AZD6244 (ARRY-142886), a Potent and Selective MEK1/2 Inhibitor Blocks the ERK1/2 Signaling Pathway, Inhibits Osteoclast Differentiation and Activation in Multiple Myeloma: Clinical Implications.
Authors: Breitkreutz Iris , Vallet Sonia , Raab Marc S. , Li Xianfeng , Raje Noopur , Hideshima Teru , Chauhan Dharminder , Munshi Nikhil , Anderson Kenneth C. , Tai Yu-Tzu 
Published: October 2019

Abstract
Multiple myeloma (MM)-associated bone disease is caused by upregulation of osteoclast (OCL) activity and constitutive inhibition of osteoblast function. The extracellular signal-regulated kinase 1/2 (ERK1/2) MAP kinase pathway contributes to cytokine-induced OCL differentiation and maturation. We hypothesized that inhibition of ERK1/2 could prevent OCL differentiation and downregulate OCL function. Here we investigate the effects of AZD6244, which blocks the ERK1/2 MAPK pathway via direct inhibition of MEK1/2, on OCL in MM. Peripheral blood mononuclear cells (PBMC) from healthy donors (n=3) and MM patients (n=11) were harvested and stimulated with RANKL (50ng/ml) and M-CSF (25ng/ml) for 2 weeks to induce OCL formation, in the presence or absence of AZD6244. OCL characteristics were measured by flow cytometric analysis of anti-alphaVbeta3 integrin expression. AZD6244 inhibited OCL differentiation in a dose-dependent manner (n=11, median control: 77.4% at 0 uM; 77% at 0.02 uM; 54% at 0.2 uM; 53% at 2 uM; 38% at 5 uM; 29% at 10 uM). TRAP staining (tartrate-resistant acid phosphatase) was performed to identify OCL and to confirm activity. Importantly, AZD6244 inhibited OCL in a dose-dependent manner, as evidenced by a marked loss of TRAP+ cells. To assess bone resorption activity, OCL were cultured with dentine discs in the presence or absence of AZD6244, followed by the measurement of soluble collagen I fragments in the supernatant. AZD6244 inhibited bone resorption in a dose-dependent manner. We next asked whether AZD6244 affects mature OCL. Mature OCL were induced by cytokine stimulation for 2 weeks and then AZD6244 was added for 3 days, followed by flow cytometric analysis. AZD6244 had no effect on total number of alphaVbeta3 integrin-expressing mature OCL (n=6). Two major myeloma growth and survival factors produced by OCL, B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), were measured in OCL culture supernatants by ELISA. AZD6244 significantly inhibited secretion of BAFF and APRIL. In addition, macrophage inflammatory protein (MIP-1alpha), an important OCL differentiation factor and MM survival factor, was inhibited. These results indicate that AZD6244 inhibits OCL differentiation induced by M-CSF and RANKL, leading to reduced bone resorption activity. Moreover, AZD6244 downregulates MIP-1alpha and BAFF, APRIL secretion by OCL, which could inhibit MM cell survival in the bone marrow microenvironment. We have also demonstrated that AZD6244 inhibits proliferation and survival of human MM cell lines, either sensitive or resistant to conventional chemotherapy, as well as freshly isolated patient MM cells (Abstract #553572 and #553605, ASH 2006). In conclusion, the present study provides a preclinical rationale for the evaluation of AZD6244 (ARRY-142886) as a potential new therapy for patients with MM.