Ribonucleotides Amplify Mitochondrial DNA‐Driven cGAS–STING Activation via FdUMP‐ribonucleotide Hybrid Particles to Potentiate Chemoimmunotherapy

Intracellular nucleotide imbalance between ribonucleotides and deoxyribonucleotides can trigger cytosolic mitochondrial DNA (mtDNA) release and subsequent activation of the cGAS-STING pathway. However, fluoropyrimidine-induced nucleotide imbalance is constrained by their metabolic instability. Here, we leverage ribonucleotides to amplify floxuridine (FUDR)-induced mtDNA release and cGAS-STING activation for enhanced cancer chemoimmunotherapy. We identified that cytidine monophosphate (CMP) significantly enhances FUDR-mediated activation of the mtDNA-driven cGAS-STING pathway, inducing over 300-fold upregulation of the interferon gene Ifnb1 and initiating STING-dependent antitumor immunity. Genome-wide CRISPR-Cas9 screening identified five pyrimidine-metabolizing enzymes (CMPK1, CMPK2, TYMP, UPP1, and UPP2) that are responsible for the metabolic instability of floxuridine monophosphate (FdUMP). CMP and its intracellular metabolites can metabolically block these enzymes, thereby increasing the peak intracellular concentration of FdUMP by over 500-fold and extending its half-life by more than 20-fold, which in turn promotes sustained intracellular nucleotide imbalance. To translate this mechanism into therapy, we co-encapsulated FdUMP and CMP into hybrid lipid nanoparticles (FC-NPs). FC-NPs robustly activated STING-dependent antitumor immunity, achieving remarkable therapeutic efficacy in multiple murine colorectal cancer (CRC) models. Our study not only addresses the long-standing issue of fluoropyrimidine metabolic instability but also reveals a novel ribonucleotide-mediated immunomodulatory mechanism, providing a promising strategy to potentiate chemoimmunotherapy in CRC.