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Background <p>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.</p> Methods <p>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.</p> Results <p>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.</p> Conclusions <p>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.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月29日

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  • Lingxiao Wang,
  • Weixing Zhang,
  • Yifan Kang,
  • Yingjia Wang,
  • Shenghuai Hou,
  • Fan Wang,
  • Yuqiao Bai,
  • Ruijun Ma,
  • Jian Yang,
  • Yaoping Li

摘要

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.

Graphical Abstract