Abstract 3483: Synergistic combination therapy with molecular targeted drugs in glioma stem-like cells
Authors: Shingu Takashi , Holmes Lindsay , Henry Verlene , Latha Khatri , Gururaj Anupama E. , Gibson Laura A. , Doucette Tiffany , Lang Frederick F. , Rao Ganesh , Yuan Liang , Sulman Erik P. , Farrell Nicholas P. , Priebe Waldemar , Hess Kenneth R. , Wang Yaoqi A. , Hu Jian , Bogler Oliver 
Published: August 2015

Abstract
[Introduction] The prognosis of patients with malignant gliomas is poor despite multimodality therapies underscoring the need for novel therapeutic strategies. The majority of glioblastomas have aberrant receptor tyrosine kinase (RTK)/RAS/phosphoinositide 3 kinase (PI3K) signaling pathways and malignant glioma cells are thought to be addicted to these aberrant signaling pathways for their survival and proliferation. However, a large number of clinical trials have demonstrated that monotherapies have limited efficacy. Tumor heterogeneities and signaling redundancy and crosstalk in intracellular signaling network may imply necessity of combination treatments. Recent studies also suggested that effective methods to personalize antitumor therapy are required. However, drug sensitivity testing using tumor cells from each patient, which is one of the potent methods for personalized tumor therapy, has been unsuccessful. One possible reason of this is a technical issue regarding evaluation of clonogenicity of glioma stem-like cells (GSCs) that are thought to be key players in gliomagenesis and the disease progression and recurrence and thus targets of glioma therapy. We previously presented an effective method to evaluate clonogenicity of GSCs by using agarose-based culture system. In this study, we tested the therapeutic effects of combination treatments on GSCs using targeted drugs that affect the signaling pathways to which most glioma cells are thought to be addicted.
[Materials and Methods] Human GSCs were cultured in agarose and treated with inhibitors of RTKs, non-receptor kinase or transcription factor. The colony number and volume were analyzed using GelCountTM colony counter system (Oxford Optronix Inc., UK) and Chou-Talalay combination index was analyzed. Phosphorylation of proteins was evaluated by reverse phase protein array and immunoblotting.
[Results] While GSCs showed diverse sensitivity to targeted therapies even in the cells of the same glioma subtype, combinations of EGFR inhibitors with sorafenib, EGFR inhibitors with MEK inhibitors, Sorafenib with U0126, and erlotinib with BKM120 showed synergy in different GSC lines, indicating effectiveness of suppressing RTK and its downstream molecule. Combination of erlotinib with sorafenib, synergistic in the GSC11 cells, induced apoptosis and autophagic cell death associated with synergistic suppression of Akt and ERK signaling pathways and with decreased nuclear PKM2 and beta-catenin in vitro, and significantly improved survival of nude mice bearing GSC11 brain tumors compared with control and monotherapy groups.
[Conclusions] Inhibition of RTK and its downstream molecule induced synergistic antitumor effects but sensitivity of GSC lines to therapies was diverse. Examining colonies initiated by GSCs obtained from individual patients may be useful for drug sensitivity testing in personalized cancer therapy.
Citation Format: Takashi Shingu, Lindsay Holmes, Verlene Henry, Khatri Latha, Anupama E. Gururaj, Laura A. Gibson, Tiffany Doucette, Frederick F. Lang, Ganesh Rao, Liang Yuan, Erik P. Sulman, Nicholas P. Farrell, Waldemar Priebe, Kenneth R. Hess, Yaoqi A. Wang, Jian Hu, Oliver Bogler. Synergistic combination therapy with molecular targeted drugs in glioma stem-like cells. [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 3483. doi:10.1158/1538-7445.AM2015-3483