Glutamine Deprivation Triggers TRIB3-Dependent G4-DNA Resolution to Maintain DNA Repair and Tumor Survival [CUT&Tag]

Glutamine, the most abundant circulating amino acid, is a central metabolite critical for the survival and growth of many tumor cells. Efforts to exploit this vulnerability have met with limited success, due to the inherent adaptability of tumor cells to withstand nutrient scarcity in the tumor microenvironment. This resilience has rendered glutamine-targeting therapies ineffective in clinical trials, demanding new mechanistic insights. 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 response (DDR) pathways. We illustrate this effect on homologous recombination (HR) pathway genes, finding that TRIB3-DDX5 preventing 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 where its elevated levels correlate with poorer patient prognosis. Together findings nominate TRIB3-DDX5-G4 axis as a therapeutically exploitable metabolic dependency in HCC and possibly other malignancies with elevated TRIB3 expression.