UFL1‐Mediated UFMylation of ENO1 Restrains Aerobic Glycolysis and Colorectal Cancer Progression

Metabolic reprogramming toward enhanced aerobic glycolysis is a hallmark of cancer, yet the contribution of ubiquitin-like modifications to this process remains poorly understood. Here, we identify the UFMylation E3 ligase UFL1 as a critical suppressor of glycolytic metabolism and colorectal cancer (CRC) progression. Mechanistically, UFL1 directly interacts with the glycolytic enzyme enolase 1 (ENO1) and catalyzes its UFMylation at lysine residues K285 and K420. This modification disrupts ENO1 dimerization, attenuates its enzymatic activity, and consequently suppresses glycolytic flux. Functionally, UFL1-mediated UFMylation inhibits tumor growth both in vitro and in vivo. Notably, pharmacological enhancement of the UFL1-ENO1 interaction using the FDA-approved antibiotic torezolid significantly potentiates the antitumor efficacy of 5-fluorouracil (5-FU) across multiple preclinical models, including CRC patient-derived organoids and xenografts, without detectable toxicity. Collectively, these findings identify ENO1 as a direct substrate of UFMylation, establish UFMylation as a previously unrecognized regulator of cancer metabolic reprogramming, and highlight the UFL1-ENO1 axis as a promising therapeutic target for colorectal cancer.