Abstract P071: Phosphoproteomics identifies Mig6 as a key mediator of adaptive resistance to ALK/ROS1 oncogene inhibition

Abstract

Abstract Tyrosine kinase inhibitors (TKIs) targeting ALK and ROS1 fusions have significantly improved outcomes for patients with non-small cell lung cancer harboring these oncogenes. However, complete responses are rarely achieved, and therapeutic resistance eventually emerges from residual tumor. To control or eradicate residual disease pre-emptively, it is important to understand the molecular mechanism contributing to the adaptive resistance under initial exposure to oncogene inhibition. Our lab previously demonstrated that EGFR mediates responses to TKIs in oncogene fusion positive cancers, but the detailed mechanism underlying adaptive EGFR signaling is still not clear. We performed global and tyrosine-specific phosphoproteomic analyses to investigate the signaling reprogramming following 2-hr treatment of crizotinib, a dual ALK/ROS1 inhibitor, in an EML4-ALK cell line (H3122) and a TPM3-ROS1 patient-derived cell line (CUTO28). Pathway analysis revealed “ErbB signaling pathway” and “regulation of ErbB signaling pathway” were enriched in the proteins with differentially expressed phosphosites under crizotinib treatment. Mig6, a protein that negatively regulates EGFR, showed a significant decrease of phosphorylation of Y394/Y395 following crizotinib treatment. Phosphorylation of these 2 residues has been shown to be essential for Mig6 binding and subsequent inhibition of EGFR, suggesting that EGFR activity may be released from Mig6 inhibition rapidly following ALK or ROS1 inhibition. Mig6 transcripts and protein levels were also reduced by following ALK/ROS1 inhibition. The MEK inhibitor trametinib decreased Mig6 transcripts and protein with a similar magnitude as ALK/ROS1 inhibition, suggesting that the regulation of Mig6 expression by ALK/ROS1 inhibition is mediated by the MAPK pathway. Mig6 knock-down was able to rescue cell survival and p-Erk suppression from ALK/ROS1 inhibition, but failed to rescue when co-treated with afatinib, a pan-HER inhibitor. Neither Mig6 knockdown nor overexpression altered the phosphorylation of EGFR itself. Instead, phosphorylation of Y239 and Y240 on Shc1, a signaling adapter shared by ALK, ROS1, and EGFR, was rescued by Mig6 knockdown under ALK/ROS1 inhibition. This is consistent with the previously reported structures showing Mig6 and Shc1 compete for the same substrate binding cleft on EGFR. Combination of afatinib and ALK/ROS1 TKIs eliminated residual colony formation in 2 weeks. Crizotinib-resistant H3122 demonstrated loss of Mig6 and resistance could be reversed using either afatinib or Mig6 overexpression. In summary, this study identified Mig6 as a novel regulator for ErbB signaling-mediated adaptive and acquired resistance to ALK/ROS1 TKIs. This resistance mechanism may have broader implications for other oncogene-driven cancers given the observed Mig6 regulation by the MAPK pathway. These data provide additional support for co-inhibition of EGFR in oncogene-driven cancers, such as ALK or ROS1, to reduce the burden of residual disease by eliminating Mig6-mediated EGFR survival pathways. Citation Format: Nan Chen, Anh T. Le, Eric A. Welsh, Bin Fang, Eric B. Haura, Robert C. Doebele. Phosphoproteomics identifies Mig6 as a key mediator of adaptive resistance to ALK/ROS1 oncogene inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P071.