Inhibition Of BTK Activity In Myeloma Cells Within a Supportive Microenvironment Promotes Their Growth But Suppresses Metastasis

Bruton’s Tyrosine Kinase (BTK), a TEC family kinase, is expressed by hematopoietic cells and is indispensable for B cell maturation and migration. We and others recently demonstrated that BTK is expressed in MM cells and mediates their homing to bone through chemotaxis to SDF-1, a chemokine highly produced in the bone marrow (BM) (Tai et al,Blood 2012; Bam et al.,AJH 2013). The aim of this study was to unravel the role of BTK in MM cell adhesion, growth and metastasis in our in vitro and in vivo experimental systems. BTK activity was suppressed using lentiviral particles containing BTK or scramble shRNA, or using the clinical BTK inhibitor, ibrutinib. Primary MM cells passaged in our animal model or IL6-dependent INA6 MM line were also infected with EGFP/luciferase construct for tracking purposes and monitoring tumor growth. Knockdown of BTK in INA6 cells diminished their homotypic cell aggregation growth pattern and reduced adhesion to fibronectin compared to control cells by 38±6% (p<0.01). In vivo we exploited our SCID-rab model whereby SCID mice were implanted with a piece of rabbit femur on each flank and luciferase-expressing INA6 MM cells containing scramble or BTK shRNA were injected directly into the right-side implanted bone. We previously demonstrated that MM cells grow restrictively in the implanted bone area and are capable of metastasis to the secondary implanted bone but not murine organs (Yata et al, Leukemia 2004). MM growth in the primary bone site of BTK shRNA group (n=13) was higher than in the scramble shRNA group (n=12) by 4±1 folds (p<0.04) assessed by live-animal imaging. Overall MM burden was also higher in the BTK shRNA group by 4.3 folds (p<0.03) and 2.3 folds (p<0.002) detected by measurement of circulating human immunoglobulins and soluble syndecan-1 levels, respectively. In contrast, metastasis to the secondary implanted bones assessed by ex-vivo imaging 10 weeks after engraftment was lower by 37±11 folds (p<0.002) in the BTK shRNA group compared to control scramble shRNA group. In both groups, metastatic tumor cells were identified only in the secondary implanted bones. MM cells extracted from the primary myelomatous bones retained their differences in BTK expression between the two groups. Cell cycle analysis on extracted MM cells revealed decreased sub-G phase (apoptotic cells) and increased in vivo BrdU labeling index in the BTK knockdown MM cells compared to control MM cells. Gene array analysis revealed BTK target genes including upregulation of factors associated with lymphoid malignancies (e.g. BCL11A, TIAM1, RAB31) and underexpression of genes mediating cell adhesion and migration (e.g. LPXN, DBN1, F11R, CT45) in the BTK knockdown cells compared to control cells. In vitro, we established a novel coculture system of MM cells with healthy adult human BM that first has been cultivated with MM patients’ serum for 7 days. Primary MM cells (n=20) were consistently capable of long-term (7 days) survival and growth in this coculture system. Growth of BTK-expressing MM cells (n=7) in the coculture was significantly increased by 61%-90% in the presence of 5 μM (p<0.02) or 10 μM (p<0.01) ibrutinib. Cocultured MM cells treated with 5 μM Ibrutinib had increased BrdU labeling index compared to control MM cells as assessed by flow cytometry. We also observed reduced expression of BTK in MM cells from focal lesion compared to interstitial marrow in paired clinical samples (n=106, p<3.24E-05). These data suggest that BTK plays a role in MM cell homotypic and heterotypic adhesion, and that inhibition of BTK activity in MM cells within a supportive microenvironment promotes their proliferation and growth but suppresses metastasis.