Protein Profiling in Myeloma In Vivo; Effects of Bortezomib in a Mouse Model of Myeloma.

The proteasome inhibitor bortezomib (VELCADE®) represents an important advance in the treatment of multiple myeloma. Despite its very significant beneficial effects, its precise mechanism of action remains unclear. There is currently evidence to suggest that bortezomib has the potential to directly affect both myeloma cells and osteoblasts. However, it is not possible to discern the precise mechanism of action of bortezomib in patients with myeloma. To address this we used a well-characterized murine model of myeloma combined with MALDI mass spectrometry (MALDI MS) to study the effect of bortezomib on

• bone formation and tumor burden in vivo and

• protein profiles of myeloma cells.

To more closely reflect the clinical setting, we used an established treatment protocol, in which bortezomib treatment was initiated upon development of myeloma. 5TGM1-GFP myeloma cells were inoculated into C57BlKaLwRij mice, resulting in tumor growth within the bone marrow and the development of an osteolytic bone disease. Tumor burden was monitored by measurement of serum IgG2bκ, and a significant increase was detected 14 days following tumor inoculation. Mice were then randomized to receive either bortezomib (0.5mg/kg, 3x week) or vehicle control for the remainder of the experiment. Treatment with bortezomib resulted in a significant reduction in tumor burden, as determined by serum IgG2bκ concentrations and by flow cytometric analysis of GFP-positive cells in the bone marrow. The tibia was analyzed by microCT, and bortezomib was found to significantly increase trabecular bone volume and reduce the number of osteolytic bone lesions. In order to study bone formation, mice were treated with 2 doses of calcein at a 7 day interval. Myeloma-bearing mice were associated with a significant reduction in rates of bone formation, which was prevented by treatment with bortezomib. GFP-positive myeloma cells were isolated from bone marrow of control- and bortezomib-treated mice and purified by fluorescence-activated cell sorting. Protein profiling by MALDI MS identified a number of proteins, both known and unknown, which were regulated by bortezomib treatment in vivo. Free ubiquitin (m/z 8565) was down-regulated, which is a known effect of proteasome inhibition resulting from the inhibition of protein degradation and release of free ubiquitin. Regulated proteins also included the up-regulation of thymosin beta-10 (m/z 4936) following bortezomib treatment. Thymosin beta 10 was undetectable in myeloma cells from control-treated mice. In addition, the calcium binding protein calgranulin A (S100A8) (m/z 10163) was up-regulated in myeloma cells isolated from bortezomib-treated mice. Neither thymosin-beta 10 nor calgranulin A have been previously linked to the activity of the proteasome, and thus may indicate novel molecular mechanisms involved in the anti-myeloma effect of bortezomib. Our data demonstrate that treatment with bortezomib from the time of established myeloma not only reduces myeloma tumor burden but also significantly increases bone formation in vivo. In addition, proteomic analysis of myeloma cells following in vivo treatment with bortezomib reveals a number of regulated proteins, providing novel insights into the molecular mechanism of action of proteasome inhibition in multiple myeloma in vivo.