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. 2009 Jan;84(1):6-14.
doi: 10.1002/ajh.21310.

The proteasome inhibitor, bortezomib suppresses primary myeloma and stimulates bone formation in myelomatous and nonmyelomatous bones in vivo

Angela Pennisi  1 , Xin Li, Wen Ling, Sharmin Khan, Maurizio Zangari, Shmuel Yaccoby
Affiliations

The proteasome inhibitor, bortezomib suppresses primary myeloma and stimulates bone formation in myelomatous and nonmyelomatous bones in vivo

Angela Pennisi et al. Am J Hematol. 2009 Jan.

Abstract

Multiple myeloma (MM), a hematologic malignancy of terminally differentiated plasma cells is closely associated with induction of osteolytic bone disease, induced by stimulation of osteoclastogenesis and suppression of osteoblastogenesis. The ubiquitin-proteasome pathway regulates differentiation of bone cells and MM cell growth. The proteasome inhibitor, bortezomib, is a clinical potent antimyeloma agent. The main goal of this study was to investigate the effect of bortezomib on myeloma-induced bone resorption and tumor growth in SCID-rab mice engrafted with MM cells from 16 patients. Antimyeloma response of bortezomib, which was evident in >50% of 16 experiments and resembled clinical response, was associated with significant increased bone mineral density (BMD) and osteoblast numbers, and reduced osteoclast numbers in myelomatous bones. This bone anabolic effect, which was also visualized on X-ray radiographs and confirmed by static and dynamic histomorphometric analyses, was unique to bortezomib and was not observed in hosts responding to melphalan, a chemotherapeutic drug widely used to treat MM. Bortezomib also increased BMD and osteoblasts number and reduced osteoclasts number in nonmyelomatous implanted bones. In vitro bortezomib directly suppressed human osteoclast formation and promoted maturation of osteoblasts. We conclude that bortezomib promotes bone formation in myelomatous and nonmyelomatous bones by simultaneously inhibiting osteoclastogenesis and stimulating osteoblastogenesis. As clinical and experimental studies indicate that bone disease is both a consequence and necessity of MM progression our results suggest and that bortezomib's effects on bone remodeling contribute to the antimyeloma efficacy of this drug.

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Figures

Figure 1
Figure 1
In vivo antimyeloma response of bortezomib (BOR) is associated with increased bone mass. SCID-rab mice engrafted with myeloma cells from 16 patients were treated with saline (control) or bortezomib. (A, B). Changes from pretreatment levels (pre-Rx) of MM burden (A) and levels BMD of the implanted bone (B) at pretreatment (pre-Rx) and experiment’s end (final) in all mice (total), responders/partial responders (n = 11) and nonresponders (n = 5, see Results for definition of response). (C) A representative experiment demonstrating the in vivo inhibitory effect of bortezomib on growth of myeloma cells from patient 1. (D) X-ray radiographs of myelomatous bones engrafted with myeloma cells from patient 1 and taken pre-Rx and at experiment’s end. Note that treatment began when bone disease was evident and that, whereas bone loss continued in the control bone, bone mass markedly increased following bortezomib treatment. (E) H&E staining of histological sections of myelomatous bones engrafted with myeloma cells from patient 1 (×10 original magnification). Note increased myeloma cell infiltration and osteoclast (OC) activity in control bone. In contrast, myelomatous bone from a host treated with bortezomib had no apparent myeloma cells but possessed increased trabecular bone and osteoblast (OB) numbers. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 2
Figure 2
Bone anabolism is unique to bortezomib. Myeloma cells recovered from hosts engrafted with cells from patient 1 were passaged into newly constructed SCID-rab mice, subsequently established as a stroma-dependent BN cell line, infected with luciferase-expressing lentivirus, and then injected into newly constructed SCID-rab mice. On establishment of myeloma growth, hosts were treated with saline (CONT, n = 5), melphalan (MEL, n = 3), or bortezomib (BOR, n = 5). A: Human Ig levels (top panel) and live animal imaging of luciferase expression (lower panel) at the end of the experiment. B: Percent change of BMD of the myelomatous bones from pretreatment levels. C: Representative live animal imaging demonstrating luciferase expression before initiation of treatment and at the end of the experiment. D: X-ray radiographs visualizing changes in bone mass during the experimental period. Note that, while bortezomib and melphalan inhibited tumor growth, increased bone mass of myelomatous bones was evident only in hosts treated with bortezomib. E: In vitro melphalan had no cytotoxic effect on osteoblasts and MSCs at doses that inhibit growth of BN myeloma cells. These data suggest that melphalan’s lack of bone-anabolic effect in vivo was not a consequence of cytotoxic effect on osteoblasts. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 3
Figure 3
Bortezomib promotes bone formation in myelomatous bones through simultaneous stimulation of osteoblastogenesis and inhibition of osteoclastogenesis. (A-D) Static (A) and dynamic (B-D) histomorphometry demonstrating increased bone mass and bone formation by bortezomib. (E) Quantification of numbers of osteocalcin-expressing osteoblasts and TRAP-expression in multinucleated osteoclasts in myelomatous bones from control and bortezomib-treated hosts. (F) Myelomatous bone sections from control and bortezomib-treated hosts immunohistochemically stained for osteocalcin and histochemically stained for TRAP. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 4
Figure 4
Bortezomib stimulates osteoblastogenesis and inhibits osteoclastogenesis in nonmyelomatous implanted bones and in vitro. Nonmyelomatous SCID-hu mice were treated with saline (control) or bortezomib (n = 10) for 4 weeks. (A) Levels of BMD of the implanted bones at pretreatment (pre-Rx) and experiment’s end. Bortezomib significantly increased BMD of the implanted bone. (B) Bortezomib had no effect on BMD of the murine femur in these mice. (C) Bortezomib reduced the numbers of TRAP-expressing osteoclasts and increased the numbers of osteocalcin-expressing osteoblasts in the implanted bones. (D-G) Static (D) and dynamic (E-G) histomorphometry demonstrating increased bone mass and bone formation by bortezomib.
Figure 5
Figure 5
Bortezomib directly inhibits osteoclastogenesis and promotes osteoblastogenesis in vitro. (A) Bortezomib inhibited growth of BN myeloma cells in vitro in a dose-dependent manner. (B) Effects of bortezomib on differentiation of osteoclast precursors into TRAP-expressing multinucleated cells in vitro. (C) Effects of bortezomib on differentiation of MSCs into osteoblasts, as assessed by quantification of Alizarin Red deposition in vitro. Note that bortezomib inhibited osteoclast formation and stimulated osteoblast differentiation at doses (2.5-5 nM) that inhibited BN myeloma cell growth.
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