Potent Activity of a Novel, Irreversible Inhibitor of the Ubiquitin-Proteasome Pathway Against Pre-Clinical Models of Multiple Myeloma.
Authors: Kuhn Deborah J. , Voorhees Peter M. , Strader John S. , Shenk Kevin D. , Sun Congcong M. , Demo Susan D. , Bennett Mark K. , Orlowski Robert Z. 
Published: April 2020

Abstract
The proteasome is a multi-catalytic proteinase complex that is integral to intracellular proteolysis, and plays a key role in many cell functions. Targeting the proteasome with small molecule inhibitors has been validated as a rational therapeutic strategy for patients with relapsed/refractory myeloma with the approval of the first proteasome inhibitor, bortezomib (VELCADE®), for this indication. Additional studies are ongoing to better define the role of this agent in myeloma and other diseases, including non-Hodgkin’s lymphoma. Since bortezomib is a reversible proteasome inhibitor, we considered the possibility that an irreversible agent might have novel, potentially attractive properties. To begin to evaluate this hypothesis, we have studied the efficacy of a novel epoxomicin-related proteasome inhibitor, PR-171, which binds irreversibly and with a high degree of specificity in vitro to the chymotrypsin-like subunit of the proteasome. PR-171 was able to inhibit proliferation of both interleukin (IL)-6-dependent ANBL-6 and KAS-6 cell lines, as well as IL-6-independent models, including RPMI 8226 and U266 cells, in a concentration- and time-dependent fashion. IL-6-dependent cells generally displayed a greater sensitivity to PR-171-mediated effects than IL-6-independent cells. Experiments modeling the in vivo pharmacokinetics of proteasome inhibitors, with a one-hour pulse of drug followed by a washout, showed that PR-171 indeed inhibited the chymotrypsin-like activity of the proteasome without effects on other proteasome proteases. Inhibition of cell proliferation was associated with an induction of programmed cell death, as judged by the appearance of apoptotic oligonucleosome DNA fragments, as well as by the activation of caspase-3. This common effector caspase was activated by both the extrinsic and intrinsic pathways, in that both caspase-8 and caspase-9 were potently induced. Additionally, pulse treatment of PR-171 induced activation of c-Jun-N-terminal kinase, a key signaling molecule in stress-induced and proteasome inhibitor-induced apoptosis. Other members of the stress response-signaling pathway, including heat shock protein-70 and mitogen activated protein kinase phosphatase-1, were induced as well. Finally, both continuous and pulse treatment with PR-171 was also able to inhibit proliferation in freshly purified patient-derived multiple myeloma plasma cells, including isolates from patients with both newly diagnosed, previously untreated disease, as well as isolates from patients who had progressed on other standard therapies, including bortezomib. Importantly, PR-171 was active in both myeloma cell line models and patient-derived samples with chromosome 13 abnormalities. Taken together, these data indicate that PR-171 is a promising, novel proteasome inhibitor with activity against models of multiple myeloma, providing a rational basis for its translation into the clinic.