New aspects of bone loss in multiple myeloma patients

Hertha Firnberg Position T 95 New aspects of bone loss in multiple myeloma patients Inge TINHOFER 27.06.2000 Multiple myeloma is a clonal B-cell neoplasm of terminally differentiated B cells which accumulate in the bone marrow and, beside the displacement of the normal bone marrow hematopolesis, cause severe osteoporosis and osteolesions. The ineffectiveness of immune surveilling effector cells to suppress the outgrowth of the neoplastic clone might partially be caused by the expression of Fas ligand on multiple myeloma cells and its immune suppressive effects toward T-cells. The longevity of myelorna cells also results from the production of survival factors by the myeloma cells themselves or by cells of the microenvironment like interleukin-1 beta, interieukin-6, transforming growth factor beta, tumor necrosis factor alpha, granulocyte colony-stimulating factor or macrophage colony-stimulating factor. Our recent data on the expression of interleukin-15 receptor (IL-15R) and intracellular IL-15 mRNA and protein in myeloma cells and the anti-apoptotic signalling capacity of IL-15R stimulation demonstrated a further autocrine/paracrine cytokine loop in multiple myeloma. An important role of both the Fas/FasL system as well as of the humoral factor IL-15 in bone resorption and bone formation has alraedy been suggested by others. Based on these aspects this research program aims to study the biological consequences of FasL expression and autocrine production of IL-15 by myeloma cells or bystander cells in the bone marrow. The program will focus sharply on three aspects: (1) the quantitation of FasL expression on neoplastic plasma cells of multiple myeloma patients in bone marrow specimens and its relationship to apoptosis induction in surrounding tumor-infiltrating cells, (2) the role of FasL expressed by myeloma cells in the decrease in osteoblast attraction and matrix deposition and (3) the quantitation of IL-15 produced by myeloma cells or by cells from the bone marrow microenvironment and its impact on the increased bone-resorbing activity of osteoclasts resulting in bone loss in multiple myeloma.

In patients with multiple myeloma, apart from increased bone resorption by osteoclasts, a decrease of bone formation by osteoblasts is found with high frequency. In this project, we addressed the question whether myeloma cells are able to induce cell death (=apoptosis) in bone-forming cells (the so-called osteoblasts) and whether this mechanism might contribute to the development of bone lesions in this disease. In particular, we examined whether the neoplastic clone which has been shown to express the specific ligands for the death receptors Fas and TRAIL- R, might kill osteoblasts via these signals, thereby actively suppressing bone formation. The characterization of osteoblasts from healthy donors showed significant expression of Fas and TRAIL-R2, and these cells underwent cell death when cultured in the presence of recombinant FasL or TRAIL. Thus, osteoblasts could be identified as potential targets of FasL+ and TRAIL+ myeloma cells. In the next step, we examined whether coculture of myeloma cells and osteoblasts would lead to an increase in the proportion of osteoblasts undergoing apoptosis and whether this would be blockable by neutralizing antibodies to FasL or TRAIL. These studies revealed that myeloma cells were indeed capable of directly killing osteoblasts and this suppression was partly mediated by TRAIL. The only partial inhibition of osteoblast killing by blocking TRAIL signaling suggested that other cytotoxic mechanisms were involved. For identification of these cytotoxic mechanisms we performed experiments in which we cultured osteoblasts in conditioned medium from myeloma cells. By this, we addressed the question whether soluble or membrane-bound factors were involved. These experiments revealed that the suppressive factor produced by myeloma cells and killing osteoblasts is a soluble one. In addition, when supernatants from myeloma cells were subjected to size exclusion centrifugation, no reduction in the killing efficacy of these supernatants was observed suggesting a small factor (<10 kD) to be involved. Beside direct induction of apoptosis of osteoblasts by supernatants of myeloma cells, these cells were sensitized to the cytotoxic effect of recombinant TRAIL when cultured in these supernatants. This finding is of high clinical relevance since recombinant TRAIL has been suggested as potential tumor-specific therapeutic tool and is already in clinical testing in several neoplastic diseases. These results were submitted for publication. Further analyses identified nitric oxide (NO) as a potential candidate factor for osteoblast suppression in multiple myeloma. NO plays an important role in normal bone metabolism. It regulates the delicate balance between formation and degradation of bone material. Increased production of NO disturbs this balance and leads to bone destruction. We found that myeloma cells express the relevant enzyme necessary for NO production and are able to produce NO and its metabolites. By this mechanism, they might kill osteoblasts and/or sensitize them to death receptor signals. By the same mechanism, they could also activate osteoclasts. In summary, we identified induction of apoptosis in osteoblasts by myeloma cells as a potential mechanism contributing to the development of bone lesions in this disease. The apoptotic signals are partially mediated by TRAIL, and involve further soluble cytotoxic factors including NO.