Deubiquitinase Usp9X Regulates Cell Survival and Metabolic Signal Transduction in Multiple Myeloma Cells

Abstract 1835

Multiple myeloma (MM) therapy has significantly improved with the addition of a number of new agents that target the proteasome and other pathways. However, most patients relapse and eventually become resistant to any drug therapy, leaving limited or no treatment options. As a result, there is tremendous interest in understanding drug resistance and regulation of MM cell survival. Toward this goal, a number of studies have assessed differential gene expression and proteome profiles between normal cells, primary myeloma and drug-resistant disease. Recent reports suggest that increased expression of the deubiquitinase (DUB) Usp9x is associated with poor prognosis of MM patients. Additional analyses indicate that Usp9x regulates a number of survival and signaling proteins including Mcl-1, a Bcl-2-family pro-survival protein previously associated with drug and apoptotic resistance in myeloma patients, and 5'-AMP kinase (AMPK), a negative regulator of the mTOR cascade. We previously described WP1130, a small molecule with Usp9x DUB inhibitory activity. This compound induced apoptosis in MM cell lines and primary cells from drug-resistant MM patients (IC50 ∼1 μM) but not in normal CD34 cells even at 5-fold higher concentrations, suggestive of a favorable therapeutic index. To determine the role of Usp9x in MM cell survival and signaling, we silenced Usp9x expression in MM cells and assessed changes in Usp9x DUB activity, Mcl-1 levels, cell survival and mTOR signaling. We also compared these results with the effects of WP1130 treatment. Constitutive knockdown of Usp9x by shRNA in H929, MM1.S and RPMI-8226 cells reduced Usp9x protein levels by >90%, but reduced Usp9x DUB activity by no more than 40%. Importantly, although Usp9x silencing reduced MM cell survival by 5–40%, major changes in Mcl-1 protein levels were not detected. In contrast, treatment of control or Usp9x-silenced cells with WP1130 resulted in a rapid reduction in both Usp9x DUB activity and Mcl-1 protein levels. These results suggest that Usp9x DUB activity was not solely dependent on Usp9x protein levels and that cells with shRNA-mediated Usp9x knockdown may activate other compensatory mechanisms to stabilize Usp9x activity. We examined the effects of Usp9x knockdown on the mTOR negative regulator AMPK, whose activation requires deubiquitination by Usp9x. Both Usp9x shRNA and WP1130 treatment stabilized ubiquitination of AMPK, resulting in inhibition of AMPK kinase activity. Such AMPK inhibition led to activation of the mTOR cascade, thereby increasing phosphorylation of Akt, S6 and GSK3β. These observations suggested that activation of mTOR through chronic Usp9x knockdown suppresses apoptosis in MM cells. Inhibition of mTOR activity (with Torin-2) in Usp9x-silenced cells rapidly induced apoptosis with a corresponding reduction in Mcl-1 protein levels. This Mcl-1 reduction was associated with decreased GSK3β activity and suppression of Mcl-1 phosphorylation at Thr159, a GSK3β phosphorylation site that negatively regulates Mcl-1/Usp9x association. Dissociation of Mcl-1/Usp9x complexes increased ubiquitination of Mcl-1 and its destruction by the proteasome. Together, these results suggest that Usp9x regulates multiple substrates with differential effects on mTOR signal transduction and apoptotic proteins. This compound impact of Usp9x on Mcl-1 and mTOR regulation may complicate the assessment Usp9x as a potential therapeutic target in MM, but may help explain the sensitivity of MM cells to WP1130. Specifically, WP1130 increases the formation of Mcl-1/Usp9x complexes by inhibiting AMPK and thereby activating mTOR/Akt/GSK3β. The Mcl-1 in complex with Usp9x becomes ubiquitinated due to rapid inhibition of Usp9x activity by WP1130. Our results predict that Usp9x inhibitors will have therapeutic activity in MM cells by modulating at least two separate pathways that regulate metabolic signaling and MM cell survival. Considering that increased Usp9x expression has been associated with short survival and poor prognosis in MM patients, Usp9x inhibitors like WP1130 could show therapeutic promise for this patient population with limited treatment options.