Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a 
multisubunit complex comprising a proteolytic 20S core particle capped by 19S 
regulatory particles. The approval of bortezomib for the treatment of multiple 
myeloma validated the 20S core particle as an anticancer drug target. Here we 
describe the small molecule b-AP15 as a previously unidentified class of 
proteasome inhibitor that abrogates the deubiquitinating activity of the 19S 
regulatory particle. b-AP15 inhibited the activity of two 19S 
regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 
(UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation 
of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to 
TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that 
treatment with b-AP15 inhibited tumor progression in four different in vivo 
solid tumor models and inhibited organ infiltration in an acute myeloid leukemia 
model. Our results show that the deubiquitinating activity of the 19S regulatory 
particle is a new anticancer drug target.