The MEK1/2 Inhibitor AZD6244 (ARRY-142886) Downregulates Constitutive and Adhesion-Induced c- MAF Oncogene Expression and Its Downstream Targets in Human Multiple Myeloma.

Human multiple myeloma (MM), an incurable plasma cell tumor, is highly dependent on its bone marrow (BM) microenvironment which secretes cytokines and growth factors (i.e., IL-6, IGF-1, BAFF) for myeloma cell growth, survival, and resistance to chemotherapeutic drugs. Studies of interactions of MM and BMSCs have demonstrated that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mediates these processes. Recently, gene expression profiling studies have revealed that the c- MAF oncogene, which is overexpressed in approximately 50% of myeloma cell lines and patient MM cells, not only stimulates cell cycle progression but also promotes pathological interactions between tumor and BMSCs. In this study, we examined effects of the MEK1/2 inhibitor AZD6244 (ARRY-142886) on the c-MAF signaling and adhesion-induced c-MAF expression in MM cells. We first demonstrated that AZD6244 inhibits growth of INA6, MM1S, MM1R and MCCAR MM cells in a dose-dependent manner (see also Abstract #553572, ASH 2006). We next found that AZD6244 significantly downregulates c- MAF mRNA and c-MAF protein expression in these MM lines, as early as 2h and this was sustained until 24h after drug treatment. Hypoxia-induced factor 2α (HIF-2α), a pro-angiogenic mediator, which regulates c- MAF gene expression and is regulated by ERK1/2, was significantly inhibited in AZD6244-treated MM cell lines. Since three c-MAF target genes, integrin β7, the C-C chemokines receptor-1 (CCR1), and cyclin D2, were recently identified in MM, we next asked whether the expression of these genes was concomitantly inhibited by AZD6244 in INA6 MM cells that lacks c- MAF translocation. Integrin β7 and CCR1 were inhibited by AZD6244 in a time-dependent manner, whereas cyclin D2 was not expressed in INA6 MM cells. Importantly, downregulation of c-MAF and integrin β7 by AZD6244 is associated with decreased MM cell adhesion to BMSCs and reduced VEGF secretion. Although the role of CCR1 in MM pathophysiology remains elusive, AZD6244 significantly decreased the expression of CCR1 as well as its cognate ligand MIP-1α in INA6 MM cells, which might inhibit a potential autocrine loop in MM cells. We next determined whether MM adhesion to BMSCs could upregulate the c-MAF pathway in MM cells, in the presence or absence of AZD6244. Adhesion of INA6 MM cells to BMSCs strongly induced ERK1/2 and STAT3 activation and augmented expression of c-MAF and its downstream targets (cyclin D2 and integrin β7). The induction of c-MAF and its downstream targets was also seen, although with less extent, in IL-6-induced INA6 MM cells. Conversely, AZD6244 inhibited adhesion-induced c-MAF and its target protein expression (cyclin D2, integrin β7) in INA6 MM cells. AZD6244 further blocked autocrine and paracrine VEGF secretion in a dose-dependent manner. In conclusion, these results confirm a role for c-MAF in the pathological interactions between MM cells and BMSCs and define an important role of the MEK/ERK activation in the ~40% of c- MAF -overexpressing myeloma cells that lack c- MAF translocations. Moreover, this study strongly supports AZD6244 (ARRY-142886) as a promising targeted therapeutic intervention in MM.