Single High-Dose Irradiation-Induced iRhom2 Upregulation Promotes Macrophage Antitumor Activity Through cGAS/STING Signaling

The clinical application of stereotactic body radiation therapy (SBRT) allows a high dose of radiation to be safely delivered to extracranial targets within the body; however, a high dose per fraction (hypofractionation) has opened the radiation oncology field to new questions on a variety of dose-fractionation schedules, especially the immunomodulatory effects of radiation therapy, which can change after various dose-fractionation schedules. We investigated the immunomodulatory effects of different fractionation schedules.We established a subcutaneous tumor model in wild-type C57BL/6J mice and STING (stimulator of interferon genes)-deficient mice. We then compared the tumor control efficacy of 3 different fractionation schedules: 2 Gy × 8, 4.5 Gy × 3, and 10 Gy × 1, which are similar biologically effective doses.We found the fractionation schedule of 10 Gy × 1 had a significantly higher antitumor effect, suggesting that a single high dose induced enhanced antitumor immunity compared with conventional fractionation (2 Gy × 8) and moderate hypofractionation (4.5 Gy × 3). However, in STING-deficient mice, differential tumor control was not observed among the 3 dose-fractionation schedules, suggesting that cGAS (cyclic GMP-AMP synthase)/STING signaling is involved in the antitumor immune effects of single high-dose schedules. Mechanistically, we found that conventional fractionation induced apoptosis; by comparison, a single high dose was more attuned to induced necroptosis, leading to the release of intracellular irradiation-induced double-stranded DNA (dsDNA) due to the loss of plasma membrane integrity, which then activated the dsDNA sensing signaling cGAS/STING in the recruited macrophage. Furthermore, iRhom2, a member of the conserved family of inhibitory rhomboid-like pseudoproteases, was upregulated in infiltrated macrophages in the single high-dose irradiation microenvironment. Therefore, iRhom2 positively regulates STING and directly promotes tumor necrosis factor α secretion. This exacerbates necroptosis of irradiated tumor cells, leading to continuous dsDNA release and enhancement of cGAS/STING signaling antitumor immunity in a positive feedback loop.iRhom2 amplifies antitumor signaling in a positive feedback loop mediated by cGAS/STING signaling and tumor necrosis factor-driven necroptosis after single high-dose radiation.