Abstract 4832: Regulatory role of EF-2 kinase in crosstalk between autophagy and apoptosis and its impact on the activity of a novel AKT inhibitor, MK-2206, against human glioma cells

Abstract

Abstract MK-2206 is the first allosteric AKT inhibitor that has entered clinical development as an anticancer agent. Although MK-2206 has been shown to possess a promising anti-tumor activity both in vitro and in vivo, how to fully achieve therapeutic benefits of this agent in treatment of cancer remains to be explored. AKT/protein kinase B, an onco-protein with serine/threonine kinase activity, plays a central role in cell signaling downstream of growth factors. Aberrant activation of AKT promotes cell growth, survival and proliferation, and is associated with cancer development and progression; inhibition of AKT has been known to induce apoptosis, suppress tumor growth, and more recently, activate autophagy. However, the functional association of autophagy/apoptosis to the anti-tumor effects of AKT inhibition is still elusive. In this study we sought to determine the effect of MK-2206 on autophagy and explore the roles that autophagy and apoptosis play in response of tumor cells to this AKT inhibitor. We found that treatment of human glioma cell lines, T98G and LN229, with MK-2206 caused a robust activation of autophagy, as examined by Western blot analysis of LC3-II and p62, and microscopic inspection for numbers of GFP-LC3 puncta. Silencing of elongation factor-2 (EF-2) kinase, a negative regulator of protein synthesis and a positive regulator of autophagy that was identified by our group, blunted the autophagic response to MK-2206 and to the AKT-targeted siRNA, suggesting an involvement of EF-2 kinase in the AKT inhibition-induced autophagy. Moreover, we observed that suppression of the MK-2206-induced autophagy by silencing of EF-2 kinase was accompanied by an activation of apoptosis, as evidenced by Annexing V staining, an increase in production of reactive oxygen species, and a reduction in the level of anti-apoptotic protein, survivin. Furthermore, inhibition of EF-2 kinase by RNAi potentiated the efficacy of MK-2206 against glioma cells, as measured by MTT viability assay. To confirm that the sensitizing effect of EF-2 kinase inhibition on the cytocidal activity of MK-2206 is mediated through blunting of autophagy, we tested the effects of 3-MA, a small molecule inhibitor of autophagy, and an siRNA targeting beclin 1, a key autophagy-related gene, on cytotoxicity of MK-2206. We showed that 3-MA and the beclin 1-targeted siRNA also enhanced sensitivity of glioma cells to the cytotoxic effect of MK-2206, indicating that suppression of autophagic response indeed renders tumor cells more sensitive to this AKT inhibitor. The results of this study demonstrate that blunting of autophagy and augmenting of apoptosis by inhibiting EF-2 kinase can modulate sensitivity of tumor cells to AKT inhibition, and suggest that targeting EF-2 kinase may represent an attractive approach to reinforcing the anti-cancer efficacy of AKT inhibitors such as MK-2206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4832.