Abstract 5255: NVP-BEZ235 a dual PI3K/mTOR inhibitor destabilises Mycn in vitro and is growth inhibitory in the TH-MYCN murine neuroblastoma model

Abstract

Abstract Neuroblastoma (NB) is the most common extra cranial solid tumour of childhood. MYCN oncogene amplification in NB is associated with advanced disease stage, therapy resistance and poor prognosis. Mycn protein is a critical regulator of NB tumour neuroblasts function/survival, and is specifically expressed in tumour tissue, making it an ideal candidate for targeted therapy. No existing small molecules selectively target transcription factors; we therefore examined indirect methods of targeting Mycn through inhibition of signalling pathways responsible for regulating the half-life of this oncoprotein. In our previous work we showed that broad-spectrum inhibitors of phosphatidylinositol-3-kinases (PI3K) destabilise Mycn and inhibit growth of native murine tumours (Chesler, 2006). Refined compounds with selective activity against class I phosphatidylinositol-3-kinases (PI3K) and mammalian target of rapamycin (mTOR), such as NVP-BEZ235 (Novartis Inc.) Inhibit class I PI3K isoforms and mTOR with IC50 values in the nanomolar (nM) range. We show that NVP-BEZ235 inhibits PI3K and/or mTOR-dependent phosphorylation of key PI3K pathway components including AKT, GSK3β, p70S6K and S6, in a panel of N-myc wild-type (wt) and N-myc phosphomutant expressing cell lines. N-myc mutant cell lines are resistant to N-myc destabilisation by broad spectrum PI3K blockade (Chesler, 2006). Treatment with NVP-BEZ235 destabilises wt N-myc and not N-myc phosphorylation mutants. In N-myc wt cells, cell proliferation and cycling is effectively inhibited as shown by both GI50 (MTS and SRB) and flow cytometry assays with significant apoptosis detected by caspase cleavage assay. N-myc mutants treated with BEZ235 exhibit no decrease in the steady state levels of the N-myc protein and show cell cycle and proliferation inhibition at higher GI50. BEZ235 preferentially induces autophagy in N-myc mutant cell lines. Taken together these data show that destabilisation and consequent degradation of the Mycn protein is a key mechanism by which PI3K/mTOR inhibitors inhibit MYCN expressing NB cells. To test the in vivo effectiveness of BEZ235 we used the TH-MYCN model in which MYCN overexpression induces spontaneous NB (Weiss, 1997). To measure effects on tumour growth, animals (n=8) were treated with 45mg/kg of NVP-BEZ235 PO, daily for 15 doses. Tumour progression was monitored using MRI. BEZ-235 induced good to very good partial responses against large tumours and a statistically valid regression of tumour size. In conclusion, inhibition of PI3K/mTOR signalling by BEZ235 is highly effective against Mycn protein in both in vitro and in vivo models of MYCN driven NB. It could therefore provide a promising therapeutic strategy for Mycn-targeted treatment of neuroblastoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5255.