patients with chronic lymphocytic leukemia inducer of apoptosis than bortezomib in lymphocytes from The proteasome inhibitor NPI-0052 is a more effective Updated

Proteasome inhibitors are potent inducers of apoptosis in isolated lymphocytes from patients with chronic lymphocytic leukemia (CLL). However, the reversible proteasome inhibitor bortezomib (PS-341; Velcade) did not display substantial antitumor activity in CLL patients. Here, we compared the effects of bortezomib and a new irreversible proteasome inhibitor (NPI-0052) on 20S chymotryptic proteasome activity and apoptosis in isolated CLL cells in vitro. Although their steady-state (3 hours) IC50s as proteasome inhibitors were similar, NPI-0052 exerted its effects more rapidly than bortezomib, and drug washout experiments showed that short exposures to NPI-0052 resulted in sustained (z24 hours) 20S proteasome inhibition, whereas 20S activity recovered in cells exposed to even 10-fold higher concentrations of bortezomib. Thus, brief (15 minutes) pulses of NPI-0052 were sufficient to induce substantial apoptosis in CLL cells, whereas longer exposure times (z8 hours) were required for commitment to apoptosis in cells exposed to equivalent concentrations of bortezomib. Commitment to apoptosis seemed to be related to caspase-4 activation, in that cells exposed to bortezomib or NPI-0052 could be saved from death by addition of a selective caspase-4 inhibitor up to 8 hours after drug exposure. Our results show that NPI-0052 is a more effective proapoptotic agent than bortezomib in isolated CLL cells and suggest that the chemical properties of NPI-0052 might also make it an effective therapeutic agent in CLL patients. [Mol Cancer Ther 2006;5(7):1836–43] Introduction Chronic lymphocytic leukemia (CLL) is the most common hematologic malignancy in adults in the Western world (1, 2). The disease is characterized by the accumulation of mature resting CD5 B lymphocytes in the peripheral blood and is thought to arise primarily as the result of defect(s) in the control of apoptosis rather than increased proliferation (3, 4). Thus, CLL cells express very high levels of the antiapoptotic protein, BCL-2, as the result of epigenic alterations in the regulation of the bcl-2 gene (5). Although nucleoside analogues and other agents initially display strong activity CLL patients, recent studies indicate that they do not extend patient survival (1). Thus, there is substantial interest in developing biology-based therapies for CLL that will qualitatively change the course of disease progression. The proteasome is a multisubunit proteolytic complex that is responsible for the degradation of f80% of all cellular proteins (6). It also plays a central role in cell cycle progression and apoptosis by mediating the degradation of ubiquitylated target proteins that include p53, p21, members of the BCL-2 family, and the physiologic inhibitor of nuclear factor-nB, InBa (6). These observations provided the rationale for the development of proteasome inhibitors as anticancer therapeutic agents (6). The reversible peptide boronate proteasome inhibitor, bortezomib (PS-341; Velcade), was the first such agent evaluated in clinical trials in patients with cancer (6, 7), and its promising activity in multiple myeloma (35% objective response rates; ref. 8) led to Food and Drug Administration approval in 2003. Bortezomib also displayed promising single-agent activity in other disease sites, including mantle cell lymphomas (8), non–small cell lung cancer (9, 10), and prostate cancer (11). Preclinical studies confirmed that bortezomib blocks nuclear factor-nB activation (6, 12, 13) and can bypass BCL-2-mediated apoptosis resistance (6, 14), possibly because it is a potent activator of the BCL-2-inhibiting protein kinase, c-Jun NH2-terminal kinase (JNK; refs. 15, 16). The success of bortezomib has prompted the development of chemically distinct proteasome inhibitors that display differences in their effects on 20S proteasome inhibition and their bioavailability. NPI-0052 (salinosporin A) is a novel marine-derived proteasome inhibitor (17) that, like bortezomib, has been developed for the treatment of cancer (18). However, NPI-0052 differs from bortezomib in terms of its inhibitory effects on the three major enzymatic activities of the 20S proteasome and its irreversibility (18). Furthermore, recent studies with NPI-0052 in multiple myeloma cells indicate that it has different effects on the three major activities of the 20S proteasome and induces Received 2/2/06; revised 4/12/06; accepted 5/12/06. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: David McConkey, Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Box 173, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-8591; Fax: 713-792-8747. E-mail: dmcconke@mdanderson.org Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-06-0066 1836 Mol Cancer Ther 2006;5(7). July 2006 on April 22, 2014. © 2006 American Association for Cancer Research. mct.aacrjournals.org Downloaded from apoptosis via mechanisms that are unique from those evoked by bortezomib (19). NPI-0052 has undergone extensive preclinical toxicity studies and entered phase I this year. The ability of bortezomib to bypass BCL-2-mediated resistance prompted investigators to evaluate its effects on apoptosis in isolated CLL lymphocytes (20–24). Overall, these preclinical studies confirmed that proteasome inhibitors display uniquely high potency against untreated and fludarabine-refractory CLL cells in vitro (24). However, in a recently completed phase II trial of the drug in patients with fludarabine-refractory CLL, the clinical activity of the drug failed to match preclinical expectations (21). Given that bortezomib displays a very short serum half-life, we wondered whether the lack of clinical activity of the drug might be related to the fact that bortezomib is a reversible proteasome inhibitor that is cleared from the serum within minutes. We speculated that the properties of NPI-0052 as an irreversible proteasome inhibitor might make it a more potent inducer of apoptosis in this disease model. The present study was undertaken to address this hypothesis. Materials andMethods Reagents Bortezomib was purchased from The University of Texas/M. D. Anderson Cancer Center pharmacy (Houston, TX). NPI-0052 was provided by Nereus Pharmaceuticals (San Diego, CA). The chymotryptic activity substrate (Suc-LLVY-AMC) was purchased from A.G. Scientific, Inc. (San Diego, CA), the caspase-like substrate (Z-LLE-AMC) was from A.G. Scientific, the tryptic substrate (Boc-LLRAMC) was from BioMo (Plymouth Meeting, PA), and the anti-procaspase-4 antibody was purchased from Stressgen (Victoria, British Columbia, Canada). Isolation of CLL Cells Freshly isolated peripheral blood from CLL patients were fractionated by Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ) sedimentation. The mononuclear cellular layer was then resuspended in RPMI 1640 containing10% heatinactivated fetal bovine serum, 10 mmol/L HEPES (pH 7.4), sodium pyruvate, L-glutamine, and antibiotics. Quantification of Apoptosis Apoptosis was measured by propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) analysis as described previously (24). Harvested primary CLL cells were pelleted by centrifugation and resuspended in a PBS containing 50 Ag/mL PI, 0.1% Triton X-100, and 0.1% sodium citrate. Samples were stored at 4jC for 16 hours and gently vortexed before FACS analysis (FL-3 channel). 20SProteasomeActivityAssay CLL cells were pelleted by centrifugation and washed once in PBS. Cells were then resuspended in cold lysis buffer [20 mmol/L Tris (pH 7.5), 0.1 mmol/L EDTA (pH 8.0), 20% glycerol, 0.05% NP40, 1 mmol/L 2h-mercaptoethanol, 1 mmol/L ATP] and frozen and thawed thrice on dry ice followed by centrifugation at 1,500 rpm for 1 minute. Lysates were then transferred to a 96-well plate, in which substrate buffer [50 mmol/L HEPES (pH 7.5), 5 mmol/L EGTA (pH 7-8) containing 50 Amol/L peptide substrate] was added at a 1:1 dilution and mixed by pipetting up and down. Samples were read 1 hour after addition of substrate buffer. Fluorescence was read on a SpectraMax Gemini EM fluorimeter (Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 380 nm and Table 1. Clinical characteristics of the CLL lymphocytes used in this study Patient Rai* WBC h2M ZAP-70c Previous treatmentb