Glutamine Deprivation Triggers Tribbles Homolog 3 Dependent G‐Quadruplex Resolution to Maintain DNA Repair and Tumor Survival

ABSTRACT Glutamine is an essential amino acid for tumor survival, but therapies targeting glutamine metabolism have largely failed due to adaptive resistance mechanisms. Here, we identify the pseudokinase TRIB3 as a key mediator of the metabolic adaptation of hepatocellular carcinoma (HCC) cells to limiting glutamine availability. TRIB3 is upregulated under glutamine deprivation in a c‐Jun‐dependent manner, functioning in the nucleus to safeguard DNA repair fidelity, allowing the timely resolution of DNA damage and preventing replication catastrophe. TRIB3 binds to G‐quadruplex DNA (G4‐DNA) structures throughout the genome, recruiting the helicase DDX5 to resolve them as a cooperative functional complex. Depleting TRIB3 or DDX5 in HCC cells leads to exaggerated G4‐DNA accumulation and heightened DNA damage associated with the downregulation of DNA damage repair (DDR) pathways. We illustrate this effect on homologous recombination (HR) pathway genes, finding that TRIB3‐DDX5 prevents G4‐DNA accumulation at the BRCA1 and RAD51AP1 promoter regions that would otherwise suppress transcription. In vivo, TRIB3 silencing suppresses HCC xenograft growth, patently increasing DNA damage and apoptosis when mice were maintained on glutamine‐deficient diets. Clinically, TRIB3 is overexpressed in HCC and correlates with poor prognosis. Our results propose the TRIB3‐DDX5‐G4 axis as a therapeutic target in HCC and other TRIB3‐high malignancies.