Pivotal Role of OCL-Derived IGF1 in Drug Resistance and Bone Destruction in MM

Multiple myeloma (MM) is largely incurable, and is characterized by devastating bone destruction caused by increased osteoclast (OCL) differentiation and bone resorption in more than 85% of MM patients. OCLs in MM not only promote bone resorption but also increase MM cell growth and drug resistance. Despite recent advances in anti-myeloma treatment, development of anti-MM drug resistance is a major limitation of MM therapy. Therefore, new treatment modalities are urgently needed to overcome drug resistance and decrease bone resorption. IGF1 is a crucial factor for tumor cell growth and survival of malignant cells, especially in MM. IGFI also contributes to development of drug resistance of MM cells to anti-MM agents, including proteasome inhibitors and immunomodulatory agents, but how OCLs contribute to drug resistance is still not clearly delineated. We found that IGF1 was highly expressed in OCLs attached to bone and bone marrow myeloid cells in vivo, and the expression levels of IGF1 in OCLs from MM bearing mice is higher than in normal OCLs. Intriguingly, OCLs produced more IGF1 (0.8 ng/ml/protein) than MM cells (not detected) and bone marrow stromal cells (BMSCs) (0.4 ng/ml/protein) in vitro. In addition, IGF1 protein expression in OCLs was upregulated (1.8 fold) by treatment with conditioned media (CM) from 5TGM1 murine MM cells, TNF-α or IL-6, major paracrine factors that are increased in the bone marrow microenvironment in MM. These results suggest that OCLs are a major source of local IGF1 in the MM bone marrow microenvironment. To further characterize the role of OCL-derived IGF1, we generated a novel mouse with targeted deletion of Igf1 in OCLs (IGF1^-/--OCL), and assessed the role of OCL-derived IGF1 in drug resistance of MM cells and bone destruction. Treatment of 5TGM1 cells with bortezomib (BTZ) (3 nM, 48 hours) decreased the viability of 5TGM1 cells by 50%. Importantly, the cytotoxic effects of BTZ on MM cells were decreased (by 5%) when MM cells were cocultured with OCLs from wild type (WT) mice. In contrast, coculture of MM cells with IGF1^-/--OCLs or WT-OCLs treated with IGF1 neutralizing antibody (IGF1-ab) did not block BTZ's effects on MM cell death. Consistent with these results, coculture of MM cells with IGF1^-/--OCLs or WT-OCLs treated with IGF1-ab resulted in BTZ-induced caspase-dependent apoptosis in MM cells. We next examined the effects of OCLs on the signaling pathways responsible for MM cell survival. WT-OCL-CM promptly induced the phosphorylation of Akt and activation of p38, ERK and NF-κB in MM cells. However, these pathways were not activated by MM cells treated with IGF1^-/--OCL-CM or IGF1-ab-treated WT-OCL-CM. Since adhesion of MM cells to BMSCs via interaction of VLA-4 and VCAM-1 plays a critical role in cell adhesion-mediated drug resistance (CAMDR) in MM, we tested if treatment of human BMSCs with human OCL-CM upregulated VCAM-1 expression. We found that OCL-CM upregulated VCAM-1 expression on BMSCs (x fold). In contrast, treatment of BMSCs with OCLs treated with IGF1-ab blocked VCAM-1 induction. These data suggest that OCL-derived IGF1 can contribute to MM cell drug resistance in the bone marrow microenvironment. We then examined the role of IGF1 inhibition on osteoclastogenesis and the bone resorption capacity of OCLs. RANK ligand induced the expression of cathepsin K and NFATc1 in CD11b⁺ bone marrow cells from WT mice, differentiation markers of OCLs, and the formation of TRAP-positive multinucleated OCLs. However, OCLs formed by RANK ligand treatment of CD11b⁺ bone marrow cells from IGF1^-/- mice had markedly decreased cathepsin K and NFATc1 expression and OCL formation. Next, we tested the bone resorption capacity of OCLs formed by CD11b⁺ bone marrow cells from IGF1^-/- mice vs. WT mice. Similar numbers of OCLs were cultured with RANK ligand on bone slices for 72 hours. The bone resorption activity of Igf1^-/--OCLs was significantly decreased (70%) compared with WT-OCLs. These results suggest that OCL-derived IGF1 plays a critical role in MM drug resistance and bone destruction, and that inhibition of the effect of IGF1 in OCLs should decrease MM drug resistance and bone destruction.