Recent evidence suggests that several deubiquitinases (DUB) are overexpressed or activated in tumor cells and many contribute to the transformed phenotype. Agents with DUB inhibitory activity may therefore have therapeutic value. In this study, we describe the mechanism of action of WP1130, a small molecule derived from a compound with Janus-activated kinase 2 (JAK2) kinase inhibitory activity. WP1130 induces rapid accumulation of polyubiquitinated (K48/K63-linked) proteins into juxtanuclear aggresomes, without affecting 20S proteasome activity. WP1130 acts as a partly selective DUB inhibitor, directly inhibiting DUB activity of USP9x, USP5, USP14, and UCH37, which are known to regulate survival protein stability and 26S proteasome function. WP1130-mediated inhibition of tumor-activated DUBs results in downregulation of antiapoptotic and upregulation of proapoptotic proteins, such as MCL-1 and p53. Our results show that chemical modification of a previously described JAK2 inhibitor results in the unexpected discovery of a novel DUB inhibitor with a unique antitumor mechanism. Cancer Res; 70(22); 9265–76. ©2010 AACR.