Mechanism of Action of a Small Molecule Inhibitor That Targets Usp9x Deubiquitinase Activity in Multiple Myeloma and Mantle Cell Lymphoma

Abstract 1415

A growing number of compounds with proteasome and E3-ligase inhibitory activity have demonstrated clinical activity against multiple myeloma (MM) and mantle cell lymphoma. Some of these compounds are now part of the therapeutic regimen. In addition, there is a growing body of evidence that specific deubiquitinases (DUBs) are overexpressed in hematologic malignancies and may be appropriate myeloma/lymphoma therapeutic targets. However, only a limited number of compounds have been described with cell-permeant and selective activity for DUBs. We previously reported the DUB inhibitory activity of small molecule WP1130. This compound caused rapid inactivation of Usp9x, a DUB reported to be overexpressed in MM and some lymphoma primary specimens. Usp9x associates with the anti-apoptotic protein Mcl-1 and protects it from proteasomal destruction by deubiquitination. We have shown that WP1130 inhibits Usp9x activity (and some additional DUBs) and stimulates proteasomal destruction of Mcl-1. However, the mechanism of Usp9x inhibition by WP1130 has not been described. To investigate this, we synthesized a biotinylated derivative of WP1130 that retains cell-penetrant Usp9x DUB inhibitory activity. Using this as a probe, we demonstrate that WP1130 directly and stably interacts with Usp9x in high salt and ionic detergent, suggesting high affinity complex formation. Proteomic assessment revealed that other previously defined cellular targets of WP1130 (Usp5, Usp14, UCH-L5) also formed high affinity complexes with WP1130-biotin. Additional studies suggested that WP1130-biotin covalently modifies Usp9x through a Michael addition reaction with critical Usp9x cysteine residues that result in the inhibition of DUB activity. To further map the sites of the Usp9x-WP1130 interaction, we produced a recombinant catalytic domain of Usp9x (Usp9xCD) and verified that it retained enzymatic activity that could be inhibited by WP1130, albeit at higher concentrations than that required to inhibit the enzyme in intact cells. Mass spectral analysis of Usp9xCD/WP1130 complexes demonstrated formation of covalent WP1130 adducts with a concentration and time dependent stoichiometry. The catalytic domain was also used to determine the affinity and reversibility of WP1130 binding to Usp9x by solution-phase analysis using Bioforte instrumentation. These studies suggested a slow but high-affinity (<1 μM), irreversible binding of WP1130 to the catalytic domain in addition to a second, low-affinity, reversible binding mode. Molecular simulation was used to identify accessible and energetically favorable Cys residues that could represent the location(s) of high-affinity WP1130 binding. These sites are being confirmed by proteomic assessment of WP1130-biotin-retaining peptides derived from trypsinization of Usp9x/WP1130 adducts. Further structural simulation of Usp9x using homology modeling suggested that the catalytic domain of Usp9x contains a unique lobe (aa1576–1642), which lies close to recently described sites of mTOR phosphorylation. Our initial studies suggest that inhibition of mTOR (with Torin-2) in MM cells results in activation of Usp9x activity. The role of phosphorylation in the regulation of Usp9x activity and stability is currently being investigated but our initial observations suggest that Usp9x plays an important regulatory role in the mTOR/5'-AMP kinase cascade. Collectively, our studies suggest that WP1130 inhibits Usp9x activity through covalent modification of critical Cys residues important for Usp9x catalytic activity. Identification of these sites and how phosphorylation influences Usp9x catalytic activity are critical for developing more potent and selective Usp9x inhibitors. Since Usp9x expression has been associated with short survival and poor prognosis in MM patients, potent and selective Usp9x inhibitors could show therapeutic promise for MM patients and other cancers with up-regulated Usp9x activity.