Inhibition of Usp9x Deubiquitinase Activity by WP1130 Reduces Mcl-1 Levels and Induces Apoptosis In Cells From Patients with Plasma Cell Dyscrasia and Drug-Refractory Multiple Myeloma

Abstract 3005

Multiple myeloma (MM) patients are typically treated with a number of combinations of unconventional drugs that provide excellent and sometimes durable responses. However, most patients are expected to relapse or progress and relapsed disease is usually less sensitive to chemotherapy. Therefore, drug resistance remains a key concern in MM therapy. Some important clues regarding MM drug and apoptotic resistance have been obtained from biochemical and molecular studies of pro-survival genes. Mcl-1 is one member of the Bcl-2 family with strong anti-apoptotic activity. Mcl-1 binds pro-apoptotic proteins such as Bim and Bak, forming hetero-dimers to suppress apoptosis. Specific knockdown of Mcl-1 but not other anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL) induces apoptosis in MM cells and a critical level of Mcl-1 is essential to MM cell viability. Also, drug resistant MM patients are reported to express higher levels of Mcl-1 when compared to drug naïve patients. These observations suggest that Mcl-1 plays a key role in MM drug and apoptotic resistance. Mcl-1 is unique among survival proteins as its cellular protein level is primarily controlled by its ubiquitination and proteasomal degradation. Recently Usp9x, a high MW de-ubiquitinase (DUB), was shown to bind and stabilize Mcl-1 by mediating its de-ubiquitination and loss of recognition by the proteasome. Usp9x was also shown to be highly expressed in MM patients with short term progression-free survival. Analysis of Usp9x gene expression in MM patients treated at our center supports a correlation between elevated Usp9x expression (>4-fold over normal PBMCs) and poor patient prognosis. These observations suggest that agents that inhibit Usp9x activity may be effective in controlling Mcl-1 levels and have therapeutic impact in MM patients. Here we describe a small molecule Usp9x inhibitor, WP1130, that causes a rapid reduction in Mcl-1 protein levels and stimulates apoptosis in MM cells. We further demonstrate that the anti-tumor activity of WP1130 is mediated through inhibition of Usp9x and other active DUBs in B-cell tumors. WP1130-mediated inhibition of Usp9x activity promotes Mcl-1 ubiquitination leading to its rapid (1-4 hours) proteasomal degradation with subsequent induction of apoptosis. Pretreatment of MM cells with proteasome inhibitors (MG-132 or bortezomib) prior to WP1130 treatment blocked the down-regulation of Mcl-1, demonstrating proteasome involvement in regulation of Mcl-1 levels by WP1130. Primary MM tumor cells (bone marrow aspirates) derived from newly diagnosed and advanced therapy refractory patients were also screened for their responsiveness to WP1130 by DUB activity assays. Samples were obtained from a newly diagnosed plasmacytoma/plasma cell leukemia patient and 4 MM patients refractory to the Velcade/Doxorubicin/Dexamethasone (VDD) regimen. Usp9x activity was detected in 4 of 5 patient specimens and WP1130 treatment reduced Mcl-1 protein levels and induced apoptosis only in samples with active Usp9x. Furthermore, WP1130 completely eliminated CD138+ cells after 24 hours in MM patient specimens with active Usp9x while only an 11% reduction was measured in MM cells that did not exhibit Usp9x activity. Cytogenetic prognostic predictors (del13) did not distinguish between WP1130-responsive and non-responsive cells. Our results suggest that inhibition of Usp9x activity by WP1130 is associated with Mcl-1 down-regulation in MM cells. These results also suggest that drug-refractory MM patients with elevated Usp9x activity would likely benefit from Usp9x inhibitor-based therapy. Preliminary MM tumor studies have shown that WP1130 suppresses tumor growth in vivo and can be administered safely. Together, these results suggest that WP1130-mediated Usp9x inhibition may be an effective and novel approach in treating progressive or drug-refractory MM patients.