MLN3897, a Novel CCR1 Antagonist, Inhibits Osteoclastogenesis by Blocking Early ERK Activation.

Osteolytic lesions are a hallmark of myeloma bone disease and other metastatic cancers, resulting in significant morbidity. In order to successfully target skeletal disease, it is critical to identify the mediators of osteoclastogenesis. Osteoclastogenesis is a multistep process regulated by receptor activator of nuclear factor κ B ligand (RANKL) and monocyte colony stimulating factor (mCSF). These cytokines recruit monocyte precursors, stimulate their fusion into multinucleated cells, and finally induce osteoclast (OC) formation and activation. Recent data suggest that several chemokines in combination with RANKL and/or vitamin D3 (Oba et al., Exp. Hematology 2005) modulate osteoclastogenesis. Moreover, the autocrine secretion of RANTES and MCP-1 mediate monocyte multinucleation (Kim et al., J. Biol. Chem. 2005), the initial step towards osteoclastogenesis. Since CCR1 is one of the main chemokine receptors expressed on monocytes and OC, we here studied the effects of CCR1 inhibition on osteoclastogenesis. MLN3897 is a small molecule, specific antagonist of the chemokine receptor CCR1. In order to analyze the effects of MLN3897 on osteoclastogenesis, we generated OC from peripheral blood mononuclear cells (PBMC) from healthy donors by stimulation with RANKL and M-CSF (50 ng/ml) for three weeks, in the absence or presence of MLN3897. Mature OC were multinucleated TRAP+ cells, and their functional activity was confirmed by a pit formation and a collagen release ELISA assay. Our data demonstrates that MLN3897 inhibits osteoclastogenesis in a dose and time-dependent fashion. MLN3897 at 10 nM concentration decreases OC number to 40% (range 20 to 70%) compared to untreated controls (p<0.05). Time course experiments demonstrate that MLN3897 inhibites OC formation if added at the beginning of culture, whereas no inhibition is noted after 7 or 14 days. The reduced OC number is associated with decreased OC activity as demonstrated by a decrease in collagen fragments (control 82 +/−8 vs treated 25+/− 19 nM). Because the effects of MLN3897 are noted at early time points, we hypothesized that MLN3897 interfers with the monocyte multinucleation process. Our data demonstrates that MLN3897 induces a 60% reduction in the multinucleated cell number at one week (control 61+/−14 vs treated 35+/−9), associated with decreased nuclei per cell at two weeks. This correlates with an early induction of CCR1 expression on monocytes by RANKL and mCSF. Flow cytometry confirms an increment of double-stained CD14/CCR1 monocyte population (from 20% to 50%) associated with increased ERK phosphorylation at 36 hours of stimulation, which is completely abrogated by MLN3897. Taken together, these data therefore suggest that CCR1 inhibition by MLN3897 blocks ERK pathway activation and impairs the monocyte multinucleation process, an early step in osteoclastogenesis. These studies delineate a novel mechanism of action of MLN3897 on osteoclastogenesis, and provide the rationale for its clinical evaluation to treat osteolytic bone disease.