UDP-GlcUA triggers PKR kinase activity to promote liquid-liquid phase separation of TOP2A and enhances radioimmunotherapy resistance.

To enhance the effectiveness of irradiation (IR) and optimize PD-1/PD-L1 blockade therapy for hepatocellular carcinoma (HCC), we conducted in vivo CRISPR-Cas9 metabolic sublibrary screening, pinpointing UDP-glucose dehydrogenase (UGDH) as a key target. Post-IR, protein kinase R (PKR) translocates to the nucleus. There, elevated UGDH produces UDP-GlcUA, which binds PKR's dsRNA-binding domain (dsRBD). This binding triggers PKR dimerization, autophosphorylation (T451), and activation. Activated PKR then phosphorylates TOP2A at S1467, inducing its liquid-liquid phase separation (LLPS) and enhancing topoisomerase activity, and finally protects tumor cells from IR-induced DNA damage. Disrupting the UDP-GlcUA/PKR complex reduces TOP2A-S1467 phosphorylation, lowers topoisomerase activity, increases cGAS-STING signaling, and improves anti-PD-L1 immunotherapy efficacy. Clinical HCC sample analysis confirmed the relevance of UDP-GlcUA and the phosphorylation of PKR and TOP2A in response to radiation. Critically, blood UDP-GlcUA may serve as a biomarker for PKR/TOP2A axis activation, guiding patient suitability for anti-PD-L1 immunotherapy after IR and enabling personalized treatment strategies.