The radiotherapy induced cyclic GMP-AMP-synthase re-localization and immune signature predicts HNSCC treatment efficacy.

BACKGROUND: Radiotherapy (RT) is a crucial treatment for head and neck squamous cell carcinoma (HNSCC). Recent pre-clinical studies highlight the potential of combining RT with immunotherapy due to RT-induced activation of the cyclic GMP-AMP-synthase (cGAS)/Stimulator of Interferon (IFN) Genes (STING) pathway, yet the clinical implications remain unclear. METHODS: In this study, we utilized a cGAS-mNeongreen knock-in tumor model, human HNSCC cell lines and serial tumor biopsies from HNSCC patients to investigate RT-induced cGAS activation and re-localization. cGAS positive micronuclei and IFN stimulated gene (ISG) induction were quantified. In HNSCC patient biopsies, cGAS positive micronuclei were identified in different cellular compartments and IFN/ISG and NF-κB serum levels were correlated to survival. Differences were tested using paired t-tests, Wilcoxon test, One-way and two-way ANOVA or Kaplan Meier curves. RESULTS: Here we show that cGAS positive foci present in micronuclei increase post-RT in both mouse tumor models (P = 0.0305; t-test) and HNSCC patient biopsies (P = 0.0012; Wilcoxon test), concurrently with higher ISG expression. Notably, cGAS re-localization in HNSCC patient biopsies is most prominent in immune cell-infiltrated tumor regions (P = 0.0061; Wilcoxon test). Enhanced systemic type I IFN inducing activity is observed post-RT (P = 0.0040; mixed-effects analysis) and correlates with improved survival (OS P = 0.0146; log-rank test; DFS P = 0.0256; Gehan-Breslow-Wilcoxon test), particularly in HPV positive patients (OS P = 0.0017; log-rank test; DFS P = 0.043; log-rank test). CONCLUSIONS: These findings suggest that cGAS re-localization and systemic IFN responses may serve as biomarkers for RT efficacy, providing a basis for optimizing HNSCC treatment protocols, especially when integrated with immunotherapy.