Original Article

Leukemia (2009) 23, 1894–1903; doi:10.1038/leu.2009.121; published online 4 June 2009

Myeloma

The effects of JNJ-26481585, a novel hydroxamate-based histone deacetylase inhibitor, on the development of multiple myeloma in the 5T2MM and 5T33MM murine models

S Deleu1, M Lemaire1, J Arts2, E Menu1, E Van Valckenborgh1, P King2, I Vande Broek1, H De Raeve3, B Van Camp1, P Croucher4 and K Vanderkerken1

  1. 1Department of Hematology and Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium
  2. 2Ortho Biotech Oncology Research and Development Department, Johnson & Johnson Pharmaceutical R & D, Beerse, Belgium
  3. 3Department of Pathology, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium
  4. 4Section of Musculoskeletal Science, University of Sheffield Medical School, Sheffield, UK

Correspondence: Professor Dr K Vanderkerken, Department of Hematology and Immunology, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: Karin.Vanderkerken@vub.ac.be

Received 21 November 2008; Revised 10 April 2009; Accepted 5 May 2009; Published online 4 June 2009.

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Abstract

Multiple myeloma (MM) is a B-cell malignancy, which often remains incurable because of the development of drug resistance governed by the bone marrow (BM) microenvironment. Novel treatment strategies are therefore urgently needed. In this study, we evaluated the anti-MM activity of JNJ-26481585, a novel 'second-generation' pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor, using the syngeneic murine 5TMM model of MM. In vitro, JNJ-26481585 induced caspase cascade activation and upregulation of p21, resulting in apoptosis and cell cycle arrest in the myeloma cells at low nanomolar concentrations. Similar results could be observed in BM endothelial cells using higher concentrations, indicating the selectivity of JNJ-26481585 toward cancer cells. In a prophylactic and therapeutic setting, treatment with JNJ-26481585 resulted in an almost complete reduction of the tumor load and a significant decrease in angiogenesis. 5T2MM-bearing mice also developed a MM-related bone disease, characterized by increased osteoclast number, development of osteolytic lesions and a reduction in cancellous bone. Treatment of these mice with JNJ-264815 significantly reduced the development of bone disease. These data suggest that JNJ-26481585 has a potent anti-MM activity that can overcome the stimulatory effect of the BM microenvironment in vivo making this drug a promising new anti-MM agent.

Keywords:

multiple myeloma, 5TMM model, histone deacetylase inhibitor, JNJ-26481585

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Introduction

Multiple myeloma (MM) is a B-cell neoplasm characterized by an accumulation of monoclonal plasma cells in the bone marrow (BM), secreting high levels of monoclonal immunoglobulins. The MM cells closely interact with the BM microenvironment, which supports their growth, survival and drug resistance through cell–cell adhesion and release of growth factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1).1, 2 Neovascularization and osteolysis are processes in the BM microenvironment that are induced by the MM cells, as well as through mutual interactions with the microenvironment and that contribute to the pathogenesis of MM. Neovascularization is induced by an alteration in the balance of pro- and anti-angiogenic factors secreted by both the MM cells and the tumor microenvironment. This neovasculari-zation has been shown to correlate with the prognosis of patients and often parallels responses to therapy.3, 4 MM cells also promote the development of bone disease, which results from a change in the balance between bone formation and bone resorption in favor of bone destruction. Multiple factors (for example, receptor activator NFkappaB ligand (RANKL), macrophage inflammatory protein-1 alpha (MIP-1alpha), MIP-1beta, vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1-alpha) have been reported to stimulate osteoclastic resorption, whereas others (for example, dickkopf-1, secreted Frizzled-related protein-2 and IL-7) may contribute to the inhibition of osteoblastic bone formation.5, 6, 7, 8, 9

New active drugs, including thalidomide, lenalidomide and the proteasome inhibitor, bortezomib, are able to overcome resistance of MM cells to conventional chemotherapy and prolong the survival of MM patients.10 Despite recent progress with these novel treatments and high-dose chemotherapy with stem cell transplantation, MM remains an incurable disease. Therefore, the identification of new key targets in both the MM cells and the BM microenvironment is crucial for the development of new therapeutic strategies.11, 12, 13

Histone deacetylase inhibitors (HDACi) are drugs with promising activity against hematological and solid malignancies. Cell cycle progression, differentiation and cell death are controlled by the acetylation state of histones. The turnover of histone acetylation is regulated by histone acetyl transferases14 and histone deacetylases (HDAC). Inhibition of HDAC results in an accumulation of acetylated histones and transcriptional alterations to specific genes contributing to tumorigenesis.15, 16 In addition to their anti-cancer activity, HDACi have also been reported to have anti-angiogenic activity both in vitro and in vivo by modulating angiogenesis-related genes, altering signalling cascades and inhibiting the proliferation of endothelial cells.17, 18, 19, 20 Recent reports also show that HDAC have a role in regulating bone turnover. Inhibition of HDAC activity with broad-spectrum inhibitors (that is, trichostatin, valporic acid and sodium butyrate) promotes osteoblast differentiation and maturation in vitro, although osteoclast survival and maturation is inhibited.21, 22, 23, 24

Several broad-spectrum HDACi have already been shown to increase cell cycle inhibitors (p21, p27), inhibit the production of cytokines, such as IL-6 and VEGF, and induce apoptosis of MM cells in vitro.25, 26, 27, 28 Phase I clinical trials have also shown that some HDACi, such as suberoylanilide hydroxamic acid (SAHA), panobinostat and belinostat, are able to reduce tumor burden in patients with MM.29, 30, 31

However, it is unclear, besides the direct in vitro effects on MM cells, how the BM microenvironment is affected by these HDACi, and more specifically how crucial processes like induction of bone disease and angiogenesis are affected.

In this study, we examined the effects of a new hydroxamate compound, JNJ-26481585, in the murine 5T2 and 5T33MM models of MM. These models originated spontaneously in elderly C57BL/KaLwRij mice and have since been propagated by intravenous (i.v.) transfer of the diseased marrow in young syngeneic, immunocompetent, mice.32, 33 Both models mimic the human disease closely, with the selective localization of cells in the BM, the presence of a serum M component, increased BM angiogenesis and induction of osteolytic bone disease (5T2MM).34, 35 The MM-related bone disease is characterized by the development of osteolytic lesions, increased osteoclast resorption and a suppression of osteoblasts. The 5T33MM model represents an aggressive form of MM with rapid tumor growth, and in this model mice were treated in a prophylactic setting. As the tumor growth of 5T2MM mice has a moderate growth, we were able to treat these mice from a time point with measurable MM disease, representing a more therapeutic setting.

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Materials and methods

Mice

C57BL/KalwRij mice were purchased from Harlan (Horst, the Netherlands). They were housed and treated following conditions approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (license no. LA1230281).

Drug

JNJ-26481585 (Janssen Pharmaceutica N.V., J&J PRD, Beerse, Belgium) was prepared in a solvent containing 10% hydroxypropyl-beta cyclodextrin, 0.8% HCl (0.1 N), 0.9 % NaOH (0.1 N), 3.4% mannitol and pyrogen-free water. Compared with the HDAC inhibitor, R306465 (a first-generation inhibitor), JNJ-26481585 has significantly improved pharmacodynamic properties with a high solubility, an increased metabolic stability in rat hepatocytes, a good oral availability in non-rodents and a long-lasting pharmacodynamic response, that is, histone 3 (H3) acetylation.36

For in vitro studies, JNJ-26481585 stock solutions were prepared at 1 mM and 5 mM in tissue-culture grade dimethylsulfoxide.

5TMM models

The in vivo growing 5T2MM and 5T33MM cells originated spontaneously from elderly C57BL/KaLwRij mice and have since been propagated by i.v. transfer of the diseased marrow in young syngeneic mice.32, 37 MM cells were isolated from diseased mice by flushing the BM out of the femurs and tibiae and crushing the vertebrae to release BM. BM mononuclear cells were purified by Lympholyte M (Cedarlane, Hornby, ON, Canada) gradient centrifugation at 1000 g for 20 min, generating enriched 5TMM cells. The purity was determined by assessing plasmacytosis on May-Grünwald Giemsa-stained cytospins. For in vivo experiments, the isolated 5T33MMvv cells were injected i.v. at 5 times 105 cells/mice, whereas the 5T2MM cells were injected at 2 times 106 cells/mice.

The isolated MM cells also grow stroma-dependently and survive only for a short period in vitro. In contrast, the 5T33MMvt cell line is a clonally identical variant that originated spontaneously from 5T33MMvv cells in vitro and grows stroma-independently.34 The 5T33MMvt cells were cultured at 37 °C in a humidified incubator with 5% CO2 in RPMI 1640 medium (Bio-Whittaker, Verviers, Belgium), supplemented with 10% fetal calf serum (Fetal Clone I; Hyclone, Logan, UT, USA), 1% sodium pyruvate, 100 U/ml penicillin, 100 mug/ml streptomycin, 2 mM l-glutamine and 1% minimum essential medium (supplements from Bio-Whittaker).

The 5T33MM in vivo model induces MM growth in the BM and angiogenesis (as assessed by microvessel density (MVD), see below). In addition to these features, the 5T2MM model also induces bone disease similar to that seen in the human disease.33, 38

STR-10 cell line

The murine BM endothelial cell line, STR-10, initially immortalized with SV40, was cultured in RPMI-1640 medium supplemented as described above (a gift from Dr Kobayashi, Japan).39

Western blot analysis

Downstream effects of JNJ-26481585 in 5T33MMvt cells and BM endothelial STR-10 cells were detected by western blotting. Samples were prepared from whole-cell pellets as described earlier.2 The amount of total protein was quantified using the Bicinchoninic acid protein assay kit (Perbio Science, Bonn, Germany). In all, 40 mug of protein lysates were separated on a 15% SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with TBS containing 5% low-fat milk and 0.1% Tween 20, and incubated overnight at 4 °C with primary antibodies against acetyl-H3 (Upstate Biotechnology, Lake Placid, NY, USA), acetyl-histone 4 (H4), caspase-3, caspase-8, caspase-9, Poly ADP ribose polymerase (PARP), beta-actin (loading control) (Cell Signalling, Danvers, MA, USA), p21Waf1, p27Kip1 (eBioscience, Halle, Belgium) and Acetyl tubulin (Sigma, Steinheim, Germany). After washing, the membrane was incubated with peroxidase-conjugated secondary antibody (Cell Signalling). The bands were visualized using the enhanced chemiluminescence system (Amersham, Buckinghamshire, United Kingdom).

MTS

The MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) colorimetric assay (Promega, Madison, WI, USA) was used to assess the viability of 5T33MMvt cells and STR-10 cells. 5T33MMvt cells were cultured in a 96-well plate at 4.104 cells/well and test compound was added (total volume 200 mul/well). For the STR-10 cells (adherent cell line), test compound was added the day after culturing the cells in a 96-well plate at 8.103 cells/well. After 72 h incubation, MTS was added according to the manufacturer's protocol and cells were incubated for 2 h at 37 °C. Dye absorbance was measured spectrophotometrically at 490 nm by using an ELISA reader (Bio-Rad Microplate Reader, Bio-Rad Laboratories). The IC50 was calculated using Graphpad Prism (Graphpad Software, La Jolla, CA, USA).

Assessment of apoptosis

Apoptosis was evaluated after dual staining of the 5T33MMvt cells with Annexin V-fluorescein isothiocyanate (FITC) and 7-amino-actinomycin D (7-AAD) (BD Pharmingen, Franklin Lakes, NJ, USA). 5T33MMvt cells were cultured in a 24-well plate at 5 times 105 cells/well and JNJ-26481585 was added at indicated concentrations. After 24 h and 72 h incubation, cells were collected and washed with cold phosphate-buffered saline. The cells were then resuspended in 100 mul of binding buffer and incubated with 5 mul 7-AAD and 5 mul Annexin V-FITC for 15 min in the dark at room temperature. Finally, cells were analyzed with a FACSCanto flow cytometer (BD Pharmingen) using the FACS Diva software.

Cell cycle analysis

5T33MMvt cells were collected and stained with popidium iodide (PI) solution containing 5 mug/ml PI (BD Pharmingen), 0.1 mg/ml sodium citrate and 0.01% Triton X-100 and incubated for at least 4 h in the dark. Cells were analyzed with a FACSCanto flow cytometer (BD Pharmingen) using CellQuest and FACS Diva software.

Prophylactic treatment of 5T33MM-bearing mice with JNJ-26481585

Two groups of 10 mice were injected i.v. with 5T33MMvv cells, whereas one group of 10 naive non-tumor-bearing mice was included. The day after the injection of MM cells, the tumor-bearing mice were treated with JNJ-26481585 (20 mg/kg, every other day, n=10) or vehicle (containing 10% hydroxypropyl-beta cyclodextrin, 0.8% HCl (0.1 N), 0.9 % NaOH (0.1 N), 3.4% mannitol and pyrogen-free water, n=10), until the first mice showed signs of morbidity (at week 3). As preliminary results showed that subcutaneous (s.c.) administration was as effective as intraperitoneal administration, the s.c. administration route was used (results not shown). Mice were killed and BM was isolated from the hind legs and tumor burden assessed. One femur was fixed and further processed to assess BM angiogenesis as described below.

Therapeutic treatment of 5T2MM-bearing mice with JNJ-26481585

Mice were injected with 5T2MM myeloma cells (n=20) or were included as a naive non-tumor-bearing group (n=10). From the time serum paraprotein was detectable by electrophoresis (at week 7), 5T2MM-bearing mice were treated with either JNJ-26481585 (20 mg/kg, every other day, subcutaneously, n=10) or vehicle (n=10, control group). After 3.5 weeks, all animals were killed. Four and two days before killing, mice were injected with calcein. BM was isolated to determine tumor load. The tibiae and femura were dissected and processed for radiographic, micro-computer tomography (micro-CT) and histological analysis of bone disease and angiogenesis (see below).

Assessment of tumor burden

Tumor burden was assessed by measurement of serum paraprotein by protein electrophoresis and determining plasmacytosis on May-Grünwald Giemsa-stained cytosmears of BM, isolated from one hind leg.

Assessment of microvessel density

Microvessel density was determined by CD31 staining as described earlier.38 Briefly, one femur was fixed in zinc fixative, decalcified, embedded in paraffin and 3-mum sections cut. After incubation with the primary antibody, rat anti-CD31 antibody (PECAM-1, 1:10; Pharmingen, San Diego, CA, USA) staining was detected with a secondary biotin-conjugated goat anti-rat antibody (1:75; Pharmingen). In the area with the highest blood vessels density (hot spot), the number of blood vessels was counted per 0.22 mm2.3, 38

Assessment of bone disease

Tibiae were dissected free of soft tissues and scanned using a micro-CT scanner (Skyscan, Kontich, Belgium) at 50 kV and 100 mA with a pixel size of 4.3 mum. Scanned images were reconstructed and analyzed using the Skyscan Recon and Skyscan CT analysis software, respectively. Trabecular volume, thickness and number, and osteolytic lesions were determined in a 1-mm volume of tissue, starting at 0.2 mm from the growth plate. The right tibia was then decalcified, embedded in paraffin and 3-mum sections cut, and reacted for tartrate-resistant acid phosphatase (TRAP) to identify osteoclasts. The number of osteoclasts (per mm) and the surface occupied by these cells (%) was measured using the Osteomeasure bone histomorphometry software (Osteometrics, Decatur, GA, USA). The left tibia was fixed in 70% ethanol, embedded in LRWhite (Agar Scientific, Stansted, UK) and 3 mum sections cut, and mounted unstained for examination under UV illumination using a DMRB fluorescence microscope (Leica, Cambridge, UK). The proportion of bone surface labeled with calcein (mineralizing surface, MS, %) and the distance between the two labels was measured using the Osteomeasure software. Mineral apposition rate (mum/day) and bone formation rate (BFR, mm2/mm3/day) were calculated from the length of calcein-labeled surface, the separation between labels and the bone surface length.

Statistical analysis

All in vitro experiments were repeated in triplicates. Values represent the meansplusminuss.d. The significance between variables from in vivo and in vitro experiments was determined using Mann–Whitney test. The results were considered significant if P<0.05.

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Results

5T33MMvt cells are more sensitive for histone acetylation and inhibition of proliferation by JNJ-26481585 compared to BM endothelial STR-10 cells.

To understand the selectivity of JNJ-26481585 of normal vs cancer cells, we compared 5T33MMvt cells and BM endothelial STR-10 cells, both treated with different concentrations of JNJ-26481585, by assessing histone acetylation and proliferation.

We first examined whether JNJ-26481585 induces histone hyperacetylation in vitro through analysis of H3 and H4 acetylation by western blotting. After 24 h incubation with different concentrations of JNJ-26481585, we observed in the 5T33MMvt cells, an induction of H3 hyperacetylation starting at 1 nM concentrations, which increased with higher concentrations of JNJ-26481585, whereas in the STR-10 cell line, induction of H3 hyperacetylation started at 10 nM. Further in the 5T33MMvt cells and in the STR-10 cells, we could show that H4 hyperacetylation started at 30 nM and was markedly increased in the 5T33MMvt cells at 100 nM.

To assess the effect of JNJ-26481585 on the proliferation of 5T33MMvt cells and STR-10 cells, we first used an MTS assay. As shown in Figure 1b, treatment with JNJ-26481585 for 72 h reduced cell growth in a concentration-dependent manner, with an IC50 of 2.43 nM for 5T33MMvt cells and with an IC50 of 52.92 nM for STR-10 cells.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Effect of JNJ-26481585 on histone acetylation and proliferation in 5T33MMvt and STR-10 cells in vitro. (a) Western blots represent the acetylation status of histone 3 (H3) and H4 in 5T33MMvt and STR-10 cell incubated with indicated concentration of JNJ-26481585 for 24 h. Equivalent amounts of lysates were immunoblotted with anti-Ac-H3 or anti-Ac-H4 and reblotted with beta-actin to confirm equal loading. One experiment representative of three is shown. (b) Anti-proliferative activity of JNJ-2681585 on 5T33MMvt and STR-10 cells incubated with JNJ26481585 at 3 times 10-10, 10-10; 3 times 10-9, 10-9; 3 times 10-8, 10-8; 3 times 10-7, 10-7; 3 times 10-6, 10-6 M. After 3 days incubation, the number of viable cells was assessed by a standard 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS) colorimetric assay.

Full figure and legend (70K)

JNJ-26481585 induces apoptosis and cell cycle arrest in 5T33MMvt cells

To elucidate whether the reduction in proliferation of the 5T33MMvt cells is due to cell cycle arrest and/or apoptosis, FACS analysis was carried out after 24 h and 72 h incubation. Treatment of JNJ-26481585 at different concentrations for 24 h had no significant effect on the number of early apoptotic cells (Annexin V+/7-AAD – stained cells) and necrotic cells (7-AAD positive stained cells), whereas the necrotic-late apoptotic cells (Annexin V+/7-AAD + stained cells) increased after 24 h from 30 nM onwards, although not significant (Figure 2a). After 72 h incubation, treatment of JNJ-26481585 significantly increased the number of necrotic-late apoptotic cells, whereas no significant effect could be shown in the number of early apoptotic and necrotic cells (Figure 2b).

Figure 2.
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Effect of JNJ-26481585 on apoptosis and cell cycle in the 5T33MMvt cells. Apoptosis of the 5T33MMvt cells was determined after 24 h (a) and 72 h (b) incubation with an Annexin V/7-amino-actinomycin D (7-AAD) staining, and cell cycle was evaluated after 24 h (c) and 72 h (d) incubation with propidium iodide staining. Both stainings were followed by FACS analysis. Mean valuesplusminuss.d. for three independent experiments are shown. Asterisks indicate significant difference from control (*P<0.05).

Full figure and legend (117K)

As indicated in Figure 2c, after 24 h incubation a significant G1 arrest could be observed at 10 and 30 nM, whereas at 100 nM the sub-G0 phase increased significantly, which is an indicator of cell death. After 72 h incubation, no cell cycle arrest could be shown. The S-phase (DNA synthesis), G0-G1 phase and the G2-M (Mitosis) phase decreased, and the sub-G0 phase increased significantly in a concentration-dependent manner after 72 h incubation (Figure 2d).

Downstream effects of JNJ-26481585 in 5T33MMvt and STR-10 cells

Western blots analysis was carried out to study downstream effects of JNJ-26481585 with the emphasis on apoptosis, cell cycle arrest and HDAC6 inhibition in 5T33MMvt cells, and BM STR-10 cells treated with different concentrations of JNJ-26481585 for 24 h (Figure 3).

Figure 3.
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Downstream effects of JNJ-26481585 on apoptosis, cell cycle inhibitors and aggresome function in the 5T33MMvt and STR-10 cells. Cells were cultured for 24 h with indicated concentrations of JNJ-26481585.60 Whole-cell lysates were subjected to western blotting with primary antibodies against caspase 8, 9 and 3, Poly ADP ribose polymerase (PARP), p21Waf1, p27Kip1, Ac-alpha-tubulin, alpha-tubulin and beta-actin. Western blots are shown representative for three independent experiments.

Full figure and legend (131K)

To understand the mechanism of action of JNJ-26481585-induced apoptosis in 5T33MMvt cells, we examined caspase activation and further compared it with non-malignant cells using the STR-10 cell line. In the 5T33MMvt cells caspase-8 was activated at 10 nM, whereas caspase-9 and -3 were activated at 30 nM. In contrast, only caspase-9 cleavage could be observed in STR-10 cells at the highest concentration tested (100 nM). Further, PARP cleavage started in 5T33MMvt cells at 30 nM and in STR-10 cells at 100 nM.

As several reports have shown that cell cycle inhibitors, p21Waf1 and p27Kip1, play a key role in cell cycle arrest, we evaluated the levels of p21Waf1 and p27Kip1. The p21Waf1 level increased in 5T33MMvt cells, as well as in the STR-10 cells, when treated with JNJ-26481585 at 30 nM or higher. At 30 nM, a slight increase in the p27Kip1 level could be observed in the 5T33MMvt cells, but this was not relevant. Furthermore, at 100 nM the p27Kip1 level decreased. In the STR-10 cells, the p27Kip1 level did not alter when cells were treated with JNJ-26481585 in the concentration range tested.

Interrupting the aggresomal protein degradation system by inhibiting HDAC6, which deacetylates alpha-tubulin and thereby regulates transport of protein aggregates, could also be involved in HDACi-induced apoptosis. Therefore, we analyzed the acetylation status of alpha-tubulin in 5T33MMvt and STR-10 cells treated with JNJ-26481585. Induction of alpha-tubulin acetylation could be observed in the 5T33MMvt cells treated from 30 nM onwards, whereas in the STR-10 cells induced alpha-tubulin acetylation only at 100 nM treatment. Further we observed in the 5T33MMvt cells a decrease in alpha-tubulin when treated with 100 nM JNJ-26481585. This observation is in line with the data reported by Vávrová et al.,40 describing a markedly decrease of tubulin in leukemia cells treated with the HDACi valproic acid.

The effect of JNJ-26481585 on tumor burden in vivo in a prophylactic and therapeutic setting

We then evaluated whether JNJ-26481585 had anti-MM properties in vivo using the 5T33MM and 5T2MM model. In a prophylactic setting, 5T33MM mice were treated with 20 mg/kg of JNJ-26481585 every other day s.c. or with vehicle, from the day after the injection of 5T33MM cells. As shown in Figure 4a and b, treatment resulted in a dramatic reduction of MM disease, as shown by a statistically significant reduction of serum M component (to a level that was undetectable using serum electrophoresis) and BM plasmacytosis (P<0.05). In a second series of in vivo experiments, the 5T2MM model was used in a therapeutic setting to determine whether JNJ-26481585 had similar anti-MM effects as seen in a prophylactic setting. In this setting, mice were treated once the MM disease was established, as determined by an increase of serum paraprotein, 8 weeks after injection of the 5T2MM cells. Treatment resulted in a statistically significant decrease in tumor burden as compared with the vehicle group (P<0.05) (Figures 4c and d).

Figure 4.
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Prophylactic effect and therapeutic effect of JNJ-26481585 (20 mg/kg, every other day, subcutaneously) in the 5T33MM model and 5T2MM model, respectively, in vivo. Serum paraprotein concentrations from 5T33MM mice (a) and 5T2MM mice (c) as determined by serum electrophoresis. Bone marrow plasmacytosis of 5T33MM mice (b) and 5T2MM mice (d). Mean values, plusminuss.d. and three groups of 10 mice (naive, 5T33MM+vehicle, 5T33MM+JNJ-26481585) are shown (*P<0.05).

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The effect of JNJ-26481585 on angiogenesis in vivo in a prophylactic and therapeutic setting

In both prophylactic and therapeutic setting, the MVD was significantly increased in tumor-bearing mice compared with naive mice, which confirms the stimulating effect of myeloma cells on angiogenesis in vivo, reported earlier.38 Treatment with JNJ-26481585 decreased the newly formed blood vessels by 50 and 35% compared with vehicle group in the 5T33MM and 5T2MM model, respectively (5T33MM- naive: 19.5plusminus2.5/vehicle: 25.8plusminus1.8/JNJ-85: 22.6plusminus2.6, 5T2MM- naive: 15.7plusminus1.4/vehicle: 24.2plusminus1.8/JNJ-85: 21.2plusminus2.9 number of blood vessels, P<0.05).

The effect of JNJ-26481585 on the MM-related bone disease in the 5T2MM model

The development of the MM-related bone disease in 5T2MM-bearing mice (vehicle control group) was characterized by an increase of osteolytic lesions (Figures 5a and b; P<0.05), a decrease in percentage trabecular bone volume (Figure 5c; P<0.05), a decrease in trabecular number (Figure 5d; P<0.05) and an increase in the percentage osteoclasts (Figure 5e; P<0.05). In addition, we observed a decrease in mineralization surface (MS) and BFR in the 5T2MM-bearing mice compared with naive mice, which was significant for MS but not for BFR (MS—19.19plusminus3.982 to 5.106plusminus0.5563%, P<0.05; BFR—0.3122plusminus0.09358 to 0.04515plusminus0.02777 mm2/mm3/day, P>0.05).

Figure 5.
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Effect of JNJ-26481585 (20 mg/kg, twice weekly, subcutaneously) on the development of bone disease and osteoclast number in 5T2MM-bearing mice. (a) Images obtained by computer tomography of the tibia (A1) naive, (A2) 5T2MM, (A3) 5T2MM-bearing mice treated with JNJ-26481585. (b) Osteolytic lesions count. (c) Percentage bone volume/trabecular. (d) Trabecular number obtained by hematoxylin-eosin (HE)-stained paraffin sections and (e) number of tartrate-resistant acid phosphatase (TRAP)+ cells. Mean values, plusminuss.d. and three groups of 10 mice (naive, 5T2MM+vehicle, 5T2MM+JNJ-26481585) are shown (*P<0.05).

Full figure and legend (167K)

As shown in Figure 5a, micro-CT images indicated that formation of the osteolytic lesions was dramatically reduced by 70% in mice treated with JNJ-26481585 (Figures 5a and b. P<0.05). Furthermore, trabecular bone volume, trabecular number and the percentage osteoclasts in mice treated with JNJ-26481585 were significantly reduced when compared with vehicle-treated mice, and returned to similar level to that seen in naive non-tumor-bearing mice (Figures 5c–e; P<0.05). We also observed in mice treated with JNJ-26481585 that the mineralizing surface and BFR were increased compared with the 5T2MM vehicle control group (MS—5.106plusminus0.5563 to 16.80plusminus4.101%; BFR—0.09146plusminus0.04409 mm2/mm3/day BFR vs 0.2098plusminus0.08044 mm2/mm3/day BFR; P>0.05), although this was not statistically significant.

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Discussion

Several HDACi have been extensively studied in neoplastic disorders for their profound anti-proliferative and pro-apoptotic properties.16 In this study, we evaluated a novel 'second-generation' pyrimidyl-hydroxamate-based pan-HDAC inhibitor, JNJ-26481585, in vitro using the murine 5T33MMvt cell line and the BM endothelial cell line, STR-10, and in vivo using the 5T2MM and 5T33MM models.34 In vitro, JNJ-26481585 showed anti-proliferative activity against the 5T33MMvt cells at low nanomolar concentrations, which is in line with results described by Janine Arts et al.41 (submitted), demonstrating that JNJ-26481585 has high potency towards all class I HDAC enzymes (IC50 values of 0.11, 0.33 and 4.8 nM, for HDAC 1, 2 and 3, respectively). This suggests that hydroxamate-based HDAC inhibitors, JNJ-26481585 together with LBH589,28 have higher anti-myeloma activity in vitro, working at nanomolar concentrations compared with others such as suberoylanilide hydroxamic acid25 and NVP-LAQ824,42 which are only effective at micromolar concentrations. By comparing the JNJ-26481585-induced histone acetylation and anti-proliferative effects in 5T33MMvt cells and STR-10 cells, we could conclude that the myeloma cells were more sensitive to the HDACi than BM endothelial cells. At 3 and 100 nM, a clear induction of histone 3 and histone 4 hyperacetylation, respectively, could be observed in the 5T33MMvt cells, whereas in the STR-10 cells at 30 nM only a modest increase of histone 3 and 4 acetylation could be shown. Furthermore, the IC50 of the BM endothelial cells was twenty times higher than that of the 5T33MMvt cells. These data suggest the specificity of JNJ-26481585 towards cancer cells.

At earlier time points (24 h) a G1 arrest could be observed using 10–30 nM of JNJ-26481585. At these concentrations western blot analysis indicated an upregulation in the level of the cyclin-dependent kinase inhibitor, p21Waf1, having important roles in blocking cell cycle in the G1 phase. This is in line with other studies, demonstrating that upregulation of p21Waf1 is a hallmark for the HDACi-induced cell cycle arrest.25, 26, 28, 43, 44, 45 This corresponds with the anti-proliferative activity of JNJ-26481585. However at 72 h incubation, cell cycle arrest is lost, most likely because of the large increase of apoptotic cells. Especially because even at 24 h the apoptotic pathways are already activated, as we demonstrated by western blotting for cleaved caspase-8, -9 and –3, which indicates that JNJ-26481585 activates the extrinsic as well as the intrinsic apoptotic pathway. Induction of PARP cleavage (a substrate of caspase-3 and a biochemical marker of the rate of apoptosis) could be shown using a JNJ-26481585 concentration of 30 nM and more.

Interestingly, JNJ-26481585 also induced hyperacetylation of alpha-tubulin at 10 nM. This means that JNJ-26481585 inhibits HDAC6, which contains a tubulin deacetylase active site. It has already been reported that HDAC6 could be targeted by tubacin, a specific HDAC6 inhibitor,46 or by hydroxamate HDACi such as suberoylanilide hydroxamic acid or LBH589, resulting in hyperacetylation of alpha-tubulin, accumulation of polyubuitinated proteins and induction of apoptosis.47, 48 In addition, further investigations have shown that HDAC6 plays a key role in the synergism between HDACi and bortezomib through aggresome–proteasome deregulation.47, 48, 49

Treating BM endothelial cells with JNJ-26481585 at 100 nM also activated the extrinsic and intrinsic apoptotic pathways, increased the p21Waf1 level and induced hyperacetyltion of alpha-tubulin, which is similar to what we saw in the myeloma cells when they were treated at much lower concentrations. This indicates that the JNJ-26481585 targets similar pathways in BM endothelial cells as in myeloma cells, but with a lower sensitivity towards the non-malignant cells.

To evaluate the potential value of JNJ-26481585 in the development of the MM disease, the in vivo anti-tumor activity of JNJ-26481585 was first evaluated in a prophylactic setting using the 5T33MM murine MM model. Treatment resulted in 80% reduction in tumor burden and complete reduction of serum paraprotein. As treatment started the day after the 5T33MM cells were injected, JNJ-26481585 could not have any influence on the MM cells during the selective homing towards the BM. Simultaneous injection of MM cells and JNJ-26481585 would potentially result in less MM cell occupation in the BM due to HDACi-induced apoptosis during the homing process. Normally this selective homing of the 5TMM cells towards the BM takes up to 16 h.50 The 5T2MM model was used in a therapeutic setting starting with an established MM disease, as evidenced by the presence of MM cells in the BM, detectable serum paraprotein and activation of angiogenesis and bone disease.51, 52 In this setting, JNJ-26481585 also affected the development of MM disease. Tumor burden in 5T2MM-bearing mice treated with JNJ-26481585 was reduced in a similar manner to that seen in the prophylactic setting using the 5T33MM model. This suggests that JNJ-26481585 inhibits MM cell survival in vivo, even in an established setting, where MM cells are actively interacting with their BM microenvironment from which they receive protective signals. The ability to overcome the protective stimulatory effect of the BM microenvironment can be due to the downregulation of cytokine receptors like CD138 (syndecan-1), IGF-1R, IL-6R and CXCR4 that trigger MM proliferation, survival and/or migration.44 We assume no direct effects on the survival of stromal cells, as normal cells are far less sensitive to HDACi than malignant cells.53, 54 On the other hand, we cannot exclude the possibility that the HDACi directly affects cytokine production of the stromal cells in the BM microenvironment. It has been shown earlier in vitro that HDACi do inhibit the production of IL-6,25 VEGF and interferon-gamma55 by stromal cells at concentrations which are cytotoxic for MM cells, but not for the stromal cells.

In the 5T2MM-bearing mice (vehicle treated), we further observed an induction of osteolysis similar to the human disease, as a consequence of the interactions between the MM cells and the BM microenvironment. When the 5T2MM-bearing mice were treated with JNJ-26481585, there was a reduction in osteolytic lesions and percentage osteoclasts, and an increase in the trabecular bone volume, trabecular number, BFR and mineralization surface, indicating that JNJ-26481585 prevents bone loss in 5T2MM-bearing mice. In contrast to earlier studies, which have shown that some HDACi promote osteoblast differentiation and maturation in vitro, we observed that the percentage bone surface covered by osteoblasts, identified on the basic characteristic morphology, was not different to the vehicle control group (results not shown).

This reduction in MM-related bone disease is likely to be caused indirectly by the reduction in tumor load, as most of the bone parameters reached the level of naive non-tumor-bearing mice. However, the possibility of directly affecting osteoclasts cannot be excluded. Earlier studies have shown that broad-spectrum HDACi decrease survival and maturation of osteoclasts by inducing p21 expression without alterations to the p27 level,23 blocking the RANKL signalling pathway or by increasing the mRNA level of IFN-beta expression, an inhibitor of the osteoclastogenesis.56 Furthermore, the HDACi, PDX101, in combination with bortezomib synergistically inhibits osteoclast formation in human BM cultures. The underlying mechanism of this synergism in vitro in osteoclast cells remains to be elucidated.57 Bortezomib, a potent and reversible proteasome inhibitor, is currently approved as first-line treatment in MM patients. Pre-clinical and clinical data have shown that bortezomib has a positive effect on bone remodeling by inhibiting osteoclast formation and stimulating osteoblast differentiation.58, 59, 60, 61 As bortezomib influence osteoclasts as well as osteoblasts and targets different mechanisms as HDACi, combining bortezomib with JNJ-26481585 would be a promising therapeutic strategy to improve the reducing effects of bortezomib on the development of MM-related bone disease.

As 5T33MM and 5T2MM cells are able to induce angiogenesis in vivo by secreting several pro-angiogenic factors, we were able to evaluate the anti-angiogenic properties of JNJ-26481585 in both models.38 We observed in both prophylactic and therapeutic treatment with JNJ-26481585 a similar significant reduction in the MVD, suggesting that JNJ-26481585 inhibits tumor-induced angiogenesis irrespective of whether the MM disease is already established in the BM or not. As some HDAC inhibitors have the ability to reduce VEGF production, one of the most prominent angiogenic factors in MM, it is possible that JNJ-26481585 not only reduces the MVD through the inhibition of the tumor burden, but also by reducing the VEGF secretion from the MM cells as well as the stromal cells.43, 55, 62, 63, 64, 65 On the other hand, JNJ-26481585 might also affect MVD directly as several HDAC inhibitors, such as valproic acid, trichostatin, NVP-LAQ824 and LBH589, have shown anti-angiogenic properties in vitro and in vivo by affecting endothelial cell survival and function directly. Several in vitro assays showed that HDACi inhibit endothelial cell proliferation, induce cell cycle arrest and inhibit endothelial cell function by decreasing endothelial cell migration, invasion and tube formation. The ability of HDACi to inhibit endothelial cell signalling by, for example, inhibiting the AKT and Erk 1/2 phosphorylation and to modulate the gene expression of some angiogenesis-related genes like survivin, could be responsible for the inhibitory effect on survival and activity of the endothelial cells, but the underlying mechanisms of these modulations remain to be elucidated.19, 20, 65

In conclusion, we have shown that JNJ-26481585 has a potent anti-myeloma activity in vitro and in vivo. Higher concentrations are needed to inhibit proliferation and to induce apoptosis in BM endothelial cells, suggesting that JNJ-26481585 exerts less toxic effects towards the BM microenvironment than towards myeloma cells, and the observed decrease in angiogenesis is likely a result of decreased tumor burden. This makes JNJ-26481585 a promising novel anti-MM agent, and highlights the importance of its further clinical evaluation in MM.

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Acknowledgements

We thank A Willems and C Seynaeve for expert technical assistance, Professor F Gorus (AZ VUB, Brussels) for serum paraprotein analysis and Dawn Emerson for the bone analysis. The work was financially supported by the Stichting tegen Kanker, the Onderzoeksraad Vrije Universiteit Brussel (OZR-VUB; GOA48), FWO-Vlaanderen and the Leukeamia Research Fund. Eline Menu is a Postdoctoral Fellow and Isabelle Vande Broek is a Senior Clinical Investigator of the Research Foundation Flanders (FWO).