MMP14-mediated proteolytic cleavage of LAMC2 domain III fragment drives osimertinib resistance in EGFR-mutant non-small cell lung cancer.

Acquired resistance to osimertinib remains a critical clinical challenge in EGFR-mutant non-small cell lung cancer (NSCLC), with 40%-60% of progressing patients lacking an identifiable genetic resistance driver. Through quantitative secretomic profiling of paired osimertinib-sensitive and -resistant NSCLC cell lines, we identified laminin subunit γ2 (LAMC2) as markedly enriched in resistant cell secretomes. Single-cell RNA sequencing reanalysis revealed a LAMC2-high drug-tolerant persister subpopulation that expanded under osimertinib pressure, with pseudotime analysis suggesting progressive co-upregulation of LAMC2 and matrix metalloproteinase 14 (MMP14) along the treatment-to-resistance trajectory. Across four independent cohorts, MMP14 exhibited the strongest co-expression with LAMC2 among all metalloproteinases; the two proteins also physically interacted, and their concurrent high expression predicted the worst overall survival. Mechanistically, MMP14 cleaved LAMC2 to release a bioactive Domain III (DIII) fragment harboring EGF-like repeats, which reactivated EGFR/AKT/ERK signaling despite continued osimertinib exposure. Exogenous DIII conferred resistance in sensitive cells, whereas an anti-DIII blocking antibody reversed MMP14-driven resistance in cell lines and patient-derived organoids. The selective MMP14 inhibitor NSC405020 restored osimertinib sensitivity in vitro and suppressed xenograft growth. In residual tumor tissues from neoadjuvant osimertinib-treated patients, non-responders exhibited higher LAMC2/MMP14 co-expression and more prominent spatial exclusion of CD8⁺ T-cells from the tumor core compared with major pathological responders. These findings identify the MMP14-LAMC2-DIII proteolytic axis as a non-genetic resistance mechanism and suggest that MMP14 inhibition or DIII neutralization may represent a rational strategy to overcome acquired osimertinib resistance.