Abstract 674: Cooperative induction of apoptosis through p53 signaling and mTOR inhibition in neuroblastoma

Abstract

Abstract The use of MDM2 inhibitors (e.g. Nutlin 3a) to stabilize p53 and induce apoptosis is an attractive therapeutic approach for neuroblastoma which is predominantly p53 wild-type at diagnosis and relapse. We sought to identify additional pathways to potentiate the p53 response. Mammalian target of rapamycin (mTOR) signaling pathway is active in neuroblastoma and mTOR inhibition has antiproliferative effects in vivo in neuroblastoma. It was recently demonstrated that two p53 target genes, Sestrin1 and Sestrin2, inhibit mTOR activity, linking genotoxic stress, p53 and the mTOR signaling pathway. We investigated the role of Sestrin1 and Sestrin2 in p53-mediated apoptosis in neuroblastoma and the interaction of mTOR and p53 pathways after their simultaneous blockade using the mTOR inhibitor, Temsirolimus and the MDM2 inhibitor, Nutlin 3a. We show here that stabilization and activation of wt-p53 through MDM2 inhibition result in significant and rapid p53-dependent apoptosis. Using microarray analysis in primary tumor line and RT-PCR in several neuroblastoma lines, we demonstrate that the global transcriptional response is p53-dependent and that Sestrin1 and Sestrin2 are significantly up-regulated in response to Nutlin. With MTT and Tunnel assays we demonstrate a p53-dependent synergistic effect of combined Nutlin 3a and Temsirolimus treatment on cell growth and apoptosis. Flow cytometric analysis of the phospho-S6 ribosomal protein demonstrates a profound dephosphorylation of S6 in vitro when low dose Nutlin 3a is combined with Temsirolimus. Additional in vivo studies suggest that mTOR inhibition reduces tumor burden and proliferation of neuroblastoma xenografts in nude mice. We conclude that MDM2 inhibition and p53 driven Sestrin1 and Sestrin2 activation enhance the apoptotic response to mTOR inhibition. Thus the therapeutic strategy of combined MDM2 and mTOR inhibition may represent a clinically potent approach to de novo and relapsed neuroblastoma. 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 674.