Abstract Ubiquitin-specific protease 8 exerts multiple cellular functions and was identified as a potential target in a multiple myeloma vulnerability screen. Here we characterized the function of USP8 in B cells and multiple myeloma, and analyzed its impact on the global and ubiquitin-modified proteome. Usp8 deletion in mice starting at the the pre-B cell stage caused a partial block in B cell development favoring immature and innate-like B cells, as well as germinal center and plasma cells. This was accompanied by elevated immune-responses and Roquin depletion. Accordingly, correlation analyses in multiple myeloma patients revealed that low USP8 expression at diagnosis correlates with decreased survival. B cells expressing catalytically inactive USP8 accumulate protein modified with mixed ubiquitin/NEDD8 chains as hallmarks of proteotoxic stress, which we identified as favored USP8 substrates. USP8 knockdown reduced survival of bortezomib-resistant multiple myeloma cells in a lysosomal dysfunction-dependent manner. In contrast, the inhibitor DUB-IN-2 resensitized bortezomib-resistant multiple myeloma cells to treatment in a bortezomib-synergistic manner. Hence, our analyses uncovered the therapeutic potential of USP8 inhibition and of DUB-IN-2 in multiple myeloma.
