β-Lapachone Antagonizes Ubiquitin-Specific Protease 18 Activity to Elicit Anti-Neoplastic Effects

The deubiquitinase (DUB) Ubiquitin-Specific Protease 18 (USP18) is as an antineoplastic target. USP18 inhibitors are needed. To address this unmet need, we engineered an enzymatically active human recombinant USP18 protein for high-throughput screening. This found β-lapachone was an inhibitor of USP18-ISG15 activity. Independent β-Lapachone treatments of murine and human lung cancer cell lines statistically-significantly reduced proliferation and increased apoptosis. Gain of USP18 expression opposed these effects. β-Lapachone treatments statistically- significantly repressed growth of H1299 human lung cancer xenografts in athymic mice and also in syngeneic mice transplanted with murine 344SQ lung cancers. β-Lapachone increased reactive oxygen species (ROS) but antineoplastic effects proceeded at concentrations having little ROS production. ROS scavenger treatments did not rescue anti-cancer effects, indicating an ROS-independent anti-neoplastic mechanism was engaged. To identify this an Interferon-Stimulated Response Element (ISRE) reporter assay was used. β-Lapachone treatment activated this reporter activity as expected for a USP18 inhibitor. β-Lapachone treatment increased global ISG15 conjugation. Proteomic analysis and immunoprecipitation assays revealed known and new ISG15ylated proteins. RNA sequencing of parental lung cancer cells engineered with or without gain of USP18 expression found differentially expressed genes and pathways after β-lapachone treatments. Taken together, β-lapachone is a novel USP18 inhibitor and cancer biology tool.