T. Yin1,2, P. Wang3, Y. Li2, J. Yu3, T. Zhang4, and F. Teng2; 1Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China, 2Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, 3Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China, 4Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
Purpose/Objective(s): Necroptosis, recognized for its potent immunogenicity, plays a significant role in shaping tumor development and cellular immune responses. The necroptosis mediator, Receptor-interacting protein kinase 1 (RIPK1) orchestrates a balance between inflammatory signaling, cellular demise, and tumor immune surveillance. However, the effects of RIPK1 on enhancing anti-tumor immunity in the context of radiotherapy (RT) remains elusive. This study aims to elucidate the mechanisms through which RIPK1 enhances immune response following RT and explore how this knowledge could improve treatment outcomes.Materials/
Methods: To elucidate the underlying mechanisms, MC38 and B16/F10 cells overexpressing RIPK1 or kinase domain (K45A or RHIM) mutant of RIPK1 and the corresponding isogenic cell lines expressing an empty vector were used to assess response to radiotherapy (RT) in vitro and in vivo. The molecular signaling and cytokine regulation in response to RT were analyzed by transcriptome sequencing. Subsequent validations were carried out via a suite of molecular techniques, including qRT-PCR, ELISA, Western blotting, and histological examination. Additionally, to delve deeper into the signaling pathways activated during treatment, assays such as protein pulldown, CHIP, and functional rescue experiments were executed. Results: Here, we observed that RIPK1 plays a vital role in enhancing antitumor immunity following irradiation of tumor cells. Notably, tumors overexpressing RIPK1 exhibited a marked reduction in growth when subjected to RT. Furthermore, RIPK1 expression augmented the type I interferon (IFN) response and facilitated the secretion of the cytokine CCL2 subsequent to tumor cell irradiation. Mechanistically, RIPK1 in irradiated tumor cells activate and intensify cGAS-STING signaling through interaction with Z-DNA-binding protein 1 (ZBP1), thereby perpetuating inflammation within the tumor microenvironment. Of note, mutations in the RHIM domain, in contrast to K45A mutations, negated these effects, indicating the critical functional importance of the RHIM domain in RIPK1 and ZBP1 interaction. Conclusion: Collectively, our results reveal that RIPK1 is instrumental in orchestrating antitumor immunity by engaging the RHIM-ZBP1 and cGAS-STING pathways. This discovery pinpoints a mechanism that amplifies IFN production in response to radiation, presenting a potential novel approach to enhance the efficacy of RT. These findings open avenues for novel therapeutic strategies combining RIPK1 modulation with RT to improve patient outcomes.
