Antibody-based inhibition of DKK1 suppresses tumor-induced bone resorption and multiple myeloma growth in vivo

Abstract

Dickkopf-1 (DKK1), a soluble inhibitor of Wnt signaling secreted by multiple myeloma (MM) cells contributes to osteolytic bone disease by inhibiting the differentiation of osteoblasts. In this study, we tested the effect of anti-DKK1 therapy on bone metabolism and tumor growth in a SCID-rab system. SCID-rab mice were engrafted with primary MM cells expressing varying levels of DKK1 from 11 patients and treated with control and DKK1-neutralizing antibodies for 4 to 6 weeks. Whereas bone mineral density (BMD) of the implanted myelomatous bone in control mice was reduced during the experimental period, the BMD in mice treated with anti-DKK1 increased from pretreatment levels (P < .001). Histologic examination revealed that myelomatous bones of anti-DKK1-treated mice had increased numbers of osteocalcin-expressing osteoblasts and reduced number of multinucleated TRAP-expressing osteoclasts. The bone anabolic effect of anti-DKK1 was associated with reduced MM burden (P < .04). Anti-DKK1 also significantly increased BMD of the implanted bone and murine femur in nonmyelomatous SCID-rab mice, suggesting that DKK1 is physiologically an important regulator of bone remodeling in adults. We conclude that DKK1 is a key player in MM bone disease and that blocking DKK1 activity in myelomatous bones reduces osteolytic bone resorption, increases bone formation, and helps control MM growth.

Figure 1

DKK1-neutralizing antibody promotes bone formation in myelomatous bones. SCID-rab mice engrafted with myeloma cells from 11 patients were treated with anti-DKK1 or control IgG antibody. (A) Changes in BMD (mean ± SEM) of the implanted bones from pretreatment levels. (B) X-ray radiographs of the implanted bones at pretreatment and end of experiment. Note that although BMD in control bones was reduced, anti-DKK1 treatment resulted in increased BMD during the experimental period.

Figure 2

DKK1-neutralizing antibody promotes bone formation in nonmyelomatous bones. SCID-rab mice were engrafted with primary myeloma cells through a direct injection of tumor cells into the implanted rabbit bone (myelomatous hosts) or left uninjected (nonmyelomatous hosts). We measured BMD of the experimental SCID-rab mice femur and implanted rabbit bones. (A-B) Changes in BMD (mean ± SEM) of the implanted rabbit bones (A) and mouse femur (B) in nonmyelomatous SCID-rab mice treated with control IgG and anti-DKK1–neutralizing antibody for 4 weeks. (C) Changes in BMD of the uninvolved mouse femur in myelomatous SCID-rab mice treated with control IgG and anti-DKK1 neutralizing antibody.

Figure 3

DKK1-neutralizing antibody stimulates osteoblastogenesis and reduces osteoclastogenesis in myelomatous bones. (A-D) Decalcified, sequential, myelomatous bone sections from control (A-B) and anti-DKK1–treated hosts (C,D) were stained for TRAP (A,C) and immunohistochemically stained for osteocalcin (B,D). Whereas in control bones TRAP-expressing osteoclasts covered the whole bone surface and no osteocalcin-expressing osteoblasts were detected, anti-DKK1 treatment resulted in marked reduction in number of osteoclasts and increased number of osteoblasts. OB indicates osteoblasts; OC, osteoclasts. (E-F) Numbers of multinucleated, TRAP-expressing osteoclasts and osteocalcin-expressing osteoblasts (E) and ratio of osteoblasts to osteoclasts in myelomatous bones (F). Data are expressed as mean ± SEM. Original magnification, 200×. Images were obtained using an Olympus BH2 microscope equipped with a 160 ×/0.17 numerical aperture objective (Olympus, Melville, NY). Images were acquired using a SPOT2 digital camera (Diagnostic Instruments, Sterling Heights, MI), and were processed using Adobe Photoshop version 10 (Adobe Systems, San Jose, CA).

Figure 4

DKK1-neutralizing antibody reduces primary myeloma burden in SCID-rab mice. (A) Changes in levels of light-chain immunoglobulin (mean ± SEM) from pretreatment in control IgG and anti-DKK1 neutralizing antibody treated hosts. (B-D) Three representative experiments demonstrating heterogeneous response (A, tumor reduction; B, growth delay; C, no response) of anti-DKK1 in SCID-rab mice.