Usp9x Silencing and Enzyme Inhibition Suppress Myeloma Cell Survival and in Vivo Tumor Growth.

Abstract 2936

Due to clinical success with proteasome inhibitors and E3 ligase modulators, proteins of the ubiquitin/proteasome system have emerged as novel therapeutic targets in several hematological malignancies. Deubiquitinases (DUBs) play a key role in regulating all aspects of this pathway and are aberrantly expressed or activated in several hematological malignancies. Usp9x is a high MW DUB, which has previously been shown to alter many signaling pathways, and its overexpression has been associated with drug resistance and poor prognosis in myeloma patients. Usp9x is reported to control cell survival through deubiquitination of Mcl-1 and other substrates, thereby reducing their proteosomal degradation. Since Mcl-1 plays a major role in myeloma cell survival and drug resistance, we compared Usp9x gene expression in myeloma cell lines to primary myeloma specimens. Usp9x gene expression varied among myeloma cell lines by 1.5 to 2.5-fold and endogenous Usp9x protein levels were even more variable (range 1.5 to 8-fold). Elevated Usp9x protein expression was not consistently associated with elevated Usp9x enzymatic activity as myeloma cells with the highest Usp9x gene and protein expression (KMS-11) had minimal Usp9x DUB activity. Moreover, Mcl-1 levels did not consistently correlate with Usp9x gene, protein or DUB activity, suggesting that a more complex mechanism regulates Usp9x activity and Mcl-1 stability in myeloma cells. To further examine the relationship between Usp9x and Mcl-1, we suppressed Usp9x expression (with siRNA and shRNA) and inhibited Usp9x activity with small molecule DUB inhibitors, WP1130 and VM030. Silencing Usp9x expression in H929 and MM1.S cells with shRNA vectors resulted in a reduction in Mcl-1 levels and induction of apoptosis, which approached the level of cell death achieved with direct Mcl-1 silencing. Similar results were obtained with siRNA-based Usp9x silencing in RMPI-8226 and KMS-11 cells; however, this approach led to the activation of a homologous DUB, Usp24, primarily through an increase in its protein stability. Usp24 silencing led to a decrease in myeloma cell survival, suggesting that Usp9x and Usp24 are coordinately regulated and play a role in myeloma cell survival. Measurement of Usp9x and Usp24 gene expression levels in primary myeloma samples and cell lines demonstrated that myeloma cell lines express 2 to 100-fold lower levels of these DUBs compared to primary tumors. Immuno-depletion studies illustrated that both Usp9x and Usp24 were activated in myeloma cells, and targeting both DUBs with WP1130 or VM030 led to a rapid reduction in Mcl-1 protein levels and the onset of apoptosis in both primary myeloma cells and cell lines. Further, treatment of NSG mice bearing MM1.S or RPMI-8226 tumors with VM030 resulted in Usp9x and Usp24 inhibition, reduction in Mcl-1 protein levels and suppression of myeloma tumor growth with limited toxicity. Together, these results suggest that Usp9x and Usp24 are highly expressed and activated in myeloma cells and both DUBs contribute to cell survival, but through different mechanisms. These results support the use of DUB inhibitors with specificity for Usp9x and Usp24 in the treatment of myeloma.