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. 2010 Dec 2;116(23):4906-15.
doi: 10.1182/blood-2010-04-276626. Epub 2010 Aug 30.

A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma

Dharminder Chauhan  1 , Ajita V Singh, Monette Aujay, Christopher J Kirk, Madhavi Bandi, Bryan Ciccarelli, Noopur Raje, Paul Richardson, Kenneth C Anderson
Affiliations

A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma

Dharminder Chauhan et al. Blood. .

Abstract

Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM.

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Figures

Figure 1
Figure 1
Proteasome inhibitor ONX 0912 is structurally distinct from bortezomib and carfilzomib, inhibits proteasome activity in vitro, and triggers MM cell death. (A-C) Chemical structures of proteasome inhibitors bortezomib, carfilzomib, and ONX 0912. (D) ONX 0912 and carfilzomib inhibit CT-L proteasome activities in human MM cell lines. MM.1S and MM.1R cells were treated with carfilzomib (5nM) and ONX 0912 (3nM) for 48 hours and harvested; cytosolic extracts were then analyzed for CT-L proteasome activities. Results are represented as percent inhibition in proteasome activities in drug-treated versus vehicle control. (E) MM cell lines were treated with or without ONX 0912 at the indicated doses for 48 hours, followed by assessment for cell viability using MTT assays. Data presented are means ± SD (n = 3; P < .05 for all cell lines).
Figure 2
Figure 2
ONX 0912 triggers antitumor activity in MM patient cells. (A) Purified patient MM cells (CD138-positive) were treated with ONX 0912 (10nM), and cell death was measured using trypan blue exclusion assay. Data presented are mean ± SD of triplicate samples (P < .05 for all patient samples). (B) PBMCs from healthy donors were treated with indicated concentrations of ONX 0912 and then analyzed for viability using MTT assay. Data presented are mean ± SD (n = 3; P < .05).
Figure 3
Figure 3
ONX 0912 triggers apoptosis in MM cells, associated with PARP cleavage and caspase activation. (A) MM cell lines were treated with ONX 0912 for 48 hours and analyzed for apoptosis using annexin V/PI staining assay. (B-C) MM.1S cells were treated with ONX 0912 at the indicated doses for 48 hours and harvested; whole-cell lysates were subjected to immunoblot analysis with anti-PARP, anti–caspase-3, anti–caspase-8, anti–caspase-9, or anti-GAPDH Abs. FL indicates full-length; CF denotes cleaved fragment. Blots shown are representative of 3 independent experiments.
Figure 4
Figure 4
ONX 0912 blocks migration and tube formation. (A) MM.1S cells were pretreated with 10nM ONX 0912 for 24 hours; the cells were > 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 (×10 original magnification /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. Image is representative of 3 experiments with similar results. (B) Cells were plated as in panel A, and ONX 0912 effect on migration was assessed in the presence or absence of rVEGF. The bar graph represents quantification of migrated cells. Data presented are means ± SD (n = 3; P = .03 for control versus ONX 0912–treated cells). Error bars represent SD. (C) HUVECs were cultured in the presence or absence of ONX 0912 for 24 hours and then assessed for in vitro angiogenesis using Matrigel capillary-like tube structure formation assays (×4 original magnification/0.10 NA oil, media: endothelium basal medium-2 [EBM-2]). Image is representative from 3 experiments with similar results. The in vitro angiogenesis is reflected by capillary tube branch formation (dark brown). Data represent means ± SD (n = 2; P < .05). (D) In vitro angiogenesis using Matrigel capillary tube formation assay was performed using HUVECs as in panel C. The bar graph represents quantification of capillary-like tube structure formation in response to vehicle alone and ONX 0912–treated cells: Branch points in several random view fields/wells were counted; values were averaged, and statistically significant differences were measured using Student t test. Data presented are means ± SD (n = 3; P = .01 for control versus ONX 0912–treated cells). Error bars represent SD.
Figure 5
Figure 5
ONX 0912 inhibits growth of xenografted human MM cells in mice. (A) MM.1S cells alone (3 × 107 cells/mouse) were implanted in the rear flank of female beige nude xid mice (5-7 weeks of age at the time of tumor challenge). On Day 30-33, mice were randomized to treatment groups and treated intravenously with carfilzomib (5 mg/kg) or orally with vehicle or ONX 0912 (30 mg/kg). Mice were treated for 2 consecutive days and the treatment repeated weekly for 7 weeks. Data are presented as mean tumor volume ± SEM (n = 9-10/group). One of the 2 representative experiments is shown. Bars indicate means ± SD. (B) Human fetal bone grafts (size range: 0.5-1.5 cm) were subcutaneously implanted into CB-17 SCID mice. Four weeks after bone implantation, INA-6 cells (2.5 × 106) were injected directly into the human fetal bone implant in SCID mice (5 mice each group), and mouse sera samples were serially analyzed for levels of secreted human soluble IL-6R (shIL-6R) by enzyme-linked immunosorbent assay as a measure of tumor burden. Upon detection of measurable shIL-6R in mice (3-4 wks after injection of cells), mice were treated with either vehicle alone or indicated doses of ONX 0912 (orally, 2 consecutive days every week for 4 weeks); mouse serum samples were subjected to analysis for shIL-6R (mean ± SD; P = .03 for both doses; n = 2). Bars indicate means ± SD (C) Kaplan-Meier survival plot shows a significant increase (P = .03) in survival of mice receiving ONX 0912 (30 or 50 mg/kg) compared with vehicle-treated control. The mean overall survival (OS) was 47.5 days (95% confidence interval; 40-55) in the untreated versus 70 days (95% confidence interval; 65-70) in ONX 0912–treated cohorts.
Figure 6
Figure 6
Effect of ONX 0912 on apoptosis, neovascularization, and ubiquitination in vivo in xenografted MM tumors. (A-D) Human bone chips were removed from mice after the last treatment and immunostained with Abs against Caspase-3, Factor VIII, VEGFR1, and Ubiquitin. Scale bar, 10μm. Dark brown represents marker-positive cells in all cases. Micrographs are representative of bone sections from 2 different mice in each group. (E) Mice in vehicle-treated control and ONX 0912–treated groups were weighed every week. The average changes in body weight are shown.
Figure 7
Figure 7
Combination of low doses of ONX 0912 and bortezomib, MS-275, Dex, or lenalidomide enhances MM cell death. (A) Low doses of ONX 0912 and bortezomib trigger synergistic anti-MM activity in MM cells. MM.1S cells were treated for 24 hours with indicated concentrations of ONX 0912, bortezomib, or ONX 0912 plus bortezomib and then assessed for viability using MTT assays. Isobologram analysis shows the synergistic cytotoxic effect of ONX 0912 and bortezomib in MM.1S cell line. The graph (left) is derived from the values given in the table (right). The numbers 1-4 in the graph represent combinations shown in the table. CI < 1 indicates synergy. Numbers represent FA (Fraction Affected) values (eg, FA = 0.5 corresponds to 50% decrease in viability or IC50 for agent). All experiments were performed in triplicate and mean value is shown. FACom = fraction of cells showing decrease in viability with ONX 0912 plus bortezomib treatment. (B) Low doses of ONX 0912 and MS-275 trigger synergistic anti-MM activity in MM cells. MM.1S cells were treated for 48 hours with indicated concentrations of ONX 0912, MS-275, or ONX 0912 plus MS-275 and then assessed for viability using MTT assays. Isobologram analysis shows the synergistic cytotoxic effect of ONX 0912 and MS-275 in MM.1S cell line. The graph (left) is derived from the values given in the table (right). The numbers 1-6 in the graph represent combinations shown in the table. CI < 1 indicates synergy. (C) MM.1S cells were treated for 48 hours with indicated concentrations of ONX 0912, Dex, or ONX 0912 plus Dex and then assessed for viability using MTT assays. Shown is mean ± SD of 3 independent experiments. (D) MM.1S cells were treated for 48 hours with indicated concentrations of ONX 0912, lenalidomide, or ONX 0912 plus lenalidomide and then assessed for viability using MTT assays. Shown is mean ± SD of 3 independent experiments.
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