Inhibition of p38alpha 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.

The 5T2MM murine model of multiple myeloma: maintenance and analysis.

Multiple myeloma is a B-cell neoplasm characterized by the monoclonal proliferation of plasma cells in the bone marrow, the development of osteolytic lesions, and the induction of angiogenesis. These different processes require three-dimensional interactions, with both humoral and cellular contacts. The 5TMM models are suitable to study these interactions. These are murine models that originate from spontaneously developed myeloma in elderly mice. They are propagated by in vivo transfer of the myeloma cells into young syngeneic mice. We report methods involving the maintenance of the 5T2MM model and the quantification of tumor burden (by determining serum paraprotein concentration and plasmacytosis), assessment of bone lesions, and quantification of angiogenesis. The combination of these different techniques in these models not only helps in unraveling basic biological processes but also in the testing of potentially new therapeutic targets.

Role of the hypoxic bone marrow microenvironment in 5T2MM murine myeloma tumor progression.

BACKGROUND AND OBJECTIVES: Unlike most other tumors, multiple myeloma (MM) cells have to survive and to grow in a bone marrow (BM) microenvironment which is already hypoxic by nature. BM hypoxia is crucial for normal hematopoiesis. However, how BM hypoxia and MM affect each other is unknown. We addressed this topic in the 5T2MM mouse model. DESIGN AND METHODS: Levels of hypoxia in the BM of control and tumor-bearing mice were analyzed by flow cytometric analysis of pimonidazole hypoxyprobe binding and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. Micro-vessel density was measured by CD31 staining of BM sections for immunohistochemistry. Apoptosis sensitivity of CD45 5T2MM subsets in hypoxic conditions were analyzed by detection of active caspase-3. RESULTS: Analysis of control and 5T2MM diseased mice injected with pimonidazole hypoxyprobe indicated that both normal BM and myeloma-infiltrated BM are hypoxic. However, the hypoxia in the myelomatous BM was significantly decreased. Analysis of HIF-1alpha expression, a surrogate marker of hypoxia, also demonstrated significantly lower levels of hypoxia in myeloma-infiltrated BM. HIF-1alpha expression was inversely correlated with the micro-vessel density. In vitro culture of 5T2MM cells under hypoxic conditions indicated induction of apoptosis in the CD45- MM-fraction, but not in the CD45+ 5T2MM-cells. INTERPRETATION AND CONCLUSIONS: These data suggest that native BM hypoxia is advantageous for the tumor-initiating CD45+ 5T2MM-cells. Together with the decreased hypoxia in myeloma-infiltrated BM it also indicates that myeloma-associated angiogenesis is functional and permissive for the expansion of CD45- 5T2MM-cells. All together the data raise the possibility of an important role of BM hypoxia in myeloma tumor progression.

Delayed in vivo disease progression is associated with high proportions of CD45+ myeloma cells in the 5T2MM murine model.

Both CD45(+) and CD45(-) multiple myeloma (MM) cells are observed in the bone marrow (BM) of MM patients; however, their impact on the outcome of the disease is unknown. Most (92%) of the mice injected with murine 5T2MM cells develop myeloma in 10-12 weeks and show hind leg paralysis at the end phase of the disease. In the end stage 5T2MM cells are predominantly CD45(-), in analogy to the common human situation. Herein we report that 8% of the mice have a delayed tumor progression (14-24 weeks) with a complete different feature in the end stage of the disease. These animals had typically a bowed back and never got paralyzed. The MM cells in the BM of these mice were predominantly CD45(+). These data indicate that CD45 subsets are associated with the final outcome of myeloma disease.

Recombinant osteoprotegerin decreases tumor burden and increases survival in a murine model of multiple myeloma.

The aim of the present study was to determine whether modifying the local bone environment with osteoprotegerin (OPG), the soluble decoy receptor for receptor activator of nuclear factor-kappaB (RANK) ligand, could affect tumor burden and survival in the 5T33MM murine model of multiple myeloma. Treatment of mice, injected with 5T33MM cells, with recombinant OPG (Fc-OPG) caused a significant decrease in serum paraprotein and tumor burden and a significant increase in time to morbidity. This was associated with a decrease in osteoclast number in vivo but had no effect on apoptosis and proliferation of 5T33MM cells in vitro. These data indicate that targeting the bone microenvironment by inhibiting the interaction between RANK ligand and RANK with Fc-OPG not only inhibits the development of myeloma bone disease but also decreases tumor growth and increases survival.