Dasatinib Promotes Osteoprogenitor Differentiation and Inhibition of Osteoclastogenesis: Rationale for Treatment of Myeloma Bone Disease.

Multiple myeloma (MM), an hematological malignancy of terminally differentiated plasma cells, is characterized by the presence of bone disease, caused by increased osteoclast (OC) activity and differentiation as well as a reduction in osteoblast (OB) number and function. Dasatinib (BMS-354825) is an oral multitargeted tyrosin-kinase inhibitor approved for chronic myeloid leukemia which is also under clinical investigation in several other types of tumors. Preclinical data suggests that dasatinib can also be of value in MM based on its effects on myelomatous plasma cells and angiogenesis. In this study, we have further investigated the effects of dasatinib on in vitro OB differentiation and function, as well as on OC formation and resorption activity.

For studies on OB differentiation and function, human mensenchymal stem cells (hMSC) from bone marrow samples of healthy donors and MM patients were used. Alternatively, the mesenchymal hMSC-TERT, the osteoblast-like (MG-63) and multiple myeloma (MM.1S) cell lines were employed. Dasatinib mechanism of action was investigated by Western blotting, PKH67/Annexin V/7 aminoactinomycin D staining, real time RT-PCR, alkaline phosphatase (ALP) activity and quantitative mineralization assays. Receptor activator of nuclear factor κ B ligand (RANKL) and osteoprotegerin (OPG) levels in conditioned media were measured by ELISA. OCs were generated by culture of peripheral blood mononuclear cells from healthy volunteers in medium containing macrophage colony stimulating factor and RANKL. The effect of dasatinib on osteoclastogenesis was assessed by enumeration of multinucleated (≥3) tartrate resistant acid phosphatase-positive cells, whereas bone resorption was calculated by the resorbed area on calcium-coated slides.

We found that dasatinib inhibited platelet derived growth factor (PDGF)-stimulated activation of PDGF receptor β (PDGFRβ) and c-Src in hMSC-TERT and MG-63 cell lines, both tyrosin kinases implicated in OB remodelation processes. Inhibition of PDGFRβ and c-Src signalling correlated with diminished proliferation of the same cell lines without affecting cell viavility as assessed by PKH67/Annexin V/7 aminoactinomycin D staining. Also, treatment of human osteoprogenitor cells with low dasatinib concentrations (2 - 5 nM) promoted OB differentiation since ALP activity at day 7 and gene expression levels of bone formation markers (Runx2/Cbfa1, ALP, and COLIA1) at day 7 and 14 in the osteoblastic differentiation process, were elevated; besides, dasatinib also increased mineral nodular formation as per quantification of alizarin red staining. Finally, treatment with dasatinib decreased the RANKL/OPG ratio in conditioned media from co-cultures of MG-63 and MM.1S cell lines. Similar range of dasatinib concentrations markedly inhibited OC formation, both at the initial and late stages of differentiation from hemopoietic progenitor cells. Finally, a significant reduction of OC resorptive activity of a calcium-coated substrate was observed.

Our results indicate that dasatinib favours both OB differentiation and activity and markedly impairs osteoclastogenesis and OC resorption, thus providing rationale for its use to improve bone lesions in MM patients and other bone pathologies.

This work was supported by grants from Ministerio de Ciencia e Innovación - ISCIII (PI081825); Fundación de Investigación Médica Mutua Madrileña AP27262008, and Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León 07-09, Consejería Sanidad JCyL-ISCIII.

Garzon:Bristol-Myers Squibb Company: Employment.