splice variant of the autophagic gene ATG16-L1 that retains exon 8 and encodes the ß-isoform of autophagy-related protein 16-1 (ATG16-L1 ß) concurs acquired resistance to EGFR-TKI in NSCLC cells. Using matched biopsies, we found increased levels of ATG16-L1 ß at the time of progression in 3 of 11 NSCLC patients treated with EGFR-TKI. Mechanistically, gefitinib-induced autophagy was impaired in resistant cells that accumulated ATG16-L1 ß. Neutralization of ATG16-L1 ß restored autophagy in response to gefitinib, induced apoptosis, and inhibited the growth of in ovo tumor xenografts. Conversely, overexpression of ATG16-L1 ß in parental sensitive cells prevented gefitinib-induced autophagy and increased cell survival. These results support a role of defective autophagy in acquired resistance to EGFR-TKIs and identify splicing regulation of ATG16-L1 as a therapeutic vulnerability that could be explored for improving EGFR-targeted cancer therapy.