USP7-dependent stabilization of FKBP4 contributes to acquired osimertinib resistance through glycolytic remodeling in NSCLC.

Osimertinib is the standard first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) for EGFR-mutant non-small-cell lung cancer (NSCLC), yet acquired resistance remains inevitable. While metabolic adaptation and proteostasis rewiring have emerged as key contributors to EGFR-TKI resistance, the actionable regulators that integrate these processes are incompletely defined. FKBP4 expression was assessed in public NSCLC cohorts and institutional specimens and examined in acquired osimertinib-resistant cell models. Gain- and loss-of-function studies were performed to evaluate osimertinib sensitivity, proliferation, clonogenicity, migration/invasion, and epithelial–mesenchymal transition (EMT). Glycolytic remodeling was characterized by untargeted metabolomics, glucose uptake and lactate production assays, and Seahorse extracellular flux analysis. PI3K–AKT signaling was analyzed by immunoblotting and pharmacological inhibition using MK2206. Candidate deubiquitinases were prioritized in silico and validated by molecular modeling, co-immunoprecipitation, ubiquitination assays, and cycloheximide chase. Therapeutic relevance was further examined in xenograft models. FKBP4 was upregulated in NSCLC tissues and further increased in acquired osimertinib-resistant cells. FKBP4 overexpression enhanced cell viability and clonogenic survival under osimertinib and shifted dose–response curves toward higher IC50 values, whereas FKBP4 depletion partially restored drug sensitivity in resistant cells. FKBP4 also promoted migration/invasion and was associated with EMT-related changes, marked by E-cadherin downregulation and increased N-cadherin, vimentin, and Snail. Mechanistically, FKBP4 promoted glucose metabolism toward a Warburg-like phenotype, as evidenced by increased glucose uptake and lactate output, upregulation of GLUT1 (SLC2A1) and LDHA, elevated ECAR, and reduced oxidative respiration. FKBP4 further activated PI3K–AKT signaling, and MK2206 attenuated FKBP4-driven resistance. Upstream, USP7 physically interacted with FKBP4 and maintained FKBP4 protein stability through deubiquitination: USP7 depletion reduced FKBP4 protein abundance without affecting its mRNA, accelerated FKBP4 turnover, and increased FKBP4 polyubiquitination, whereas wild-type USP7—but not a catalytically inactive mutant—suppressed FKBP4 ubiquitination. In vivo, FKBP4 silencing enhanced the antitumor effect of osimertinib in resistant xenografts and mitigated EMT features. These findings support a role for the USP7–FKBP4 axis in acquired osimertinib resistance in NSCLC and suggest that FKBP4 stabilization is associated with glycolytic remodeling and pro-survival signaling in resistant cells. Our study extends current understanding of resistance-associated metabolic adaptation and identifies the USP7–FKBP4 pathway as a potential therapeutic vulnerability that warrants further investigation.