De-Ubiquitinase Inhibition by WP1130 Induces Formation of Aggresomes, Engages Autophagy and Activates Apoptosis in B-Cell Malignancies.

Abstract 3769

Poster Board III-705

Nearly all protein in mammalian cells is subject to regulation by ubiquitination and it is becoming clear that modification of this process may have therapeutic activity in several diseases, including cancer. Bortezomib and Hsp90 inhibitors affect protein behavior through ubiquitination and have therapeutic activity in specific cancers. These observations suggest that additional molecules that alter protein ubiquitination through a distinct mechanism may have therapeutic value. Previously we described a small molecule, WP1130, that inhibited cytokine and Bcr-Abl mediated signaling through a unique mechanism (Blood. 2007;109(8):3470-8, Cancer Res. 2007;67(8):3912-8). In this study we examined the mechanism of WP1130 action. Like bortezomib, WP1130 induced rapid, time and dose-dependent protein ubiquitination in treated cells. However unlike bortezomib, WP1130 treatment resulted in accumulation of ubiquitinated proteins in the detergent insoluble cell fraction. Sub-cellular fractionation studies revealed deposition of ubiquitinated proteins into the cytoskeletal-enriched fraction of intra-cellular structures called aggresomes. Since proteosome inhibition also results in aggresome formation, confocal microscopic examination of WP1130 treated cells was performed which demonstrated formation of peri-nuclear aggresomes containing ubiquitinated proteins, in complex with heat shock chaperones and other aggresome markers such as HDAC6 and γ-tubulin. However, WP1130 did not directly or indirectly inhibit the 20S proteasome, did not disrupt HSP90 activity or induce oxidative stress, distinguishing its mechanism from other agents. Since aggresomes also acts as disposal grounds for accumulated insoluble protein aggregates through autophagy, we assessed whether autophagy was engaged as a consequence of aggresome formation in WP1130 treated cells. We observed activation of the autophagy pathway, evident by the rapid conversion of LC3-I to LC3-II and decrease in p62/SQSTM1 protein levels. We also compared WP1130 to its parental compound, AG490, and found no changes in ubiquitination or an induction of autophagy with AG490. Since autophagy and aggresome formation are known to be cytoprotective measures initiated by cells to cope with unfavorable growth conditions, inhibition of autophagy may enhance cytotoxicity associated with WP1130. Co-treatment of cells with WP1130 and wortmannin, a known inhibitor of autophagy, resulted in a rapid increase of caspase-3 activity, culminating in enhanced cell death. Biochemical evidence supports a role for de-ubiquitinase (DUB) inhibition in WP1130 treated cells as a mechanism of enhanced resident ubiquitination of cellular proteins. Lysates from WP1130 treated cells displayed time- and concentration-dependent decreased DUB activity. These results suggest that WP1130 initiates rapid and robust protein ubiquitination through suppression of cellular DUB activity to initiate an alternate pathway to control protein behavior and induce apoptosis in specific B-cell malignancies which are highly sensitive to WP1130 and other ubiquitin content modifying agents.