Les Coulton

Inhibiting Dickkopf-1 (Dkk1) removes suppression of bone formation and prevents the development of osteolytic bone disease in multiple myeloma.

Multiple myeloma (MM) is associated with the development of osteolytic bone disease, mediated by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf‐1 (Dkk1), a soluble inhibitor of wingless/int (Wnt) signaling and osteoblastogenesis, is elevated in patients with MM and correlates with osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk1 on the development of osteolytic lesions in the 5T2MM murine model of myeloma. We showed that Dkk1 is expressed by murine 5T2MM myeloma cells. Injection of 5T2MM cells into C57BL/KaLwRij mice resulted in the development of osteolytic bone lesions (p < 0.05), mediated by increased osteoclast numbers (p < 0.001) and a decrease in osteoblast numbers (p < 0.001) and mineralizing surface (p < 0.05). Mice bearing 5T2MM cells were treated with an anti‐Dkk1 antibody (BHQ880, 10 mg/kg, IV, twice weekly for 4 wk) from time of paraprotein detection. Anti‐Dkk1 treatment prevented 5T2MM‐induced suppression of osteoblast numbers (p < 0.001) and surface (p < 0.001). Treatment increased mineralizing surface by 28% and bone formation rate by 25%; however, there was no change in mineral apposition rate. Inhibiting Dkk1 had no effect on osteoclast numbers. μCT analysis showed that anti‐Dkk1 treatment significantly protected against 5T2MM‐induced trabecular bone loss (p < 0.05) and reduced the development of osteolytic bone lesions (p < 0.05). Treatment had no significant effect on tumor burden. These data suggest that inhibiting Dkk1 prevents the suppression of bone formation and in doing so is effective in preventing the development of osteolytic bone disease in myeloma, offering an effective therapeutic approach to treating this clinically important aspect of myeloma.

Inhibiting activin-A signaling stimulates bone formation and prevents cancer-induced bone destruction in vivo.

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma‐induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer.

Role of CCR1 and CCR5 in homing and growth of multiple myeloma and in the development of osteolytic lesions: a study in the 5TMM model

Multiple myeloma (MM) is a plasma cell malignancy, characterized by the localization of the MM cells in the bone marrow (BM), where they proliferate and induce osteolysis. The MM cells first need to home or migrate to the BM to receive necessary survival signals. In this work, we studied the role of CCR1 and CCR5, two known chemokine receptors, in both chemotaxis and osteolysis in the experimental 5TMM mouse model. A CCR1–specific (BX471) and a CCR5–specific (TAK779) antagonist were used to identify the function of both receptors. We could detect by RT-PCR and flow cytometric analyses the expression of both CCR1 and CCR5 on the cells and their major ligand, macrophage inflammatory protein 1α (MIP1α) could be detected by ELISA. In vitro migration assays showed that MIP1α induced a 2-fold increase in migration of 5TMM cells, which could only be blocked by TAK779. In vivo homing kinetics showed a 30% inhibition in BM homing when 5TMM cells were pre-treated with TAK779. We found, in vitro, that both inhibitors were able to reduce osteoclastogenesis and osteoclastic resorption. In vivo end-term treatment of 5T2MM mice with BX471 resulted in a reduction of the osteolytic lesions by 40%; while TAK779 treatment led to a 20% decrease in lesions. Furthermore, assessment of the microvessel density demonstrated a role for both receptors in MM induced angiogenesis. These data demonstrate the differential role of CCR1 and CCR5 in MM chemotaxis and MM associated osteolysis and angiogenesis.

Targeting the IGF-1R using picropodophyllin in the therapeutical 5T2MM mouse model of multiple myeloma: beneficial effects on tumor growth, angiogenesis, bone disease and survival.

During the last decade, a central role for insulin‐like growth factor 1 (IGF‐1) in the pathophysiology of multiple myeloma (MM) has been well established. IGF‐I provided by the tumor–microenvironment interaction may directly and indirectly facilitate the migration, survival and expansion of the MM cells in the bone marrow (BM). The inhibition of the IGF‐1R‐mediated signaling pathway has recently been suggested to be a possible new therapeutic principle in MM. Using the mouse 5T2MM model, we now demonstrate that targeting the IGF‐1R using picropodophyllin (PPP) in a therapeutical setting not only has strong antitumor activity on the established MM tumor but also influences the BM microenvironment by inhibiting angiogenesis and bone disease, having a profound effect on the survival of the mice. At therapeutically achievable concentrations of PPP, the average survival was 180 days for the PPP‐treated mice as compared to 100 days for vehicle‐treated mice. PPP used as single drug treatment in the 5T2MM model resulted in a decrease of tumor burden by 65% while the paraprotein concentrations were reduced by 75%. This decrease was associated with a significant inhibition of tumor‐associated angiogenesis and osteolysis. The present studies on the biological effects of PPP in the 5T2MM model constitute an important experimental platform for future therapeutic implementation.

Bortezomib Alone or in Combination with the Histone Deacetylase Inhibitor JNJ-26481585: Effect on Myeloma Bone Disease in the 5T2MM Murine Model of Myeloma

The proteasome inhibitor bortezomib (Velcade) is currently approved as second-line treatment of multiple myeloma (MM). MM-related bone disease is one of the most debilitating complications of MM. Besides supportive care with biphosphonates, which have proven efficacy in reducing and delaying skeletal-related events, there is no specific treatment of lytic bone lesions. The present study investigated the effect of bortezomib alone or in combination with a hydroxamate-based histone deacetylase inhibitor, JNJ-26481585 on tumor burden, and MM bone disease in the 5T2MM model. Injection of 5T2MM cells into C57Bl/KaLwRij mice resulted in MM bone disease, characterized by an increase in the percentage osteoclasts, a decrease in osteoblasts, trabecular bone volume, trabecular number, and the development of bone lesions. Treatment of 5T2MM-bearing mice with bortezomib significantly reduced tumor burden, angiogenesis, and MM bone disease. More importantly, the combination of bortezomib with JNJ-26481585 resulted in a more pronounced reduction of osteoclasts and increase of osteoblasts, trabecular bone volume, and trabecular number compared with bortezomib as single agent. These data suggest that bortezomib has bone remodeling properties that can be improved in combination with low dose JNJ-26481585. The study indicates that this combination therapy could be a useful strategy for the treatment of MM patients, especially in those patients with skeletal complications.

Effect of 900 MHz Electromagnetic Fields on Nonthermal Induction of Heat-Shock Proteins in Human Leukocytes

Abstract Lim, H. B., Cook, G. G., Barker, A. T. and Coulton, L. A. Effect of 900 MHz Electromagnetic Fields on Nonthermal Induction of Heat-Shock Proteins in Human Leukocytes. Radiat. Res. 163, 45–52 (2005). Despite many studies, the evidence as to whether radiofrequency fields are detrimental to health remains controversial, and the debate continues. Cells respond to some abnormal physiological conditions by producing cytoprotective heat-shock (or stress) proteins. The aim of this study was to determine whether exposure to mobile phone-type radiation causes a nonthermal stress response in human leukocytes. Human peripheral blood was sham-exposed or exposed to 900 MHz fields (continuous-wave or GSM-modulated signal) at three average specific absorption rates (0.4, 2.0 and 3.6 W/kg) for different durations (20 min, 1 h and 4 h) in a calibrated TEM cell placed in an incubator to give well-controlled atmospheric conditions at 37°C and 95% air/5% CO2. Positive (heat-stressed at 42°C) and negative (kept at 37°C) control groups were incubated simultaneously in the same incubator. Heat caused an increase in the number of cells expressing stress proteins (HSP70, HSP27), measured using flow cytometry, and this increase was dependent on time. However, no statistically significant difference was detected in the number of cells expressing stress proteins after RF-field exposure. These results suggest that mobile phone-type radiation is not a stressor of normal human lymphocytes and monocytes, in contrast to mild heating.

Effect of 50 Hz Electromagnetic Fields on the Induction of Heat-Shock Protein Gene Expression in Human Leukocytes

Abstract Coulton, L. A., Harris, P. A., Barker, A. T. and Pockley, A. G. Effect of 50 Hz Electromagnetic Fields on the Induction of Heat-Shock Protein Gene Expression in Human Leukocytes. Radiat. Res. 161, 430–434 (2004). Although evidence is controversial, exposure to environmental power-frequency magnetic fields is of public concern. Cells respond to some abnormal physiological conditions by producing cytoprotective heat-shock (or stress) proteins. In this study, we determined whether exposure to power-frequency magnetic fields in the range 0–100 μT rms either alone or concomitant with mild heating induced heat-shock protein gene expression in human leukocytes, and we compared this response to that induced by heat alone. Samples of human peripheral blood were simultaneously exposed to a range of magnetic-field amplitudes using a regimen that was designed to allow field effects to be distinguished from possible artifacts due to the position of the samples in the exposure system. Power-frequency magnetic-field exposure for 4 h at 37°C had no detectable effect on expression of the genes encoding HSP27, HSP70A or HSP70B, as determined using reverse transcriptase-PCR, whereas 2 h at 42°C elicited 10-, 5- and 12-fold increases, respectively, in the expression of these genes. Gene expression in cells exposed to power-frequency magnetic fields at 40°C was not increased compared to cells incubated at 40°C without field exposure. These findings and the extant literature suggest that power-frequency electromagnetic fields are not a universal stressor, in contrast to physical agents such as heat.

A new xenograft model of myeloma bone disease demonstrating the efficacy of human mesenchymal stem cells expressing osteoprotegerin by lentiviral gene transfer

We describe a new model of myeloma bone disease in which β2m NOD/SCID mice injected with KMS-12-BM cells develop medullary disease after tail vein administration. Micro-computed tomography analysis demonstrated significant bone loss in the tibiae and vertebrae of diseased animals compared to controls, with loss of cortical bone (P<0.01), as well as trabecular bone volume, thickness and number (P<0.05 for all). Bone marrow of diseased animals demonstrated an increase in osteoclasts (P<0.01) and reduction in osteoblasts (P<0.01) compared to control animals. Both bone loss and osteoclast increase correlated with the degree of disease involvement. Mesenchymal stem cells (MSCs) were lentivirally transduced to express human osteoprotegerin (hOPG). Systemic administration of OPG expressing MSC reduced osteoclast activation (P<0.01) and trabecular bone loss in the vertebrae (P<0.05) and tibiae of diseased animals, to levels comparable to non-diseased controls. Because of its predominantly medullary involvement and quantifiable parameters of bone disease, the KMS-12-BM xenogeneic model provides unique opportunities to test therapies targeted at the bone marrow microenvironment.

Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix.

Inhibition of p38α Mitogen-Activated Protein Kinase Prevents the Development of Osteolytic Bone Disease, Reduces Tumor Burden, and Increases Survival in Murine Models of Multiple Myeloma

The bone microenvironment plays a critical role in supporting the growth and survival of multiple myeloma as well as in the development of osteolytic bone disease. Signaling through p38alpha mitogen-activated protein kinase (MAPK) mediates synthesis of multiple myeloma cell growth factors, and its inhibition reduces proliferation in vitro. However, it is unclear whether targeting p38alpha MAPK prevents multiple myeloma growth and the development of bone disease in vivo. In this study, we determined whether SCIO-469, a selective p38alpha MAPK inhibitor, inhibits multiple myeloma growth and prevents bone disease in the 5T2MM and 5T33MM models. SCIO-469 decreased constitutive p38alpha MAPK phosphorylation of both 5T2MM and 5T33MM cells in vitro. This was associated with decreased DNA synthesis and an induction of apoptosis when the cells were cultured with bone marrow stromal cells. Treatment of C57Bl/KaLwRij mice bearing 5T33MM cells with SCIO-469 inhibited p38alpha MAPK phosphorylation and was associated with a significant decrease in serum paraprotein, an almost complete reduction in tumor cells in the bone marrow, a decrease in angiogenesis, and a significant increase in disease-free survival. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in myeloma bone disease characterized by increased osteoclast occupation of the bone surface, reduced cancellous bone, and the development of osteolytic bone lesions. Treatment of 5T2MM-injected mice with SCIO-469 reduced this development of bone disease. Together, these data show that targeting p38alpha MAPK with SCIO-469 decreases myeloma burden in vivo, in addition to preventing the development of myeloma bone disease.