Distinct Dynamic Profiles for NPI-0052-And Bortezomib-Induced Apoptosis in Multiple Myeloma.
Authors: Chauhan Dharminder , Singh Ajita , Velankar Mugdha , Hideshima Teru , Ovaa Huib , Berkers Celia , Bianchi Giada , Sitia Roberto , Munshi Nikhil C. , Richardson Paul , Palladino Michael A. , Anderson Kenneth C. 
Published: October 2019

Abstract
Our recent study showed that a novel proteasome inhibitor NPI-0052, like bortezomib, triggers apoptosis in multiple myeloma (MM) cells but is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action (Chauhan et al., Cancer Cell, 2005, 8:407–419). Here we examined the time-kinetics of early events triggered by these two agents in MM.1S MM cells. We first determined the minimum exposure time of drug required to trigger an irreversible apoptosis. Cells were exposed to NPI-0052 (IC50:7 nM) or bortezomib (IC50:5 nM) for 15 min, 30 min, 1h, 3h or 6h, cultured in regular medium without drugs for an additional 24h; and analyzed for viability. The results demonstrate that 15 min of exposure to NPI-0052 is sufficient to trigger a 50% decrease in cell viability, whereas bortezomib requires 6h of exposure to trigger a similar extent of apoptosis. These in vitro data were confirmed by using a human MM-xenograft model: MM.1S cells were treated with IC50 doses of each agent for 30 min ex-vivo and injected into immunodeficient BNX mice along with untreated control cells. Examination of the tumors on day 21 showed that the mice with NPI-0052-treated cells develop no significant tumor, whereas mice injected with untreated and bortezomib-treated cells developed significant tumor. We next determined whether NPI-0052, like bortezomib, affects NF-κB signaling and ER-stress responses. NPI-0052 inhibits 50% NF-κB DNA binding activity by 1h, whereas bortezomib requires 6h exposure to cause a similar degree of inhibition. Moreover, treatment of MM.1S cells with NPI-0052 or bortezomib for 5h shows a more robust induction of GADD153 by NPI-0052 than bortezomib along with caspase-4 activation. We next examined the extent of proteasome inhibition triggered by NPI-0052 or bortezomib. Competition experiments were performed between NPI-0052 or bortezomib and dansyl-Ahx3L3VS, a proteasome inhibitor that targets all active subunits of proteasome with comparable affinity: specifically, sites that are not targeted by NPI-0052 or bortezomib are labeled by dansyl-Ahx3L3VS and visualized by subsequent immunoblotting with anti-dansyl Abs, allowing for quantitative assessment of proteasome activity. Cells were treated with NPI-0052 or bortezomib for 30 min, 1h, 3h, and 6h; and protein extracts were incubated for 2h with the dansyl-Ahx3L3VS probe at 37°C, followed by immunoblot analysis with the anti-dansyl Abs. Densitometric analysis of immunoblot showed that 1) 30 min exposure time is sufficient to decrease Chymotrypsin-like activity by both NPI-0052 and bortezomib; however, a much more robust inhibition occurs in response to NPI-0052 than bortezomib; 2) NPI-0052 inhibits caspase-like activity at 30 min, whereas a decrease in C-L was noted only after 3h exposure to bortezomib; and 3) NPI-0052, in contrast to bortezomib, inhibits trypsin-like activity. Taken together, these findings suggest that 1) although both NPI-0052- and bortezomib-triggered apoptosis is associated with sequential occurrence of proteasome inhibition>NF-κB> blockade>ER stress induction, but with differential kinetics, and 2) the cellular response to NPI-0052 occurs much earlier than to bortezomib.