Combination therapy of inhibitors of epidermal growth factor receptor/vascular endothelial growth factor receptor 2 (AEE788) and the mammalian target of rapamycin (RAD001) offers improved glioblastoma tumor growth inhibition Ranjit K. Goudar, 1 Qing Shi, 2 Mark D. Hjelmeland, 2 Stephen T. Keir, 2 Roger E. McLendon, 1 Carol J. Wikstrand, 1 Elizabeth D. Reese, 2 Charles A. Conrad, 7 Peter Traxler, 8 Heidi A. Lane, 8 David A. Reardon, 2,3 Webster K. Cavenee, 9 Xiao-Fan Wang, 4 Darell D. Bigner, 1 Henry S. Friedman, 1,2,3 and Jeremy N. Rich 2,5,6 Departments of 1 Pathology, 2 Surgery, 3 Pediatrics, 4 Pharmacology and Cancer Biology, and 5 Neurobiology and 6 Division of Neurology, Duke University Medical Center, Durham, North Carolina; 7 University of Texas M.D. Anderson Cancer Center, Houston, Texas; 8 Novartis Institutes for Biomedical Research, Oncology, Novartis Pharma AG, Basel, Switzerland; and 9 Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California Abstract Malignant gliomas are highly lethal tumors that display striking genetic heterogeneity. Novel therapies that inhibit a single molecular target may slow tumor progression, but tumors are likely not dependent on a signal transduction pathway. Rather, malignant gliomas exhibit sustained mitogenesis and cell growth mediated in part through the effects of receptor tyrosine kinases and the mammalian target of rapamycin (mTOR). AEE788 is a novel orally active tyrosine kinase inhibitor that decreases the kinase activity associated with the epidermal growth factor receptor and, at higher concentrations, the vascular endothelial growth factor receptor 2 (kinase domain region). RAD001 (everolimus) is an orally available mTOR inhibitor structurally related to rapamycin. We hypothe- sized that combined inhibition of upstream epidermal growth factor receptor and kinase domain region receptors with AEE788 and inhibition of the downstream mTOR pathway with RAD001 would result in increased efficacy against gliomas compared with single-agent therapy. In vitro experiments showed that the combination of AEE788 and RAD001 resulted in increased rates of cell cycle arrest and apoptosis and reduced proliferation more than either agent alone. Combined AEE788 and RAD001 given orally to athymic mice bearing established human malignant glioma tumor xenografts resulted in greater tumor growth inhibition and greater increases in median survival than monotherapy. These studies suggest that simultaneous inhibition of growth factor receptor and mTOR pathways offer increased benefit in glioma therapy. [Mol Cancer Ther 2005;4(1):101 – 12] Introduction Glioblastomas remain essentially universally fatal despite maximal therapy with a median survival of only 10 to 12 months (1). Traditional treatments rely on cytotoxic therapies that achieve effects through damage of DNA or disruption of the mitotic machinery. Novel therapies under development inhibit the activities of specific molecular targets that contribute to the malignancy of cancers. Current clinical studies in neuro-oncology involve new therapeutic strategies that specifically target the unique molecular properties of gliomas. The epidermal growth factor receptor (EGFR) pathway is dysregulated in the majority of glioblastomas through overexpression, ampli- fication, and/or mutation (2 – 5). EGFR plays important roles in the pathophysiology of glioblastomas, including the induction of cellular proliferation, motility, resistance to chemotherapy and radiation, and induction of neoangio- genesis. Expression of either wild-type or mutant EGFR may also contribute to tumor formation in genetic glioma models (6 – 10). EGFR function has been targeted through several techniques, prominently small molecule ATP- mimetic tyrosine kinase inhibitors (TKI). We reported recently the first completed trial of an EGFR TKI, gefitinib, in recurrent glioblastomas (11). Although a subset of patients experienced long-term control of tumor growth, Received 8/23/04; revised 10/11/04; accepted 11/3/04. Grant support: Pediatric Brain Tumor Foundation of the United States (J.N. Rich and D.D. Bigner); Accelerate Brain Cancer Cure (J.N. Rich); Southeastern Brain Tumor Foundation (J.N. Rich); NIH grants NS047409 (J.N. Rich), CA14236 (C.J. Wikstrand), NS30245-15 and CA94231-02 (H.S. Friedman), NS20023 and CA11898, and M01 RR 30; GCRC Program; National Center for Research Resources; National Cancer Institute Specialized Programs of Research Excellence 1 P20 CA096890; and FCG grants (D.D. Bigner). R.K. Goudar was a Howard Hughes Medical Institute Medical student fellow. J.N. Rich is a Damon Runyon-Lilly Clinical Investigator and a Sidney Kimmel Cancer Foundation Scholar. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: R.K. Goudar, Q. Shi, and M.D. Hjelmeland contributed equally to this work. Requests for reprints: Jeremy N. Rich, Division of Neurology, Duke University Medical Center, P.O. Box 2900, Durham, NC 27710. Phone: 919-681-1693; Fax: 919-684-6514. E-mail: rich0001@mc.duke.edu Copyright C 2005 American Association for Cancer Research. Molecular Cancer Therapeutics 101 Mol Cancer Ther 2005;4(1). January 2005