Exploitation of Novel Hyperdiploid and Nonhyperdiploid Myeloma Cell Lines for Studying Innovative Interventions for Myeloma and Its Associated Bone Disease.

Establishment of clinically relevant human myeloma (MM) cell lines is central for our understanding of MM pathogenesis and development of novel therapies for the disease. Unfortunately, most available lines were generated from extramedullary sites and categorized as nonhyperdiploid. In contrast, hyperdiploid MM cell lines are rare. We established procedures for establishment of stroma-dependent MM lines by passaging primary MM in SCID-hu or SCID-rab mice followed by maintenance in co-culture with stromal cells. We described the establishment and characterization of 2 hyperdiploid (LD, and CF) and 2 nonhyperdiploid (JB and BN) cell lines. LD, CF, JB and BN cells have approximately 51, 57, 44 and 43 chromosomes, respectively. Using our animal models, we also established bortezomib-sensitive and -resistant BN lines. These cell lines were cellularly, phenotypically and molecularly characterized using flow cytometry immunophenotyping, DNA content, G-band and multicolor spectral karyotyping (SKY), and microarray. The cell lines were infected with lentiviral-expressing luciferase for detection of tumor cells at high sensitivity level and for monitoring MM growth in co-cultures and in vivo by live animal imaging. We have reported that hyperdiploid MM cells express high level of the Wnt signaling inhibitor, DKK1, and that DKK1 expression is correlated with level of bone disease. The CF cells which typically represent hyperdiploid patients based on pattern of trisomies, DKK1 expression and induction of severe osteolysis, were engrafted in SCID-rab mice and upon establishment of MM were locally treated with control and DKK1 neutralizing antibodies (R&D Systems, 100 μg/day, S.C.) for 4 weeks. Anti-DKK1 had no significant effect on CF cell growth however; in contrast to control group, bone mineral density (BMD) of the myelomatous bone was preserved by anti-DKK. Prevention of bone loss by anti-DKK1 antibody was associated with increased number of osteocalcin-expressing osteoblasts (27±4 vs. 8±3 in control bones, p<0.003) and reduced numbers of TRAP-expressing multinucleated osteoclasts in myelomatous bones (59±6 vs. 32±3 in control bones, p<0.006). Recent clinical observations indicate bortezomib increases osteoblast activity in MM patients. For studying the effect of bortezomib on MM bone disease, SCID-rab mice engrafted with luciferase-expressing BN cells (n=5 per group) were treated with saline, bortezomib (S.C. 0.5 mg/kg) and melphalan (2.5 mg/kg) twice a week for 4 weeks. Both agents effectively inhibited MM growth as shown by live imaging and hIg ELISA, however, while in bortezomib-treated hosts myelomatous BMD increased by 17±12%, it decreased in saline- and melphalan-treated hosts by 27±9% and 20±9%, respectively (p<0.003 bortezomib vs. saline), indicating that the bone anabolic effect was unique to bortezomib. Ex vivo growth of luciferase-expressing BN cells in co-culture with osteoblasts was lowered by 53±3% (p<0.0005) than their growth rate in co-culture with the supporting mesenchymal stem cells, suggesting that increased bone formation and osteoblast activity negatively impact certain myelomas and that anti-MM mechanism of action of bortezomib involves effect on bone metabolism. These MM cell lines and the procedures used for their establishment provide essential tools for studying myeloma biology and therapy.