AV-65, a novel Wnt/β-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model

Abstract

Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that β-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/β-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished β-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery.

Figure 1

Inhibition of myeloma cell proliferation. (a) The dose-dependent effect of compounds on the proliferation of myeloma cell lines was evaluated by the modified MTT assay. Cells were incubated with serial dilutions of compounds for 72 h. Data represent the means±s.d. of three independent experiments, and four replicate experiments. (b) Time-dependent inhibition of proliferation of IM-9 cells. Cells were incubated with AV-65 for 6, 12, 24 and 48 h. Data represent the means±s.d. of three independent experiments, and four replicate experiments. (c) Effect of compounds on CD138-positive myeloma cells and CD138-positive normal plasma cells. CD138-positive myeloma cells and CD138-normal plasma cells were purified from BM mononuclear cells obtained from myeloma patients and healthy volunteers, respectively. Data represent the mean of four replicates for each concentration.

Figure 2

Alteration of β-catenin expression in myeloma cells by AV-65 treatment. (a) Cells were incubated with serial dilutions of AV-65 and harvested after 6, 12 and 18 h. Nuclear and cytoplasmic protein fractions were obtained and analyzed by western blot using an anti-β-catenin Ab. Results are representative of three independent experiments. (b) Ubiquitination of β-catenin by AV-65 treatment. Cells were incubated with the indicated concentration of AV-65 (1 × IC₅₀), and then harvested after 12 h. Whole lysate was subjected to immunoprecipitation using an anti-β-catenin Ab, and immune complexes were captured using protein A beads. The protein A slurry (30 μl) was analyzed by immunoblot using anti-mono- and anti-poly-ubiquitinyl conjugates to detect ubiquitination of β-catenin (left panel). The bands of about 60 and 30 kDa detected in the membrane using anti-β-catenin Ab are nonspecific (right panel).

Figure 3

Inhibition of β-catenin-TCF signaling by AV-65 treatment. (a) Inhibition of TCF transcriptional activity in HCT-15 colorectal cancer cells. Cells were co-transfected with a TCF/LEF firefly luciferase reporter, and a CMV-driven Renilla luciferase reporter. To normalize transfection efficiency, cells were incubated with AV-65 for 14 h, and then luciferase activity was measured. Data represent the mean±s.d. of three independent experiments. The relative TCF transcription activity represents the difference between the relative luciferase units of treated versus non-treated cells. (b) Decreased expression of TCF downstream targets in IM-9 cells. Cells were incubated with serial dilutions of AV-65 and then harvested at 24 h. Harvested cells were lysed in radioimmunoprecipitation assay buffer. Cell extracts (20 μg of protein) was analyzed by western blot using the indicated Abs. Cyclin D1 and c-myc were not expressed in IM-9 and U266 cells, respectively.

Figure 4

Enhancement of the interaction between β-catenin and β-TrCP by AV-65 treatment. (a) Cells were incubated with serial dilutions of AV-65 and then harvested after 6 h. Whole lysate was subjected to immunoprecipitation using an anti-β-catenin Ab, and immune complexes were captured using protein A beads. The protein A slurry (30 μl) was analyzed by immunoblot using anti-β-TrCP Ab to detect binding between β-catenin and β-TrCP. (b) IM-9 cells were transduced with lentivirus-based short hairpin RNAs targeting β-TrCP and control vector. β-TrCP levels were measured by real-time polymerase chain reaction. Data represent the means±s.d. of three independent experiments, and four replicate experiments. (c) The alterations of β-catenin expression in IM-9 cells (left panel) and β-TrCP-KD IM-9 cells (right panel) by AV-65 treatment. After 24 h incubation with serial dilutions of AV-65, cells were harvested, and were lysed in radioimmunoprecipitation assay buffer. Cell extracts (20 μg of protein) were analyzed by western blot using an anti-β-catenin Ab and an anti-β-TrCP Ab.

Figure 5

Induction of apoptosis in myeloma cells by AV-65. (a) Cells were incubated with serial dilutions of AV-65 and harvested after 24 h. Data are representative of three independent experiments. The numbers inside each quadrant indicate the percentage of the cell population with the quadrant characteristic. (b) Cells were incubated with serial dilutions of AV-65 and harvested after 24 h. Harvested cells were lysed in lysis buffer, and then caspase-3, -8 and -9 activity was evaluated using a fluorometric protease assay. Data represent the means±s.d. of three independent experiments. Solid, gray and open bars indicate caspase-3, -8 and -9, respectively.

Figure 6

The in vivo inhibitory effects of AV-65 on MM cells in an orthotopic mouse model. Following irradiation (2 Gy), specific pathogen-free 7- to 8-week old female NOD/SCID mice were inoculated intravenously with 5 × 10⁶ IM-9 cells in 100 μl phosphate-buffered saline through the tail vein. The day after inoculation, AV-65 was administered intravenously for four cycles of 4 days on/1 day off. (a) Survival of IM-9-bearing mice treated with higher doses of AV-65 was significantly prolonged compared with vehicle-treated mice (P=0.028). Red, green and blue lines represent the survival rates of high dose of AV-65 (8 mg/kg), low dose of AV-65 (4 mg/kg) and vehicle-treated groups, respectively (10 mice per group). (b) The complete blood counts of vehicle- of AV-65-treated mice. There was no significant difference between mice treated with the higher doses of AV-65 and vehicle. Data represent the means±s.d. of four mice in each group. (c) Influence on BM hematopoiesis in treated mice. After four courses of AV-65 treatment, NOD/SCID mice were killed, and BM cells were obtained. Mononuclear cells were obtained by density centrifugation and analyzed by colony-forming assay. Colony-forming assay was performed in duplicate for each mouse (4 mice per group). There was no significant difference between mice treated with the higher doses of AV-65 and vehicle. Data represent the means±s.e. of four mice in each group.