Targeting MEK1/2 Signaling Cascade by AS703026, a Novel Selective MEK1/2 Inhibitor, Induces Pleiotropic Anti-Myeloma Activity in Vitro and In Vivo.

Abstract 3848

Poster Board III-784

The mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in the pathogenesis of human multiple myeloma (MM) by promoting interactions of MM cells with bone marrow stromal cells (BMSCs) that secrete cytokines and growth factors for MM cell growth, survival, and resistance to chemotherapeutic drugs. Accumulating studies have supported targeting this signaling pathway in MM. Here we investigate cytotoxicity of AS703026, a novel selective MEK1/2 inhibitor with highly oral bioavailability, in MM cell lines and patient MM cells and define its mechanisms of action. AS703026, more potently (∼9-10 fold) than AZD6244, inhibits growth and survival of MM cells and cytokine-induced osteoclast differentiation. It specifically blocks baseline and adhesion-induced pERK1/2, but not pSTAT3. Selective MEK1/2 inhibition by AS703026 led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest, as shown by increased subG0 cells, and concurrently abolished S phase cells. AS703026 also reduced expression of c-maf oncogene in a time-dependent manner, suggesting a MEK1/2-dependent regulation of c-maf that may contribute MM cell growth inhibition. AS703026 further induced apoptosis in MM cells, as manifested by caspase 3 and PARP cleavages in a time-dependent manner. It blocked osteoclastogenesis in vitro, as measured by number of TRAP-positive multinuclear cells following culturing PBMCs with RANKL and M-CSF. Importantly, AS703026 sensitized drug-resistant MM cells to a broad spectrum of conventional (dexamethasone, melphalan), as well as novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Synergistic or additive cytotoxicity (combination index < 1) induced by these combinations was further validated by annexin-V/PI staining and flow cytometric analysis. Combining these agents led to a significantly increased apoptosis and cell death than AS703026 alone, confirming enhanced cytotoxicity against MM cells. In vivo studies demonstrate that treatment of MM cell line H929-bearing mice with AS703026 (n=4 at 30 mg/kg; n=6 at 15 mg/kg), but not vehicle alone (n=6), blocked MM tumor growth in a dose-dependent manner (p<0.008 at 30 mg/kg; p<0.02 at 15 mg/kg). Immunoblotting and immunohistochemistrical staining showed that AS703026-reduced tumor growth was associated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nM) triggered significant cytotoxicity against the majority of patients with relapsed and refractory MM (>84%, n=18), regardless mutation status of 3 RAS and BRAF genes. Bone marrow stromal cells-induced viability of MM patient cells is similarly blocked within the same dose range. Our results therefore strongly support clinical protocols evaluating AS703026, alone or with other anti-MM agents, to improve patient outcome in MM.