Pim Inhibition Suppresses Osteoclastogenesis and Tumor Growth in Myeloma

Devastating bone destruction in multiple myeloma (MM) still remains a significant clinical problem. In pursuing factors responsible for MM tumor expansion and bone destruction, we found that the serine/threonine kinase Pim-2 is constitutively over-expressed as an anti-apoptotic mediator, and further up-regulated in MM cells when cocultured with bone marrow stromal cells or osteoclasts (OCs) (Leukemia, 2011). We also demonstrated that Pim inhibition is able to induce bone formation while suppressing MM tumor growth (Leukemia, 2015). However, the impact of Pim inhibition on MM-induced bone resorption remains unknown. Therefore, the present study was undertaken to clarify the role of Pim-2 in osteoclastogenesis enhanced in MM and the therapeutic effects of Pim inhibition on mutual interaction between MM cells and OCs. Pim-2 was highly expressed almost exclusively in cathepsin K-positive mature OCs on the surface of bone but not in other bone marrow cells in normal mouse bone tissues. RANK ligand and TNF-α induced the expression of Pim-2 in monocytes and RAW264.7 preosteoclastic cells at mRNA and protein levels. Inhibitors of the classical NF-κB pathway, SN50 or IMG2001, abolished Pim-2 up-regulation in RAW264.7 cells by RANK ligand or TNF-α, while Pim inhibition marginally affected the nuclear translocation of NF-κB subunits, p50 and p65, as well as the promoter activity of NF-κB, suggesting Pim-2 up-regulation downstream of the NF-κB pathway. Pim-2 appeared to be up-regulated along with c-fos, NFATc1 and cathepsin K during osteoclastogenesis. The Pim inhibitor SMI-16a potently suppressed the RANK ligand-induced expression of c-fos, NFATc1 and cathepsin K in RAW264.7 cells, and abolished osteoclastogenesis and bone resorption enhanced by MM cell conditioned media on hydroxyapatite-coated dishes. Furthermore, the Pim inhibition was found to suppress [Ca²⁺]i oscillation and thereby nuclear translocation of NFATc1, a critical transcription factor for osteoclastogenesis. MM cells and acid-producing OCs are mutually interacted in bone lesions to enhance MM tumor growth and bone destruction while creating an acidic milieu, thereby forming a progressive vicious cycle. Pim-2 was also up-regulated in MM cells when cocultured with OCs as well as bone marrow stromal cells, and to lesser extent merely by acidic conditions. Interestingly, acidic conditions rather preferentially enhanced the cytotoxic effects of the Pim inhibitor SMI-16a on MM cells even in cocultures with OCs or bone marrow stromal cells. Finally, treatment with SMI-16a reduced OC numbers in bone lesions together with tumor reduction and the restoration of bone formation in mouse MM models with intra-tibial injection of murine 5TGM1 MM cells. These results collectively demonstrated that Pim-2 play a critical role in osteoclastogenesis and tumor growth in acidic bone lesions in MM, and further corroborated that Pim-2 is a pivotal therapeutic target for MM bone disease and tumor progression.