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