ALBERT

Description: The Dickkopf-1 (DKK1) gene product inhibits osteoblast activity by blocking Wnt signaling. Elevated levels of DKK1 in bone marrow plasma and peripheral blood are associated with the presence of osteolytic bone lesions in patients with multiple myeloma (Tian E, et al. N Engl J Med.2003, 349:2483), evidently by inhibiting osteoblast differentiation and, as a result, stimulating osteoclastogenesis driven by osteoblast progenitors’ enhanced production of RANKL and reduced expression of OPG. DKK1-neutralizing antibody suppressed bone resorption induced by inflammatory joint disease and myeloma in murine models (Diarra, Nature Medicine2007, 13:156, Yaccoby, Blood2007, 109:2106), and ectopic Wnt3a expression prevented myeloma induced bone resoption (Qiang, Blood2008;112:374). To further investigate whether DKK1 is essential for producing myeloma bone disease, we used 5 myeloma cell lines with various expression levels of DKK1 in our SCID-hu model. U266 and CAG cells, which express minimal amounts of DKK1 mRNA by qRT-PCR corresponding to 5ng/ml in spent media by ELISA, caused severe bone resorption, whereas ARP1, H929 and OPM2 which express 55, 61, and 320 fold higher levels of DKK1 mRNA (and 80 ng/ml protein for ARP1 cells) had no effects on the bones. OPM2 cells overexpressing DKK1 following transduction with lentiviral vector (127 fold higher than U266 cells), also did not cause bone resorption. To understand if the lack of relationship between DKK1 expression by myeloma cell lines and bone resorption reflected the level of expression, we used the prostate cancer cell line PC3 that expresses 100 times higher DKK1 mRNA than DKK1-transduced OPM2 cells. These cells caused severe bone destruction in the SCID-hu model. DKK1 expression by PC3 was inhibited by 80% by constitutive or conditional (tet-on) shRNA-lentivirus, and resulted in corresponding reduction (75%, p=0.02) in loss of bone mineral density compared with control cells transduced with conditional or constitutive control shRNA lentivirus. PC3 cells recovered from the tumors had 50% reduced levels of DKK1 mRNA. Myeloma associated osteolytic bone disease results from inhibition of osteoblast differentiation, resulting in reduced bone formation and overcharged osteoclastogenesis by osteoblast progenitors. It thus appears that a quantitative threshold exists below which DKK1 inhibition of Wnt is not sufficient to cause osteolytic bone disease. Alternatively, it is possible that the DKK1 produced by the MM cell lines is not funtional, or that factors other than inhibition of Wnt signaling by DKK1 can also be responsible for producing osteolytic bone disease. We are currently investigating whether direct stimulation of osteoclastic activity by the myeloma cell lines that cause osteolysis but express low DKK1 can explain this observed discrepancy, or if they inhibit osteoblast differentiation by other mechanisms. This work was supported in part by NIH grant CA55816.