Weijian Wu1, Huiying Xu1, Chenyang Liao1, Youqiao Wang1, Ruirui Wu1, Jiaxin Wu1, Wenlv Zheng2, Yunzhi Li1, Chaoying Jin1, Yuxuan Zhao1, Junmin Quan2, Xin Yue3,4, Xianzhang Bu5,6. 1. School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. 2. State Key Laboratory of Chemical Oncogenomics, Guangdong Provincial Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, 518055, China. 3. Department of Radiation Oncology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China. yuex5@mail.sysu.edu.cn. 4. Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China. yuex5@mail.sysu.edu.cn. 5. School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. phsbxzh@mail.sysu.edu.cn. 6. State Key Laboratory of Chemical Oncogenomics, Guangdong Provincial Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, 518055, China. phsbxzh@mail.sysu.edu.cn.
Abstract
PURPOSE: The adaptive immune responses induced by radiotherapy has been demonstrated to largely rely on STING-dependent type I interferons (IFNs) production. However, irradiated tumor cells often fail to induce dendritic cells (DCs) to produce type I IFNs. Hence, we aim to uncover the limitation of STING-mediated innate immune sensing following radiation, and identify efficient reagents capable to rescue the failure of type I IFNs induction for facilitating radiotherapy. METHODS: A targeted cell-based phenotypic screening was performed to search for active molecules that could elevate the production of type I IFNs. USP14 knockout or inhibition was assayed for IFN production and the activation of STING signaling in vitro. The mechanisms of USP14 were investigated by western blot and co-immunoprecipitation in vitro. Additionally, combinational treatments with PT33 and radiation in vivo and in vitro models were performed to evaluate type I IFNs responses to radiation. RESULTS: PT33 was identified as an enhancer of STING agonist elicited type I IFNs production to generate an elevated and durable STING activation profile in vitro. Mechanistically, USP14 inhibition or deletion impairs the deubiquitylation of K63-linked IRF3. Furthermore, blockade of USP14 with PT33 enhances DC sensing of irradiated-tumor cells in vitro, and synergizes with radiation to promote systemic antitumor immunity in vivo. CONCLUSION: Our findings reveal that USP14 is one of the major IFN production suppressors and impairs the activation of IRF3 by removing the K63-linked ubiquitination of IRF3. Therefore, blockage of USP14 results in the gain of STING signaling activation and radiation-induced adaptive immune responses.
PURPOSE: The adaptive immune responses induced by radiotherapy has been demonstrated to largely rely on STING-dependent type I interferons (IFNs) production. However, irradiated tumor cells often fail to induce dendritic cells (DCs) to produce type I IFNs. Hence, we aim to uncover the limitation of STING-mediated innate immune sensing following radiation, and identify efficient reagents capable to rescue the failure of type I IFNs induction for facilitating radiotherapy. METHODS: A targeted cell-based phenotypic screening was performed to search for active molecules that could elevate the production of type I IFNs. USP14 knockout or inhibition was assayed for IFN production and the activation of STING signaling in vitro. The mechanisms of USP14 were investigated by western blot and co-immunoprecipitation in vitro. Additionally, combinational treatments with PT33 and radiation in vivo and in vitro models were performed to evaluate type I IFNs responses to radiation. RESULTS: PT33 was identified as an enhancer of STING agonist elicited type I IFNs production to generate an elevated and durable STING activation profile in vitro. Mechanistically, USP14 inhibition or deletion impairs the deubiquitylation of K63-linked IRF3. Furthermore, blockade of USP14 with PT33 enhances DC sensing of irradiated-tumor cells in vitro, and synergizes with radiation to promote systemic antitumor immunity in vivo. CONCLUSION: Our findings reveal that USP14 is one of the major IFN production suppressors and impairs the activation of IRF3 by removing the K63-linked ubiquitination of IRF3. Therefore, blockage of USP14 results in the gain of STING signaling activation and radiation-induced adaptive immune responses.
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