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.

Identification and semisynthesis of (-)-anisomelic acid as oral agent against SARS-CoV-2 in mice.

(-)-Anisomelic acid, isolated from Anisomeles indica (L.) Kuntze (Labiatae) leaves, is a macrocyclic cembranolide with a trans-fused α-methylene-γ-lactone motif. Anisomelic acid effectively inhibits SARS-CoV-2 replication and viral-induced cytopathic effects with an EC50 of 1.1 and 4.3 µM, respectively. Challenge studies of SARS-CoV-2-infected K18-hACE2 mice showed that oral administration of anisomelic acid and subcutaneous dosing of remdesivir can both reduce the viral titers in the lung tissue at the same level. To facilitate drug discovery, we used a semisynthetic approach to shorten the project timelines. The enantioselective semisynthesis of anisomelic acid from the naturally enriched and commercially available starting material (+)-costunolide was achieved in five steps with a 27% overall yield. The developed chemistry provides opportunities for developing anisomelic-acid-based novel ligands for selectively targeting proteins involved in viral infections.

STAT3 inhibition enhances CDN-induced STING signaling and antitumor immunity.

Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a key regulator in innate immunity and has emerged as a promising drug target in cancer treatment, but the utility of this pathway in therapeutic development is complicated by its dichotomous roles in tumor development and immunity. The activation of the STING pathway and the induced antitumor immunity could be attenuated by the feedback activation of IL-6/STAT3 pathway. Here we reported that STAT3 inhibition significantly enhanced the intensity and duration of STING signaling induced by the STING agonist c-diAM(PS)2. Such sensitization effect of STAT3 inhibition on STING signaling depended on STING rather than cGAS, which was mediated by simultaneously upregulating the positive modulators and downregulating the negative modulators of the STING pathway. Furthermore, the combination treatment with the STAT3 inhibitor and STING agonist markedly regressed tumor growth in syngeneic mice by increasing CD8+ T cells and reducing regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Our work provides a rationale for the combination of STAT3 inhibitors and STING agonists in cancer immunotherapy.

A novel CAV derived cell-penetrating peptide efficiently delivers exogenous molecules through caveolae-mediated endocytosis.

Cell-penetrating peptide (CPP) is a promising cargo for delivering bioactive molecules. In this study, the N terminus of VP1 from chicken anemia virus, designated as CVP1, was found to carry enriched arginine residues with α-helix. By confocal imaging, flow cytometry and MTT assay, we identified CVP1 as a novel, safe and efficient CPP. CVP1-FITC peptide could entry different types of cells tested with dose dependence, but without cytotoxic effects. Compared with TAT-FITC peptide, the CVP1-FITC peptide showed much higher cell-penetrating activity. Moreover, CVP1 could successfully deliver ß-glycosidase, poly (I:C) and plasmid into HCT116 cells. Inhibitors and temperature sensitivity analysis further indicated that the cell-penetrating activity of CVP1 was based on ATP-dependent and caveolae-mediated endocytosis. All these data demonstrate that CVP1 has efficient cell-penetrating activity and great potential for developing a novel delivery vector.