Abstract 3494: Combined MEK1/2 and PI3K inhibition induces synergistic caspase-dependent apoptosis in neuroblastoma
Authors: Hart Lori S. , Chen Lucy , Batra Vandana , Tsang Matthew , Raman Pichai , Caponigro Giordano , Krupa Shiva , Boehm Markus , Peters Malte , Maris John M. 
Published: August 2015

Abstract
Background: Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system that is often widely metastatic at diagnosis. Despite the intensive multimodal therapy patients receive including surgery, chemoradiotherapy, stem cell transplantation and immunotherapy, cure rates remain less than 40% for patients with high-risk disease. The identification of MAPK pathway genomic aberrations (NRAS mutations, loss of NF1) in primary and relapsed disease lends relevance to the use of MEK1/2 inhibitors in the treatment of neuroblastoma. Although PI3K and PTEN mutations are rarely detected in neuroblastoma, activation of the PI3K/Akt axis is described in primary tumors and is a well-defined mechanism of resistance to MEK inhibition in other solid tumors. We hypothesize that combined inhibition of MEK and PI3K will synergize to reduce neuroblastoma cell survival and proliferation.
Methods: A panel of human-derived neuroblastoma cell lines (N = 15) was used to analyze the effect of combined binimetinib (MEK162, MEK1/2 inhibitor) and alpelisib (BYL719, alpha-specific PI3K inhibitor) treatment. Effects on cell survival were assayed with CellTiter-Glo® and pathway (MAPK, PI3K/Akt, mTOR) inhibition was analyzed following both single agent and combination treatments. Synergy was determined using the Chou-Talalay method. Additional cell death analysis included flow cytometry (sub-G1, Annexin V) and immunoblotting (BIM, cleaved-PARP). In vivo efficacy testing is ongoing in neuroblastoma xenograft models (N = 3) with defined MAPK aberrations.
Results: Treatment with both binimetinib and alpelisib in 15 neuroblastoma cell line models induced a synergistic (N = 14; CI value range 0.098-0.637) or additive (N = 1; CI = 0.951) anti-proliferative response. The mechanism for the additive cytotoxicity was the conversion of binimetinib-induced cell cycle arrest to the induction of programmed cell death, as measured by morphology, sub-G1 analysis and PARP cleavage. Furthermore, the cell death response was rescued by treatment with the caspase inhibitory peptide, QVD-OPh. By immunoblot analysis, combination treatment decreased the levels of phospho-ERK, phospho-Akt, phospho-S6K, beyond that observed with ether single agent. Neuroblastoma xenograft studies are ongoing with models selected based on the presence of MAPK pathway aberrations, including NRAS mutations and copy number loss of NF1, and in vitro sensitivity to the combination.
Conclusions: Synergistic activity of combined MEK1/2 and PI3K inhibition in human neuroblastoma cell lines via the induction of apoptosis was observed. Combined binimetinib and alpelisib treatment suppresses the activation of MAPK, PI3K/Akt and mTOR. The in vitro studies and preclinical in vivo models are expected to form the basis of testing binimetinib-alpelisib in neuroblastoma clinical trials designed to enrich for subjects with canonical MAPK activating lesions.
Citation Format: Lori S. Hart, Lucy Chen, Vandana Batra, Matthew Tsang, Pichai Raman, Giordano Caponigro, Shiva Krupa, Markus Boehm, Malte Peters, John M. Maris. Combined MEK1/2 and PI3K inhibition induces synergistic caspase-dependent apoptosis in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3494. doi:10.1158/1538-7445.AM2015-3494