CCR1 Inhibition Impairs Osteoclast Activity and Interaction with Myeloma Cells.

Multiple Myeloma (MM) is characterized by increased osteoclasts (OC) activity leading to severe bone disease. Several inducers of OC number and activity have been identified, including several key chemokines. In particular, macrophage inflammatory protein-1α (MIP-1α) and RANTES activate CCR1 and CCR5, resulting in increased osteoclastogenesis (Oba et al. Exp Hematol. 2005) and increased OC motility (Yu X. J Bone Miner Res. 2004) These studies underscore the role of CCR1 in promoting osteoclastogenesis. Here we demonstrate the effects of inhibition of CCR1 with a specific receptor antagonist (MLN3897, Millenium Pharmaceuticals) on normal mature OC. Mature OC were obtained by stimulating PBMC of healthy donors with RANKL and MCSF (50 ng/ml) for three weeks. OC expressed high levels of CCR1 and secreted both MIP-1α (1 ng/ml +/− 1.8) and RANTES (12 pg/ml +/− 0.66), suggesting an autocrine effect of these chemokines. Analyzing the bone resorptive ability with a pit formation assay, we observed that MLN3897 impaired OC function in a dose-dependent fashion (at 10 nM: 20% of reduction in resorbed area, at 100 nM: 50% of reduction). We then studied the effects of CCR1 inhibition on OC viability by analyzing nuclear integrity with Hoechst33258 staining. After 12 hours of cytokine-deprivation, MLN3897 enhanced OC nuclear fragmentation and condensation, suggesting a role for CCR1 ligands in OC survival. We next studied the interactions between OC and MM cells. Because OC secrete several chemoattractants, we studied their ability to stimulate RPMI8226 MM cell migration. We first confirmed high expression levels of CCR1 on RPMI8226 MM cells by flow cytometry (86% compared to isotype control). Inhibition of CCR1 with MLN3897 did not induce any direct cytotoxicity or growth arrest of these cells. We then studied the effects of OC on RPMI8226 MM cells. We observed that OC potently stimulated migration of RPMI8226 MM cells (8-fold increase), which was almost completely blocked by treatment with MLN3897. In contrast, MIP-1α alone induced only a modest effect on migration: the highest effective concentration (0.5 ng/ml) induced only a 2-fold increase. Neutralizing MIP-1α antibody partially reversed these effects, suggesting that other factors may contribute to this migratory effect. Our data therefore demonstrate that CCR1 inhibition by MLN3897 impairs mature OC survival and activity. Although MLN3897 does not have any direct antiMM activity, it affects MM-OC interactions and inhibits MM cell migration to OC. Ongoing studies are further characterizing the effects of CCR1 inhibition on MM-OC interactions in order to provide the framework for its clinical evaluation in MM.