Blockade of USP14 potentiates type I interferon signaling and radiation-induced antitumor immunity via preventing IRF3 deubiquitination

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.