Development Of Myeloma Bone Disease Research Articles

Inhibition of p38α MAPK Reduces Tumor Burden, Prevents the Development of Myeloma Bone Disease, and Increases Survival in the 5T2 and 5T33 Murine Models of Myeloma.

The bone microenvironment plays a critical role in supporting the growth and survival of myeloma cells and the development of osteolytic bone disease. Signalling through p38 α MAPK mediates synthesis of myeloma cell survival factors by stromal cells; whereas, inhibiting p38 α MAPK reduces myeloma cell proliferation and inhibits osteoclast formation in vitro. However, it is unclear whether p38 α MAPK inhibition will prevent the growth and survival of myeloma cells and the bone disease in vivo. The aim of this study was to determine whether SCIO-469, a selective p38 α MAPK inhibitor, would inhibit myeloma growth and prevent the development of bone disease in the 5TMM syngeneic models of myeloma.Treatment of 5TMM cells, in vitro, with SCIO-469 resulted in a clear inhibition of p38 phosphorylation, as assessed by Western blotting and an inhibition up to 35% of stromal cell induced 5T33MM proliferation.Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in the growth of myeloma in bone and the development of bone disease characterized by increased osteoclast surface (p<0.05), a reduction in cancellous bone (p<0.01) and the presence of osteolytic bone lesions on x-ray (p<0.01). Treatment of 5T2MM-bearing mice with SCIO-469 (150mg/kg in the diet, therapeutical treatment from paraprotein detection) resulted in a 42% decrease in serum paraprotein and prevented development of osteolytic lesions (p<0.01). Injection of 5T33MM cells into C57Bl/KaLwRij mice also resulted in the development of myeloma but not associated bone disease. Treatment of 5T33MM-bearing mice from the time of tumor cell injection with SCIO-469 resulted in a decrease in serum paraprotein (8.8+/−1.4g/dl to 0.04+/− 0.03g/dl, p<0.001) and a reduction in the proportion of tumor cells in the bone marrow (67 +/− 8.1% to 1.09 +/− 0.58%, p<0.001). Kaplan-Meier analysis demonstrated an increase in disease-free survival (veh=27.5 days vs 96 days, p<0.001) after treatment of the mice with SCIO-469.These data demonstrate that targeting p38 α MAPK with SCIO-469 is associated with an anti-myeloma effect, which indirectly prevents the development of myeloma bone disease.

Toward Defining the Functional Relevance of Wnt Signaling in Osteoclasts in Multiple Myeloma.

Disruption of Wnt signaling is thought to play a central role in the development of myeloma bone disease. Although Wnt signaling in osteoblasts (OB) is essential for their differentiation and Wnt signaling in OB indirectly regulates osteoclast (OC) development through the regulation of both OPG and RANKL, little is known about the direct role of Wnt signaling in osteoclastogenesis. In this study, we sought to characterize the Wnt signaling pathway and its functional role in OC formation using the macrophage cell line Raw 264.7, which can be induced to differentiate into OC, as well as human primal progenitor osteoclast cells isolated from bone marrow of normal donors and patients with MM. We first analyzed the pattern of Wnt signaling components in these cells by RT-PCR and sequence analysis. High level of expression of Wnt receptors Frizzled (Fz) Fz2, 3, 4, 5, 7, 8 and 9, and co-receptors LRP5, and LRP6, and transcription factors TCF1, TCF3, and LEF1 was observed in 10 of 10 OC cells from MM patients and 4 of 4 OC from normal donors and Raw264.7. Wnt3a conditioned medium and recombinant Wnt3a induced increased levels of total and un-phosphorylated beta-catenin in all cells. Pretreatment of OC cells with sFRP1 decreased Wnt3a induced increased beta-catenin. Pretreatment with DKK1, a specific inhibitor of the canonical Wnt pathway, blocked Wnt3a induced stabilization of beta-catenin. Additionally, the GSK3beta inhibitor lithium chloride induced stabilization of beta-catenin in OC and Raw264.7 cells in dose-dependent fashion. Wnt-3a induced TCF/LEF transcriptional activity in Raw264.7 cells transfected with TOPflash compared with Raw264.7 transfected with FOPflash, which possess mutant sequence for binding beta-catenin. These results suggest that Wnt signaling functions in primal MM OC cells, as well as OC cell lines. Finally, we sought to determine the biological function of Wnt signaling in OC cells. The results showed that Wnt-3a did not affect the proliferation and survival of OC cells, nor increased formation of TRAP positive cells. Wnt3a did not synergize with RANKL and M-CSF in induction of OC differentiation. Additional biological functional assays are underway to determine the direct role of Wnt signaling on OC. Identification of the role of Wnt signaling in osteoclastogenesis may improve our understanding of the bone lytic process in multiple myeloma.

Identification of New Targets for Therapy of Osteolytic Bone Disease in Multiple Myeloma

One of the most characteristic features of multiple myeloma is the development of osteolytic bone lesions. Myeloma-associated bone disease is caused by an increase in osteoclastic bone resorption and a decrease in osteoblastic new bone formation. Insight into the molecular mechanisms of osteoclastogenesis has been provided by the detection of receptor activator of NF-kappaB ligand (RANKL), its specific receptor (RANK) and its decoy receptor antagonist osteoprotegerin (OPG). The RANK signaling system is abnormally regulated in multiple myeloma and targeting this system may ameliorate myeloma bone disease. Less is known about the development of osteoblastic dysfunction, and further knowledge about the interaction between myeloma cells and osteoblasts is required. The aim of this review is to focus on the principles of bone biology for a better understanding of the development of myeloma bone disease and to identify possible therapeutic targets.

Evidence of a role for RANKL in the development of myeloma bone disease.

Multiple myeloma is associated with the development of a devastating bone disease mediated by increased osteoclastic activity. The ligand for receptor activator of nuclear factor-kappaB (RANKL) plays a critical role in normal osteoclast biology and is abnormally regulated in myeloma. Targeting this system with recombinant decoy receptor, osteoprotegerin, or soluble forms of the receptor activator of nuclear factor-kappaB is able to prevent myeloma bone disease in pre-clinical models. Intriguingly, inhibiting osteoclast formation and bone resorption, and altering the bone marrow microenvironment, results in an indirect anti-myeloma effect.

Zoledronic acid treatment of 5T2MM-bearing mice inhibits the development of myeloma bone disease: evidence for decreased osteolysis, tumor burden and angiogenesis, and increased survival.

Multiple myeloma is characterized by the growth of plasma cells in the bone marrow and the development of osteolytic bone disease. Myeloma cells are found closely associated with bone, and targeting this environment may therefore affect both the bone disease and the growth of myeloma cells. We have investigated the effect of the potent bisphosphonate, zoledronic acid, on the development of bone disease, tumor burden, and disease-free survival in the 5T2MM model of myeloma. 5T2MM murine myeloma cells were injected intravenously into C57BL/KaLwRij mice. After 8 weeks, all animals had a paraprotein. Animals were treated with zoledronic acid (120 microg/kg, subcutaneously, twice weekly) or vehicle, from the time of tumor cell injection or from paraprotein detection for 12 or 4 weeks, respectively. All animals injected with tumor cells developed osteolytic lesions, a decrease in cancellous bone volume, an increase in osteoclast perimeter, and a decrease in bone mineral density. Zoledronic acid prevented the formation of lesions, prevented cancellous bone loss and loss of bone mineral density, and reduced osteoclast perimeter. Zoledronic acid also decreased paraprotein concentration, decreased tumor burden, and reduced angiogenesis. In separate experiments, Kaplan-Meier analysis demonstrated a significant increase in survival after treatment with zoledronic acid when compared with control (47 vs. 35 days). A single dose of zoledronic acid was also shown to be effective in preventing the development of osteolytic bone disease. These data show that zoledronic acid is able to prevent the development of osteolytic bone disease, decrease tumor burden in bone, and increase survival in a model of established myeloma.

Bisphosphonates and osteoprotegerin as inhibitors of myeloma bone disease.

A major clinical feature in multiple myeloma is the development of osteolytic bone disease. The increase in bone destruction is due to uncontrolled osteoclastic bone resorption. Until recently the factors responsible for mediating the increase in osteoclast formation in myeloma have been unclear. However, recent studies have implicated a number of factors, including the ligand for receptor activator of NFkappaB (RANKL) and macrophage inflammatory protein-1alpha. The demonstration that increased osteoclastic activity plays a central role in this process and the identification of molecules that may play a critical role in the development of myeloma bone disease have resulted in studies aimed at identifying new approaches to treating this aspect of myeloma. Studies have been performed to determine the ability of recombinant osteoprotegerin (Fc.OPG), a soluble decoy receptor for RANKL, and potent new bisphosphonates to inhibit the development of myeloma bone disease in the 5T2MM murine model of multiple myeloma. Fc.OPG was shown to prevent the development of osteolytic bone lesions in 5T2MM bearing animals. These changes were associated with a preservation of the cancellous bone loss induced by myeloma cells and an inhibition of osteoclast formation. Bisphosphonates, including ibandronate and zoledronic acid, were also shown to inhibit the development of osteolytic bone lesions in the 5T2MM model and alternative models of myeloma bone disease. Bisphosphonates and Fc.OPG are effective inhibitors of the development of osteolytic bone lesions in pre-clinical murine models of myeloma bone disease.

Ibandronate decreases bone disease development and osteoclast stimulatory activity in an in vivo model of human myeloma.

The benefits of bisphosphonate therapy for multiple myeloma bone disease have been clearly documented. However, the effects of bisphosphonates on the osteoclast stimulatory activity (OSA) that is present in the marrow of patients with multiple myeloma, even before the bone disease is detectable, are unknown. Therefore, we examined the effects of ibandronate (IB) treatment prior to the development of bone disease in a murine model of human myeloma. Sublethally irradiated severe combined immunodeficient (SCID) mice were transplanted with ARH-77 cells on day 0. These ARH-77 mice were treated daily with subcutaneous injections of IB started before or at different times after tumor injection as follows: group 1 was started on day -7; group 2 on day 0; group 3 on day +7; group 4 on day +14 after IB administration; and group 5 (control) received no IB. Mice were sacrificed after they developed paraplegia. The onset of paraplegia was delayed in group 1 vs all other groups (mean day 27 vs day 32; p = 0.0098). The number of lytic lesions and the bone surface area of resorption (mm(2)) were significantly decreased in groups 1, 2, and 3, which were treated early with IB, when compared with groups 4 and 5 (p = 0.003 and 0.002, respectively). OSA, as measured by the capacity of bone marrow plasma from ARH-77 mice to induce osteoclast (OCL) formation in human bone marrow cultures, was decreased proportionally to the length of IB treatment. Group 1 had the lowest OSA compared with the other groups (p = 0.003). However, all mice eventually developed paraplegia, and at time of sacrifice, tumor burden was not grossly different among the groups. Interestingly, macroscopic abdominal tumors were more frequent in mice treated with IB. These data demonstrate that early treatment of ARH-77 mice with IB prior to development of myeloma bone disease decreases OSA and possibly retards the development of lytic lesions, but not eventual tumor burden.