USP48 stabilizes YBX1 to enforce a Wnt-driven stemness circuit and osimertinib resistance in EGFR-mutant NSCLC.

Osimertinib resistance remains a major obstacle to durable disease control of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC), yet the regulatory circuitry that sustains a stem-like drug-tolerant state is incompletely understood. Here, the deubiquitinase (DUB) USP48 was identified as a critical determinant of acquired resistance through stabilization of Y-box binding protein 1 (YBX1) and reinforcement of Wnt/β-catenin signaling. CRISPR-Cas9 dropout screening of patient-derived organoids under osimertinib selection, integrated with multi-omics profiling and clinical correlation analyses, identified USP48 as a functionally selected vulnerability factor in resistant NSCLC. Mechanistically, USP48 associated with YBX1 and removed K48-linked polyubiquitin chains to prevent proteasomal degradation, thereby enabling YBX1-dependent transcriptional activation of PTK7 and subsequent amplification of Wnt-driven stemness programs. Notably, the canonical Wnt effector TCF7L2 directly upregulated USP48 transcription, establishing a positive feedback loop that locked resistant cells in a stable stem-like state. Therapeutically, genetic or pharmacological disruption of this axis, including inhibition of USP48 activity, blockade of downstream Wnt transcriptional output, disruption of the USP48-YBX1 interaction with glycyrrhizic acid, and lipid nanoparticle-mediated delivery of siUSP48-restored osimertinib sensitivity, achieved robust antitumor efficacy with survival benefits in orthotopic and patient-derived xenograft models. Collectively, our findings defined a DUB-centered positive feedback circuit that sustained osimertinib resistance, and highlighted USP48 as a therapeutically useful node to overcome EGFR-TKI resistance of EGFR-mutant NSCLC.