Cell Surface CXCR4 and BTK Expression Are Associated in Myeloma Cells and Osteoclast Precursors and Mediate Myeloma Cell Homing and Clonogenicity, and Osteoclastogenesis

Abstract 884

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Clonal B-lymphocytes and plasma cells harboring lymphocytic phenotypes have a proposed role in sustaining myeloma. Bruton's tyrosine kinase (BTK) is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. The SDF-1/CXCR4 signaling pathway is reportedly involved in homing to bone of myeloma cells and osteoclast precursors. The aims of the study were to investigate the possible association between CXCR4 and BTK signaling in myeloma cells and osteoclast precursors, and the consequences of BTK inhibition on myeloma cell migration and clonogenicity, and osteoclastogenesis. By global gene expression profiling (GEP), BTK expression was moderately higher in clinical myeloma cells (n=559) than normal plasma cells (n=25, p<0.05). BTK was also detected in IL6–dependent cell lines and myeloma cell lines that can passage in vivo (n=7), using GEP, qRT-PCR and Western Blot analyses. Cell surface CXCR4 determined by flow cytometry was variably expressed in myeloma cells (5%–95%) and was significantly correlated with BTK gene expression (r=0.75, p<0.002, n=14). Most myeloma cell lines grown independently in vitro expressed very low levels of BTK; however, even in such cell lines (e.g. CAG), enrichment of myeloma cells expressing cell-surface CXCR4 by FACS analysis resulted in detectable BTK expression. BTK inhibition by shRNA in IL6–dependent INA6 cells inhibited their migration toward SDF-1 by >50% (p<0.006) and diminished their ability to form colonies in a 2-weeks clonogenic assay (p<0.0002). The BTK small molecule inhibitor LFM-A13 (25–50 μM) consistently reduced SDF-1-induced migration of primary myeloma cells and myeloma lines (n=6, p<0.03), and clonogenicity (colonies number and size) of 3 BTK-expressing myeloma lines by >50% (p<0.03). Using kinase immunoprecipitation assay, SDF-1 rapidly induced BTK phosphorylation (activation) in myeloma cells, an effect that was blocked by LFM-A13. In vivo, pretreatment of luciferase-expressing myeloma cells with LFM-A13 lessened their homing to bone following intravenous injection into SCID-rab mice as determined by live-animal imaging. BTK was highly expressed in osteoclast precursors while cell surface CXCR4 was detected in 5% of this cell population. LFM-A13 also reduced osteoclast precursor migration toward SDF-1 and suppressed osteoclast formation and bone resorption activity on dentine slices (p<0.002). In myeloma-bearing SCID-rab mice, LFM-A13 (n=10, 40 mg/kg, twice daily for 3 weeks, i.p.) reduced osteoclast number by 45% (p<0.004), prevented reduction of bone mineral density (BMD, p<0.04) and attenuated myeloma growth at near significant level (p<0.07). These data indicate that CXCR4 and BTK signaling are linked in myeloma cells and osteoclast precursors and that BTK has potential as a targeted myeloma therapy because of its roles in myeloma cell homing and clonogenicity and myeloma-induced osteolysis.