RRM1 competes with NEDD4 to stabilize USP19 by blocking K387 ubiquitination and suppresses autophagy-mediated chemoresistance in colorectal cancer

BACKGROUND: Resistance to 5-fluorouracil (5-FU) remains a major clinical challenge in colorectal cancer (CRC) treatment. This study investigates the non-canonical role of ribonucleotide reductase subunit M1 (RRM1) in regulating autophagy-mediated chemoresistance. METHODS: A comprehensive multi-omics strategy, including metabolomics, transcriptomics and proteomics analyses, was employed to study 5-fluorouracil-resistant colorectal cancer tissues from patients and matched drug-resistant cell models. Protein-protein interactions, ubiquitination events and functional consequences were validated via complementary assays including Co-IP, GST pull-down, in vivo ubiquitination, structure-guided molecular docking, site-directed mutagenesis, and subcutaneous xenograft mouse models. Structure-based virtual screening was conducted to identify candidate compounds targeting the RRM1-USP19 interaction, with in vivo anti-tumor activity verified in 5-FU-resistant CRC xenografts. RESULTS: RRM1 is significantly downregulated in 5-FU-resistant CRC tissues, and low RRM1 expression correlates with poor clinical prognosis. Mechanistically, RRM1 directly binds USP19 via conserved E647/R648 residues, competitively blocking NEDD4-mediated USP19 K387 ubiquitination and subsequent proteasomal degradation. Loss of RRM1 destabilizes USP19, impairs autophagic substrate deubiquitination, hyperactivates autophagic flux and thereby induces 5-FU resistance. Restoration of RRM1 or expression of the ubiquitination-defective USP19-K387R mutant re-sensitizes resistant CRC cells to 5-FU. The peptide GAGGVGKSAL, identified via virtual screening, specifically disrupts the RRM1-USP19 interface, inhibits excessive autophagy and potently suppresses tumor progression in 5-FU-resistant CRC xenograft models. CONCLUSIONS: This study identifies the novel RRM1-USP19-NEDD4 regulatory axis as a core mediator of autophagy-driven 5-FU resistance in CRC, uncovers a non-canonical, metabolism-independent role of RRM1 as a USP19 stabilizer via competitive inhibition of NEDD4-mediated ubiquitination, and validates GAGGVGKSAL as a promising lead compound targeting this axis to reverse 5-FU resistance. The RRM1-USP19-NEDD4 axis represents a novel therapeutic target for overcoming chemoresistance in CRC.