AT9283, a Small Molecule Multi-Targeted Kinase Inhibitor with Potent Activity Against Aurora Kinase and STAT3 In Combination with Lenalidomide Results In Synergistic Anti-Myeloma Activity

Abstract 2994

Despite recent advances with new drugs such as bortezomib, thalidomide and lenalidomide, multiple myeloma (MM) remains an incurable disease. Used as single agents, these compounds have shown marked antitumor activity, but the number of patients with relapsed and refractory disease remains high. Combination of these agents with other classes of novel drugs would offer great promise to improve patient outcome. AT9283 (Astex therapeutics, Cambridge UK) is a multi-targeted kinase inhibitor that inhibits Aurora A (AURKA), Aurora B (AURKB) and Janus Kinase (JAKs). AURKA and AURKB expression has been correlated with genetic instability and cellular proliferation in MM; therefore, Aurora kinases represent an attractive therapeutic target in MM. In addition the JAK/STAT pathway plays an important role in the survival and proliferation of MM cells. Blocking this pathway may therefore be critical for the survival of MM cells. AT9283 decreased both phospho-Histone H3 and the phosphorylation of Aurora A at Thr 288 in Nocodazole treated cells, suggesting the dual activity of AT9283 against AURKA and AURKB. Importantly, besides Aurora kinase inhibition, we observed that AT9283 inhibited STAT3 tyrosine phosphorylation within 30 minutes of treatment. The effect of AT9283 on pSTAT3 inhibition was further investigated by using U3A cells stably expressing a luciferase reporter gene under the control of a STAT-dependent promoter. AT9283 inhibited STAT3-dependent luciferase activity with an EC50 of approximately 0.125 μ M. Consistent with AT9283 induced cytotoxicity, genetic depletion of STAT3, AURKA or AURKB showed growth inhibition of MM cells, suggesting that AT9283-induced inhibition of these molecules is in part the underlying mechanism of MM cell growth inhibition. In vivo data using a xenograft mouse model of human MM show that mice treated with AT9283 demonstrated slower tumor growth compared to the control group (p=0.018) and prolongation in median overall survival (32 days in treated group versus 18 days in control group; p < 0.0001) without adverse effects. We next evaluated the activity of AT9283 in combination with established MM drugs and strong synergistic effect was found when AT9283 was combined with lenalidomide (Selleck Chemicals LLC, TX, USA) (Combination Index < 0.9). We hypothesized that the synergistic effect of this combination is due to the fact that the two drugs target different pathways and different phases of the cell cycle, thus augmenting their individual anti-myeloma activity. We examined MM cell cytotoxicity of the combination by using AT9283 and lenalidomide at concentrations lower than their maximal cytotoxic concentrations. Increasing doses of AT9283 (0 -0.125 μ M) were added to lenalidomide (0-2μ M) and a significant decrease in viability (as measured by MTT and cell growth as determined by ³H-TdR at 48 h) was observed with combined therapy compared to either agent alone. A significant increase (55.7%) in early and late apoptosis occurred after 72 hours of exposure of cells to combined therapy with associated caspase-8 and PARP cleavage. Combination treatment resulted in downregulation of pSTAT3 and pERK following 4 hours of treatment. Considering the role that the BM microenvironment plays in growth and survival of MM cells, we examined whether the combination of low dose AT9283 plus lenalidomide induced MM cell death in the context of the BM microenvironment. MM.1S cells were cultured with or without BMSCs in the presence or absence of AT9283, lenalidomide or in combination regimen. Combined therapy inhibited ³H-TdR uptake of MM.1S cells cultured in the presence of BMSCs. Interestingly, we observed that AT9283 plus lenalidomide downregulated the expression of the p-STAT3 and p-ERK when MM.1S cells were cultured with BMSCs, highlighting the role of this drug combination in overcoming the protective effect of BMSCs. These results provide the rationale for the clinical evaluation of AT9283 in combination with lenalidomide in MM patients.