Combination of novel proteasome inhibitor NPI-0052 and lenalidomide trigger in vitro and in vivo synergistic cytotoxicity in multiple myeloma

Abstract

Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade) and, importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid) induces synergistic anti-MM activity in vitro using MM-cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with (1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and poly(ADP) ribose polymerase; (2) activation of BH-3 protein BIM; (3) translocation of BIM to endoplasmic reticulum; (4) inhibition of migration of MM cells and angiogenesis; and (5) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low-dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM.

Figures

Figure 1

Combination of low doses of NPI-0052 and lenalidomide induces synergistic MM-cell death. (A-F) MM-cell lines were pretreated with or without lenalidomide for 24 hours, and then NPI-0052 was added for an additional 24 hours, followed by assessment for cell viability using MTT assays. The experiments with single agents and the respective combinations were carried out simultaneously. The concentrations of drugs, either alone or in combination, were as follows: for MM.1S, KMS12PE, and INA-6 cells: 2nM NPI-0052, 3μM lenalidomide, or NPI-0052 (2nM) plus lenalidomide (3μM); for RPMI-8226 cells: 2nM NPI-0052, 10μM lenalidomide, or NPI-0052 (2nM) plus lenalidomide (10μM); for U266 cells: 5nM NPI-0052, 10μM lenalidomide, or NPI-0052 (5nM) plus lenalidomide (10μM); and for MM.1R cells: 3nM NPI-0052, 5μM lenalidomide, or NPI-0052 (3nM) plus lenalidomide (5μM). *P < .05 for all cell lines. (G-I) MM.1S, RPMI-8226, or INA-6 cells were pretreated with or without lenalidomide for 24 hours, and then NPI-0052 was added for an additional 24 hours and harvested, and whole cell lysates were subjected to immunoblot analysis with anti-PARP, anticaspase-3, antitubulin, or anti-GAPDH Abs. FL indicates full length; and CF, cleaved fragment. Blots shown are representative of 3 independent experiments.

Figure 2

Combined NPI-0052 and lenalidomide trigger antitumor activity in MM patient cells. (A) Purified patient MM cells (CD138+) were pretreated with lenalidomide for 24 hours; NPI-0052 was then added for an additional 24 hours, followed by cell death analysis using Trypan blue exclusion assay. Data are mean ± SD of triplicate samples (P < .05 for all patient samples). (B) PBMCs from healthy donors were treated (as in panel A) with indicated concentrations of NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide, and then analyzed for viability using MTT assay. Data are mean ± SD (n = 3; P = .25 from Jonckheere-Terpstra test for trend).

Figure 3

Combined low doses of NPI-0052 and lenalidomide block migration and tubule formation. (A) For migration assay, MM.1S cells were pretreated with lenalidomide for 12 hours, and then NPI-0052 was added for an additional 6 hours; the cells were more than 90% viable at this time point. The cells were washed and cultured in serum-free medium. After 2 hours of incubation, cells (viability > 90%) were plated on a fibronectin-coated polycarbonate membrane in the upper chamber of Transwell inserts and exposed for 4 hours to serum-containing medium in the lower chamber. Cells migrating to the bottom face of the membrane were fixed with 90% ethanol and stained with crystal violet (original magnification, 10×/0.25 numeric aperture [NA] oil). A total of 3 randomly selected fields were examined for cells that had migrated from top to bottom chambers. (Left panel) Image is representative of 2 experiments with similar results. (Right panel) The bar graph represents quantification of migrated cells. Data are mean ± SD (n = 2; P < .05 for control vs NPI-0052 plus lenalidomide–treated cells). (B) HUVECs were cultured in the presence or absence of combined low doses of NPI-0052 plus lenalidomide for 48 hours, and then assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays (original magnification, 4×/0.10 NA oil, media: EBM-2). (Left panel) Image is representative from 3 experiments with similar results. The in vitro angiogenesis is reflected by capillary tube branch formation (dark brown). (Right panel) The bar graph represents quantification of capillary-like tube structure formation in response to indicated agents: Branch points in several random view fields/well were counted, values were averaged, and statistically significant differences were measured using Student t test. (C-D) MM.1S and RPMI-8226 cells were cultured for 48 hours in BMSC-coated or noncoated wells with control media, NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide. Cell proliferation was assessed by the nonradioactive WST-1 colorimetric assay. Data are mean ± SD of 2 independent experiments. Error bars represent SD.

Figure 4

Mechanisms mediating anti-MM activity of NPI-0052 plus lenalidomide. (A) MM.1S cells were pretreated with or without lenalidomide for 24 hours, and then NPI-0052 was added for an additional 24 hours. Cells were harvested, and whole cell lysates were subjected to immunoblot analysis with anticaspase-8 or anticaspase-9 Abs. FL indicates full length; and CF, cleaved fragment. Blots shown are representative of 3 independent experiments. (B) MM.1S cells were treated with indicated agents (as in panel A) in the presence or absence of biochemical inhibitors of caspase-3, caspase-8, or caspase-9, and then analyzed for apoptosis using annexin V/propidium iodide staining assay. Data are mean ± SD (n = 3; P < .005). (C) MM.1S cells were pretreated with or without lenalidomide for 24 hours, and then NPI-0052 was added for an additional 24 hours. Cells were harvested, and whole cell lysates were subjected to immunoblot analysis with anti-BIM or anti-GAPDH Abs. (D) Bar graph showing quantification by densitometry of BIM protein bands in panel C: A 2- to 3-fold increase in BIMEL and BIM(L+S), respectively, was noted in NPI-0052 plus lenalidomide–treated versus untreated cells. Samples were normalized to GAPDH. (E) MM.1S cells were transfected with siRNA BIM or scrambled siRNA for 24 hours and harvested; whole cell lysates were subjected to immunoblot analysis with anti-BIM or antitubulin Abs. Blots shown are representative of 2 independent experiments. (F) MM.1S cells were transfected with siRNA BIM or scrambled siRNA for 24 hours and treated with indicated agents (as in panel A), followed by analysis for apoptosis by annexin V/propidium iodide staining. As a control, nontransfected cells were also treated with indicated drugs and similarly analyzed. Data are mean ± SD (n = 2; *P > .004). Error bars represent SD.

Figure 5

Effects of NPI-0052 plus lenalidomide on ER stress signaling, heat shock proteins, and proteasome activity. (A) MM.1S cells were pretreated with or without lenalidomide for 24 hours, and then NPI-0052 was added for an additional 24 hours. Cells were harvested, and ER-protein fractions were subjected to immunoblot analysis with anti-BIM or antiactin Abs. Bar graph (bottom) showing quantification of BIMEL protein bands in immunoblot by densitometry: A 2.2-fold increase in BIMEL isoform was noted in NPI-0052 plus lenalidomide–treated versus untreated cells. Samples were normalized to actin. (B) MM.1S cells were treated with indicated agents (as in panel A); whole cell lysates were subjected to immunoblot analysis with anticaspase-12 or tubulin Abs. (C) MM.1S cells were treated with indicated agents (as in panel A); whole cell lysates were subjected to immunoblot analysis with anti-Hsp-70, anti-Hsp-27, anti-Hsp-90, or antitubulin Abs. Lysates from HeLA cells served as a positive control for Hsp Abs. Blots shown are representative of 2 independent experiments. (D) MM.1S cells were pretreated with or without lenalidomide for 6 hours, and then NPI-0052 was added for an additional 6 hours and harvested; cytosolic extracts were then analyzed for CT-L, C-L, and T-L proteasome activities. Results are represented as percentage inhibition in proteasome activities in drug-treated versus vehicle control. Data are mean ± SD (n = 3, P < .05).

Figure 6

Combination of low doses of NPI-0052 and lenalidomide inhibits human plasmacytoma growth in CB-17 SCID mice. (A) Average and SD of tumor volume (mm3) from group of mice (n = 5/group) versus time (days) when tumor was measured. Mice were treated with vehicle, NPI-0052 (orally), lenalidomide (orally), or NPI-0052 plus lenalidomide (orally) at the indicated doses for 24 days on a twice-weekly schedule for NPI-0052 and 4 consecutive days weekly for lenalidomide. A significant delay in tumor growth in NPI-0052 plus lenalidomide–treated mice was noted compared with vehicle-treated control mice (P = .002). Bars represent mean ± SD. (B) Micrographs show apoptotic cells in tumors sectioned on day 24 (endpoint) from untreated or NPI-0052 (0.15 mg/kg) plus lenalidomide (2.5 mg/kg)- treated mice as identified by caspase-3 cleavage (dark brown cells). Photographs are representative of similar observations in 2 different mice receiving treatment. Images were obtained with a Zeiss Axioimager M1 microscope (63×/1.4 Plan-Apochromat objective), axioCam HRc camera, a Axiovision Version 4.6 software, and permount imaging solution. (C) Kaplan-Meier plots showing survival for mice treated with NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide at the indicated concentrations. NPI-0052 plus lenalidomide–treated mice show significantly increased survival (P < .002) compared with the untreated group. The mean overall survival (OS) was 48 days (95% confidence interval, 35-60) in the untreated or single agent–treated cohorts versus 135 days (95% confidence interval, 120-150) in groups treated with combination of NPI-0052 and lenalidomide (0.25 or 0.5 mg/kg). Overall, a 67% increase in survival was observed in mice receiving combined low dose of NPI-0052 (0.15 mg/kg plus lenalidomide (0.5 mg/mg) versus mice receiving either agent alone at these doses. A statistically significant prolongation in mean OS compared with control mice was observed in animals treated with 0.25 mg/kg (P < .002) and 0.5 mg/kg (P > .007). (D) Mice in vehicle-treated controls, NPI-0052–, lenalidomide-, or NPI-0052 plus lenalidomide–treated group were weighed every week. The average changes in body weight are shown. (E) Mice were treated with vehicle, NPI-0052, lenalidomide, or NPI-0052 plus lenalidomide (as in panel A) for 24 days; blood samples were obtained and subjected to analysis for serum bilirubin, hemoglobin, and creatine levels using Quantichrom Creatinine, Bilirubin, and Hemoglobin Assay kit (BioAssay Systems).

Figure 7

Effect of NPI-0052 plus lenalidomide on neovascularization, ubiquitination, BIM, Bcl-6, and YB-1 in vivo in xenografted MM tumors. Tumor sections from mice receiving indicated treatment were immunostained with factor VIII (A), VEGFR1, or ubiquitin Abs (B). (C) Tumor lysates from control and drug-treated mice were subjected to immunoblot analysis using anti-BIM or antiactin Abs. Lanes 1 through 6 represent tumor lysates from mice receiving the following treatments: lane 1, vehicle alone (control); lane 2, NPI-0052 (0.15 mg/kg); lane 3, lenalidomide (2.5 mg/kg); lane 4, lenalidomide (5.0 mg/kg); lane 5, NPI-0052 (0.15 mg/kg) plus lenalidomide (2.5 mg/kg); and lane 6, NPI-0052 (0.15 mg/kg) plus lenalidomide (5.0 mg/kg). (D) Bar graph represents quantification of BIMEL protein bands in immunoblot shown in Figure 4C by densitometry: a 3.3-fold increase in BIMEL isoform was noted in NPI-0052 plus lenalidomide–treated versus untreated cells. Samples were normalized to actin. (E) Tumor sections from control and NPI-0052 plus lenalidomide–treated mice were immunostained with YB-1 Ab. (F) Tumor lysates from control and drug-treated mice were subjected to immunoblot analysis using anti–Bcl-6 or antiactin Abs. Lanes 1 through 6 are the same as in panel C. (A-B,E) Representative of similar observations in 2 different mice receiving the same treatment. For panels A, B, and E, images were obtained with a Zeiss Axioimager M1 microscope (63×/1.4 Plan-Apochromat objective), a AxioCam HRc camera, Axiovision Version 4.6 software, and permount imaging solution.