Abstract LB-305: The tyrosine kinase inhibitor bafetinib (INNO-406) inhibits osteoclast formation and bone resorption

Abstract

Abstract Lyn phosphorylation up-regulates several kinases including Syk, phospholipase Cγ2 and phosphatidyl inostitol-3 kinase through the immunoreceptor tyrosine-based activation motif (ITAM). Lyn plays critical roles in cell proliferation, Ca2+ mobilization and cell differentiation. This kinase also plays an essential role in transmission of inhibitory signals through phosphorylation of tyrosine residues within the immunoreceptor tyrosine-based inhibitory motifs (ITIM). ITAM-ITIM cross talk is important for macrophage differentiation and osteoclast formation. Bafetinib (INNO-406) is an inhibitor of Bcr-Abl, Fyn and Lyn kinase. No studies have evaluated potential anti-bone resorptive effects of this tyrosine kinase inhibitor. Osteoclasts are multinucleated bone-resorbing cells derived from monocytes that play critical roles in bone remodeling. To evaluate the effects of bafetinib on osteoclast formation and bone resorption, we isolated monocytes using immunomagnetic bead selection from normal or multiple myeloma (MM) patients' PBMCs. CD14+ cells (monocytes) were treated with 50ng/ml RANKL and 20 ng/ml MCSF at the beginning of the culture and during a medium change at 3 days. During the second day of culture, bafetinib or the nitrogen-containing bisphosphonate zoledronic acid, an inhibitor of osteoclast formation and bone resorption, was added into the cells. The cells were fixed and tartrate-resistant acid phosphatase staining performed on day 21. Bafetinib and zoledronic acid both markedly inhibited osteoclast cell formation of monocytes induced by RANKL and MCSF at similar concentrations in a concentration-dependent fashion in both monocytes derived from MM patients and normal subjects. Next, we assessed bone resorption using monocytes that were induced with M-CSF and RANKL and cultured on bone slides for 28 days. At that time, bone resorption was determined using toluidine blue staining. Resorption pits were measured and percentage of surface area with lacunar resorption on each bone slice was determined using an image analysis system. At a concentration as low as 5μM, bafetinib significantly inhibited bone resorption in a concentration-dependent fashion (P<0.001). We further examined the effects of bafetinib on NF-κB and JNK phosphorylation in human monocytes that were induced with RANKL and MCSF through assessment of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-κB protein levels were reduced and phosphorylation of JNKK was also decreased following exposure to bafetinib. These studies suggest that bafetinib may be a new therapeutic option to reduce skeletal complications through its ability to block osteoclast development and reduce bone resorption. We are evaluating the effects of this tyrosine kinase inhibitor on bone resorption in vivo using our SCID-hu murine models of human myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2011-LB-305