Identification and characterization of a proteasome deubiquitinase inhibitor

The 26S proteasome has emerged as an attractive therapeutic target in the treatment of cancers. Proteasome inhibitors have been shown to selectively kill cancer cells. The FDA approval of bortezomib for the treatment of multiple myeloma and mantle cell lymphoma has clinically validated the 26S proteasome as a therapeutic target in oncology. Despite the acceptable therapeutic index, patients treated with this drug manifest several toxic side effects and moreover, the poor outcome of bortezomib treatment has been associated with acquired resistance to the drug. Currently, many efforts are made to develop new proteasome inhibitors that act through mechanisms distinct from that of bortezomib. In this theses we uncover a novel proteasome inhibitor of the 19S deubiquitinase activity, b-AP15. The inhibition of the proteasome function by this small molecule inhibitor is achieved through the inhibition of deubiquitinating enzymes UCHL5 and USP14 resulting in the induction of poly-ubiquitinated proteins in cells. b-AP15 elicits antitumor effects both in vitro and in vivo that is associated with the inhibition of proteasome function. In syngenic and xenograft mouse models, representing both solid and leukemic malignancies, b-AP15 exhibited potent antitumor activity causing tumor regression, reduced tumor growth and delayed tumor onset. b-AP15 elicits similar, but yet distinct, cellular response as bortezomib. b-AP15 induces rapid apoptotic response which is associated with strong increases of chaperone expression and strong induction of oxidative stress. Notably, these responses are stronger in cells exposed to b-AP15 than bortezomib. Similarly to bortezomib, b-AP15 induced ER stress being involved in the induction of apoptosis. b-AP15 is a reversible inhibitor of deubiquitinase activity and proteasome function. Despite being reversible inhibitor, bAP15 treated cells rapidly commit to apoptosis/cell death which we here report is due to rapid uptake and enrichment of the drug into cells. Bortezomib-mediated resistance has been associated with anti-apoptotic Bcl-2 overexpression and induction of aggresome formation in cancer cells. Here we report that b-AP15 induced apoptosis is insensitive to the overexpression of Bcl-2 and that bAP15 blocks aggresome formation suggesting that bortezomib-mediated resistance could be overcome by b-AP15 treatment. Furthermore, we found that b-AP15 is more toxic to cancer cells than immortalized normal cells with differences larger than those observed for bortezomib, suggesting more favorable therapeutic window of b-AP15 compared to bortezomib. The findings in this thesis strongly suggest that b-AP15 is a good candidate for clinical drug development. LIST OF PUBLICATIONS I. D'Arcy P*, Brnjic S*, Olofsson H M, Fryknas M, Lindsten K, De Cesare M, Perego P, Sadeghi B, Hassan M, Larsson R, Linder S. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nature Medicine, 2011, 17(12), 1636-40 *Equal contribution II. Brnjic S, Mazurkiewicz M, Fryknas M, Sun C, Zhang X, Larsson R, D'Arcy P, Linder S. Induction of tumor cell apoptosis by a proteasome deubiquitinase inhibitor is associated with oxidative stress. Antioxidants & Redox Signaling, 2013, doi: 10.1089/ars.2013. 5322 III. Wang X, Mazurkiewicz M, Brnjic S, Zhang X, Fryknas M, Gullbo J, Larsson R, Stafford W, Arner E, D’Arcy P, Linder S. The 19S deubiquitinase inhibitor b-AP15 is enriched in cells and elicits rapid commitment to cell death. Manuscript IV. Brnjic S, Sun C, Mazurkiewicz M, Larsson R, Fryknas M, Swanton L, High S, Linder S, D'Arcy P. Inhibitor of proteasome deubiquitinase activity inhibits cytoprotectiveaggresome formation in cancer cells. Manuscript