Targeting the TBK1-p62 condensate axis restores sensitivity to EGFR-TKIs in resistant lung cancer

Abstract Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in non-small cell lung cancer remains a pressing clinical challenge. Liquid-liquid phase separation has emerged as a new mechanism of drug resistance, yet its role in EGFR-TKI resistance in lung cancer is largely unexplored. Herein, we prioritized Sequestosome 1 (SQSTM1/p62) as a key condensate by integrating proteomics data from EGFR-TKI resistance cell lines and clinical biopsy specimens, in which the cytoplasmic p62 condensate formation positively correlated with EGFR-TKI resistance. Domain mapping demonstrated that the PB1 and UBA domains of p62 were critical for promoting phase separation and reducing sensitivity to EGFR-TKI treatment, whereas S403 phosphorylation promoted p62 condensation and EGFR-TKI resistance. Further xenograft studies validated that reduction of p62 condensation restores EGFR-TKI sensitivity. Kinase enrichment and interaction assays identified TBK1 as an upstream regulator of p62 S403 phosphorylation to promote p62 condensation after pharmacological inhibition of EGFR. Notably, among the five compounds identified from a drug library screen that both disrupted p62 condensate formation and inhibited the viability of resistant cells, the highly selective TBK1 inhibitor GSK8612 stood out. GSK8612 inhibited p62 S403 phosphorylation and consequently, at subcytotoxic doses, synergized with EGFR-TKIs to suppress the viability of resistant cells and tumor growth in the xenograft mouse model. These findings propose TBK1-p62 axis links p62 condensate homeostasis to EGFR-TKI resistance as an underlying mechanism of action and an emerging strategy to resensitize EGFR-TKI treatment in resistant lung cancer.