Dual Inhibition of the STAT3 and Src Pathways by NS-018, a JAK2 and Src-Family Kinase Inhibitor, Reduces Myeloma Cell Proliferation and Adhesion and Suppresses Osteoclast Formation

Abstract 2900

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of malignant plasma cells in the bone marrow, and it finally leads to osteolytic bone destruction and impaired hematopoiesis. The pathophysiology of MM is closely linked with the bone marrow microenvironment, which consists of various cell components including bone marrow stromal cells (BMSCs) and bone marrow endothelial cells as well as osteoclasts and osteoblasts. Interaction between MM cells and BMSCs through cell-adhesion molecules confers drug resistance to myeloma cells and stimulates the release of cytokines such as interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) from BMSCs. IL-6 is a major cytokine which enhances cell proliferation and promotes the survival of MM cells by downstream signaling through Janus kinase (JAK) and signal transducer and activator of transcription (STAT). RANKL triggers osteoclast differentiation and activation leading to bone resorption, lytic bone lesion, and osteopenia. The Src family kinases (SFKs), c-Src and Fyn, mediate signaling by cell-adhesion molecules and the RANKL receptor, and they play important roles in cell adhesion and osteoclast differentiation.

NS-018 is a potent and selective dual JAK2/SFKs inhibitor which is under phase1/2 clinical development for the treatment of myelofibrosis. A previous study has shown that NS-018 inhibits JAK2, c-Src and Fyn kinases with IC₅₀ values of 0.72, 6.0 and 7.3 nmol/L, respectively. NS-018 also inhibits JAK2 and SFKs in cells as evidenced by its antiproliferative effect against Ba/F3 cells expressing constitutively activated JAK2 and NIH3T3 cells transformed by v-Src.

In the present study, the ability of NS-018 to inhibit JAK2/STAT3 signaling was examined in IL-6-responsive human MM cell lines such as U266, RPMI 8226, and PCM6. NS-018 suppressed IL-6-induced phosphorylation of STAT3 in a dose-dependent manner at concentrations greater than 100 nmol/L. In addition, NS-018 inhibited the IL-6-enhanced the proliferation of PCM6 cells at concentrations similar to those required to inhibit STAT3 phosphorylation.

To assess whether SFKs inhibition by NS-018 could contribute to an improvement in MM pathology, we next investigated the effect of NS-018 on the adhesion of myeloma cells to cell-adhesion molecules and on osteoclast formation. NS-018 (100 nmol/L) inhibited the adhesion of RPMI 8226 cells to collagen type 1 and VCAM-1 by about 40%. NS-018 also suppressed RANKL-induced differentiation of human osteoclast precursor cells to mature osteoclasts. The numbers of TRAP-positive multinucleated osteoclasts were reduced to about one-half at 100 nmol/L NS-018 and none were observed at 1000 nmol/L. NS-018 similarly suppressed the differentiation of murine RAW264.7 cells to mature osteoclasts. The suppression of cell adhesion and osteoclast formation by NS-018 could both be mediated by c-Src and/or Fyn inhibition, because both inhibitory effects were observed with a typical SFKs inhibitor but not with a typical JAK2 inhibitor.

In conclusion, NS-018 reduced IL-6-enhanced myeloma cell proliferation through inhibition of the JAK2/STAT3 signaling pathway and suppressed cell adhesion and osteoclast formation through inhibition of c-Src and/or Fyn. These results suggest that NS-018 has a dual mechanism of action in MM by simultaneously blocking the JAK2/STAT3 and SFKs pathways. Treatment with NS-018 is a potential new therapeutic option to improve the complex pathological condition of patients with MM.