Philipp Schenk, Shane M. Devine, Simon A. Cobbold, Niall D. Geoghegan, Elizabeth L Kyran, Ching‐Seng Ang, Jack A Alexandrovics, Dale J. Calleja, Dylan H. Multari, Vineet Vaibhav, Bernadine G.C. Lu, Theresa Klemm, Laura F. Dagley, Kym N. Lowes, Nicholas A. Williamson, Pieter J.A. Eichhorn, Ashley P. Ng, Rebecca Feltham, David Komander
The ubiquitin-specific protease (USP) USP9X is a human deubiquitinase (DUB) with a large number of described targets and cellular roles. In cancer, USP9X is found as an oncogene or as a tumour suppressor depending on context, and its utility as a target for cancer therapy remains unclear. We here describe WEHI-092, a piperazine-based USP9X-specific small-molecule inhibitor, which binds to a unique region in the USP9X Fingers-subdomain, distinct from known DUB-inhibitor binding sites. Using proteomics and ubiquitinomics, we show that USP9X targets distinct substrates compared to USP7, yet the substrate profile of USP9X varies significantly across cancer cell lines. We reveal a core set of 17 proteins commonly regulated by USP9X in most cell lines, which we consider as proximal biomarkers for USP9X inhibition. Consistent with proteomics, we show in unrelated cell lines that WEHI-092 treatment arrests the cell cycle in metaphase without inducing cell death. This explains growth suppression in long-term clonogenic assays in most cancer cell lines, and positions USP9X inhibitors as a new class of selective mitotic poisons.