The Mechanisms of PIM Kinase Inhibitor, SGI-1776 Free Base, in Treating Myeloma-Associated Bone Disease

Background and Purpose: The PIM kinase family, which includes PIM1, 2 and 3 and is expressed in a variety of solid and hematological malignancies, has an essential role in cell growth and survival. Multiple myeloma remains an incurable hematologic cancer which always companions with osteolytic bone disease. In this work, we investigated the mechanisms of a PIM kinase inhibitor, SGI-1776 free base, in anti-myeloma-associated osteolytic bone disease in vitro and in vivo.

Experiment Design: CCK-8 and flow cytometry were used to assess the cytotoxicity of SGI-1776 free base on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, H929, LP-1, OPM2) . The effect on RANKL-induced osteoclasts was assessed in osteoclastogenic and osteoblastic cultures. Western blot and immunofluorescence were used to investigate the mechanisms of SGI-1776 free base. SiRNAs were used to mimic the inhibition of PIM kinase by the drug. IVIS in vivo imaging system, ELISA (human Igk, CTX-I, PINP) and micro-CT (parameters such as trabecular numbers and trabecular space) were used to surveillance the tumor burden and examine the bone destruction in murine models.

Results: SGI-1776 free base induced apoptosis and cell cycle arrest in the G1 phase in myeloma cells mediated by decreasing the phosphorylation of PIM kinase targets C-Myc, Bad, 4EBP1 and H3. Also, the expression of Mcl-1, C-Myc, cyclin D2 and cyclin E1 were reduced, and the inhibition of NF-kB and STAT3 pathways also observed. The IC50 varies from 2 to 6 uM in different cells. Expression of apoptosis related proteins Cleaved Caspase-3, 7, 8, 9 and PARP-1 were increased. Importantly, SGI-1776 free base could overcome the protective effect of bone marrow stromal cells and osteoclasts on myeloma cells. Knocking down PIM kinase (PIM-1, 2, and 3) by SiRNAs slowed down the proliferation rates of myeloma cells (8226 and MM.1s), indicating the pro-growth impact of PIM kinase on myeloma. Notably, SGI-1776 free base inhibited osteoclast formation and resorption though decreasing PIM-1, c-fos, NFATc1, Cathepsin K and DUSP-1. Calcium coated slide assay, TRAP staining and F-acting ring formation also proved the inhibition effect. Also, phosphorylation levels of Erk, p-38, p-65, AKT and AMPK were reduced in the cells treated with SGI-1776 free base. Knocking down PIM-1 impaired the F-acting ring formation and reduced OC numbers compared with controls, and STAT3 pathway was inhibited as well. ALP staining, ALP activity and alizarin red staining showed SGI-1776 would not suppress osteoblasts formation and its mineralization. Finally, SGI-1776 free base reduced the tumor burden in subcutaneous and disseminated myeloma murine models and prevented myeloma associated bone loss with serum PINP (bone formation) and trabecular numbers increased while serum monoclonal Igk (tumor burden), CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group. Moreover, SGI-1776 free base could directly reduce the RANLK-induced rapid bone loss in non-tumor bearing mouse model.

Conclusion: Our investigation reveals the mechanisms of SGI-1776 free base in treating myeloma associated bone disease. The drug has a dual effect of anti-myeloma and bone protection. These results support the clinical use of this drug in multiple myeloma.