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

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. 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. 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). 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. Mekers et al. examine how the immune sensor cGAS responds to radiotherapy in mouse models, human cancer cell lines, and patient tumor samples. Radiotherapy enhances cGAS activity and immune signaling, which is linked to improved survival, particularly in HPV positive patients. Radiotherapy (RT) is a common treatment for head and neck cancer, but patients do not all benefit equally. Research suggests that RT can also stimulate the immune system, which may improve the effectiveness of treatment. In this study, we examined how a protein involved in the immune system called cGAS behaves after RT, using mouse models, human cancer cells, and samples from people with cancer. We found that RT increased cGAS activity and boosted the activity of the immune system in both mice and patients. Patients who showed stronger immune responses after treatment tended to live longer. These results indicate that measuring immune activity could help guide treatment and improve outcomes, especially in combination with immunotherapy.