Targeting the adaptor protein Gab1 overcomes MET/Src-driven alectinib resistance in ALK-rearranged NSCLC.
BACKGROUND In non-small cell lung cancer (NSCLC) with anaplastic lymphoma kinase (ALK) rearrangement, bypass signaling activation commonly leads to resistance against alectinib. Identifying key molecular targets that integrate signals from resistance-driving kinases is crucial for overcoming this resistance. However, these targets have not yet been identified. METHODS We generated alectinib-resistant (AR) subclones from H3122 and H2228 cells. Subsequently, we analyzed bypass signaling pathways using western blotting, evaluated drug sensitivity with Cell Counting Kit-8 (CCK-8) assays. To assess the function of Grb2-associated binder 1 (Gab1), we employed siRNA-mediated knockdown and lentiviral shRNA both in vitro and in xenograft models. Additionally, we determined the stability of Gab1 through cycloheximide chase assays. RESULTS AR cells demonstrated concurrent activation of the mesenchymal epithelial transition receptor tyrosine kinase (MET) and Rous sarcoma oncogene cellular homolog non-receptor tyrosine kinase (Src), accompanied by upregulation of Gab1. The combined inhibition of MET and Src, as opposed to single-agent blockade, effectively suppressed Gab1/protein kinase B (AKT) signaling and restored sensitivity to alectinib. Gab1 knockdown mirrored the effects of dual kinase inhibition, by disrupting MET/Src/AKT signaling and resensitizing cells to alectinib. Mechanistically, the elevation of Gab1 resulted from post - translational stabilization, with a significantly extended half - life in resistant cells. In vivo, silencing Gab1 inhibited the growth of H3122-AR2 xenografts without causing systemic toxicity, which correlated with decreased phosphorylation of MET, Src and AKT in tumor tissues. CONCLUSION In ALK-positive NSCLC with acquired resistance to alectinib, Gab1 emerged as a crucial downstream signaling convergence target of MET and Src co-activation. Targeting this adaptor protein presented a promising therapeutic strategy to overcome bypass-mediated resistance.