Abstract 1100: miR205 mediates acquired resistance to ALK inhibition via targeting Mig6 expression and enhancing EGFR signaling

Abstract

Abstract Complete responses to ALK tyrosine kinase inhibitors (TKIs) are rare and resistance eventually develops in ALK fusion-positive non-small cell lung cancer patients. To overcome resistance and improve therapeutic outcomes, it is crucial to understand the molecular mechanisms contributing to resistance. Our lab has previously shown EGFR signaling mediates adaptive resistance to ALK inhibitors. We demonstrated that RNA and protein levels of Mig6, an endogenous protein inhibitor of EGFR, were suppressed rapidly following ALK inhibition, thus unlocking EGFR from inhibition to support cell survival. In this study, we asked whether EGFR signaling activation and Mig6 suppression persist once acquired resistance is established. An EML4-ALK cell line, H3122, was continuously exposed to a fixed dose of the ALK inhibitor crizotinib to generate three resistant lines (H3122-CR1, -2, and -3). All H3122-CR lines lacked ALK kinase mutations and were also cross-resistant to other ALK TKIs including ceritinib and alectinib. We found phosphorylation and total EGFR were upregulated while Mig6 protein was attenuated across all resistant lines compared to their parental counterpart. Afatinib, a pan-ERBB family inhibitor, or Mig6 overexpression, was able to re-sensitize those resistant cells to ALK inhibition. Interestingly, we did not find Mig6 overexpression altered phosphorylation of EGFR. Previously reported data suggest that Mig6 competes with Shc1, a critical signaling adapter shared by ALK and EGFR, for the same substrate-binding cleft on EGFR. We then hypothesized Mig6 could block EGFR-Shc1 binding and downstream signaling transduction without directly impacting EGFR phosphorylation. Indeed, the co-immunoprecipitation assay showed Mig6 knockdown in H3122 cells enhanced Shc1 binding to EGFR without altering phosphorylation of EGFR itself, suggesting a novel mechanism in regulating EGFR signaling by impairing the signaling adapter binding. We then investigated the mechanism responsible for Mig6 protein attenuation in resistant lines. By examining the RNA-seq data for all the CR lines compared to the parental H3122, we found MIR205HG was substantially upregulated in resistant cells. MIR205HG is the host gene for miR-205, which was known to target ERRFI1 gene that encodes Mig6. Stem-loop RT-qPCR confirmed that miR-205 was increased ~5 fold in all CR lines. Overexpressing miR-205 in H3122 cells could downregulate Mig6 expression in a dose-dependent manner. Herein we presented a novel resistance mechanism to ALK inhibition by which miR205 upregulation attenuates Mig6 expression, releasing EGFR-Shc1 signaling transduction from inhibition to support cell survival. This study also provides additional support for targeting EGFR signaling to overcome ALK TKI resistance to improve patient survival. Citation Format: Nan Chen, Robert C. Doebele. miR205 mediates acquired resistance to ALK inhibition via targeting Mig6 expression and enhancing EGFR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1100.