ZBP1-MLKL-dependent Necroptosis Triggers STING-mediated Cytosolic DNA Sensing and Promotes Antitumor Immunity of Radiation

Ionizing radiation promotes cytosolic DNA sensing and consequent antitumor immune responses. But how tumor cell-intrinsic cytosolic DNA sensing is initiated by radiation remains poorly defined. Here, we demonstrated that STING-mediated type I interferon production in tumor cells after radiation relied on the engagement of MLKL-mediated necroptosis, which was elicited by the ZBP1-RIPK3 signaling axis. Physiologically, tumor cell-intrinsic ZBP1-MLKL cascade augmented antitumor immune responses after radiation largely by regulating STING signaling. Mechanistically, ZBP1-MLKL-dependent necroptosis maintained the enrichment of mitochondria DNA inside the cytosol of tumor cells after radiation in a cell-density dependent fashion, contributing to type I interferon responses. In contrast, ablation of caspase-8 unleashed ZBP1-MLKL cascade to gain enhanced cytosolic DNA sensing, and in turn potentiated therapeutic effects of radiation. Thus, our findings uncover an unanticipated mechanism that ZBP1-MLKL-dependent necroptosis drives cytosolic DNA sensing-mediated antitumor immunity after radiation, and provide new strategy to improve radiotherapy by inhibiting caspase-8 cascade. WT-MC38 (CRISPR-Ctrl) or MLKL-KO MC38 (MLKL-gRNA3) tumor cells were subjected to single fraction of X-ray (15 Gy). 60 hours later, total RNA from each sample was extracted using Trizol Reagent following standard protocols and mRNA sequencing was performed.