Abstract
Glioblastoma multiforme (GBM) is the most common intracranial cancer but despite recent advances in therapy the overall survival remains about 20 months. Whole genome exon sequencing studies implicate mutations in the receptor tyrosine kinase pathways (RTK) for driving tumor growth in over 80% of GBMs. In spite of various RTKs being mutated or altered in the majority of GBMs, clinical studies have not been able to demonstrate efficacy of molecular targeted therapies using tyrosine kinase inhibitors in GBMs. Activation of multiple downstream signaling pathways has been implicated as a possible means by which inhibition of a single RTK has been ineffective in GBM. In this study, we sought a combination of approved drugs that would inhibit in vitro and in vivo growth of GBM oncospheres. A combination consisting of gefitinib and sunitinib acted synergistically in inhibiting growth of GBM oncospheres in vitro. Sunitinib was the only RTK inhibitor that could induce apoptosis in GBM cells. However, the in vivo efficacy testing of the gefitinib and sunitinib combination in an EGFR amplified/ PTEN wild type GBM xenograft model revealed that gefitinib alone could significantly improve survival in animals whereas sunitinib did not show any survival benefit. Subsequent testing of the same drug combination in a different syngeneic glioma model that lacked EGFR amplification but was more susceptible to sunitinib in vitro demonstrated no survival benefit when treated with gefitinib or sunitinib or the gefitinib and sunitinib combination. Although a modest survival benefit was obtained in one of two animal models with EGFR amplification due to gefitinib alone, the addition of sunitinib, to test our best in vitro combination therapy, did not translate to any additional in vivo benefit. Improved targeted therapies, with drug properties favorable to intracranial tumors, are likely required to form effective drug combinations for GBM.
Citation: Joshi AD, Loilome W, Siu I-M, Tyler B, Gallia GL, et al. (2012) Evaluation of Tyrosine Kinase Inhibitor Combinations for Glioblastoma Therapy. PLoS ONE 7(10): e44372. doi:10.1371/journal.pone.0044372 Editor: Maciej S. Lesniak, The University of Chicago, United States of America Received December 9, 2011; Accepted August 6, 2012; Published October 2, 2012 Copyright: ?2012 Joshi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by National Institutes of Health (NIH) Grant R01 NS052507, the Virginia and D.K. Ludwig Fund for Cancer Research, and the Children’s Cancer Foundation. GJR is supported by the Irving J. Sherman M.D. Neurosurgery Research Professorship. AJ was supported by Matt Trainham ABTA basic research fellowship (http://www.abta.org/). WL was supported by the Faculty of Medicine at Khon Kaen University, Thailand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Introduction
Improving therapy for patients with Glioblastoma multiforme (GBM) is one of the biggest challenges in oncology. Although molecular targeting has shown success in many cancers, targeted therapy for GBM has yet to demonstrate an appreciable clinical survival benefit [1,2]. For example, targeting of Epidermal Growth Factor Receptor (EGFR) with small molecules or monoclonal antibodies has been reported to offer no survival benefit [1], despite the fact that EGFR is the most common genomically altered oncogene in GBM, and targeting EGFR has shown benefit in other cancers. So an important question is: can targeted therapy provide a benefit to GBM patients? The oncogenic receptor tyrosine kinases (RTKs) that are mutated in GBM are obvious molecular targets and many small molecule inhibitors of the RTKs are available. A mutation analysis of over 20,000 gene coding regions in GBM genomes confirmed that the RTK/PI3K/AKT pathway is one of the most frequently altered groups of genes in GBM [3]. The commonly altered genes include EGFR (40% approximate frequency), PTEN (37%), PIK3CA(13%), PIK3R1 (8%) and PDGFRA (8%) [3,4]. Over 80% of glioblastomas have an acquired alteration in the RTK/PI3K/AKT pathway with about 40% of tumors having some alteration in EGFR [3,5] suggesting that scarcity of a prevalent alteration is not the problem with targeted therapy in most GBMs. However, in spite of recent advances in development of targeted therapies, RTK inhibitors have shown negligible success against GBMs. Lack of successful therapies against GBMs using RTK inhibitors raises several questions. Are the molecular targeting agents reaching and inhibiting the presumed target effectively in GBM? What are the resistance mechanisms involved if the inhibitors are reaching the tumor in effective concentrations? Growth signaling through alternate pathways, as well as tumor heterogeneity could be two of many factors involved in tumor resistance mechanisms. In the following study, we tried to evaluate a series of RTK inhibitors in GBM systems in vitro and in vivo to determine if we could find a combination of RTK inhibitors that would be more successful than a single agent. The premise of the work was to evaluate approved inhibitors designed to target the most frequently activated tyrosine kinases in GBMs. The best in vitropair of drugs inhibited GBM oncospheres synergistically was gefitinib and sunitinib. However, the improved activity of RTK combination did not perform as predicted in vivo. Gefitinib alone had a significant but modest survival benefit in a GBM xenograft mouse model mouse model. Moreover, in vivo evaluation of the same drugs in a syngeneic rat model of GBM failed to provide any survival benefit. Although the single agent therapy might show activity in certain genetic backgrounds, combinations that effectively target multiple RTK pathways in an intracranial target are needed.

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