Biological aspects of altered bone remodeling in multiple myeloma and possibilities of pharmacological intervention.

Multiple myeloma is a fatal B cell neoplasm often resulting in focal and in some cases more diffuse destruction of bone. The bone destruction is a result of increased activity of bone resorbing cells--multinucleated osteoclasts emerging through of multiple fusions. In multiple myeloma, clonally expanding cancer cells provide a stimulatory signal for osteoclast recruitment, differentiation and excessive bone resorption. The stimulatory actions of myeloma cells are believed to be mediated via the production of cytokines and local factors or by modulating bone microenvironment in order to stimulate osteoclastic bone resorption. However, our recent study revealed potentially a novel and more intimate contribution of myeloma cells to the bone destruction. Our analysis of the bone biopsies from myeloma patients showed fully integrated malignant nuclei inside osteoclasts, which were transcriptionally active. As a result, about 30% of the osteoclasts in the bone marrow biopsies from myeloma patients were in fact osteoclast-myeloma cell hybrids. As the functional relevance of this novel cell type remained uncertain, the aim of my PhD study became to 1) strengthen the evidence of the existence of hybrid cells, 2) elucidate the functional differences between hybrid cells and non-hybrid OCs and 3) relate these findings to the pathogenesis of osteolytic disease in multiple myeloma. To this end, I developed a culture model of osteoclast-myeloma cell fusion between (pre)osteoclasts already committed to fuse and myeloma cells selected for adherence. The model was applied for testing of the bone resorptive properties of hybrid cells identified by labelling with green fluorescence. When comparing the highly fluorescent and non-fluorescent OCs on bone slices, it seemed that the frequency of highly fluorescent osteoclasts actively resorbing bone was increased as compared with non-fluorescent osteoclasts. This was assessed in two independent ways. Furthermore, these fluorescent osteoclasts appear to resorb deeper compared to non-fluorescent osteoclasts. The preliminary data that need to be confirmed suggest that formation of hybrid cells by fusion of myeloma cells with osteoclasts may result in reprogramming of the osteoclasts and contribute to the more aggressive bone resorption by osteoclasts as it is typically seen in myeloma patients. Another aspect of multiple myeloma and associated bone disease is the unmet need for novel and more efficient therapeutic regiments. Resveratrol (trans-3, 4', 5-trihydroxystilbene; RSV) is a natural compound shown to target the key players of myeloma bone disease: bone resorbing osteoclasts, bone forming osteoblasts and myeloma cells. Our in vitro study on RSV showed that it possessed this ideal triad of properties appearing and thus might be of interest as a potential drug for the treatment of multiple myeloma. RSV suppresses the growth and survival of myeloma cells, inhibits osteoclasts and stimulates the formation of osteoblasts. However, the need for high concentrations combined with low biological availability after oral administration and risk of important side effects stimulated a search for RSV derivates with the same spectrum of actions but safer and with better bioavailability. As the other task of my PhD, I screened structurally modified RSV analogues in cultures of myeloma cells, osteoblasts and osteoclasts. Compared to resveratrol, some analogues showed an up to 5,000-times increased potency to inhibit osteoclast differentiation and could still promote osteoblast maturation but they did not antagonize myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but effect on bone loss could not be detected. During my PhD, I also participated in the studies of the effect of the proteasome inhibitor - bortezomib on osteoclasts conducted at the department. Based on its potent activity in multiple myeloma, bortezomib was accepted as a front-line treatment of myeloma patients by EMEA for the European Union. In our study we assessed the effect of bortezomib on osteoclasts in cultures under the conditions that mimic the pulse-treatment regime used for myeloma patients. The pulse administration of bortezomib significantly inhibited OC activity and, moreover, significantly but transiently reduced levels of two bone resorption markers measured in serum of treated myeloma patients. In MM the clonal expansion of malignant plasma cells results in the unbalanced bone remodelling, therefore it is essential to understand the molecular mechanisms governing the actions of osteoclasts and osteoblasts. During my PhD, I was involved in the investigations of mesenchymal stem cells over-expressing delta like protein - 1(Dlk-1) previously shown to inhibit the differentiation of mesenchymal stem cells (MSC) into osteoblasts. In results, the over-expression of Dlk-1 evoked pro-inflammatory phenotype in MSC suggesting the involvement of Dlk-1 in the immune response.