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A Novel Oncolytic Herpes Simplex Virus That Synergizes with Phosphoinositide 3-Kinase/Akt Pathway Inhibitors to Target Glioblastoma Stem Cells

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A Novel Oncolytic Herpes Simplex Virus That Synergizes with Phosphoinositide 3-Kinase/Akt Pathway Inhibitors to Target Glioblastoma Stem Cells Published OnlineFirst April 19, 2011; DOI: 10.1158/1078-0432.CCR-10-3142 Clinical Cancer Cancer Therapy: Preclinical Research A Novel Oncolytic Herpes Simplex Virus that Synergizes with Phosphoinositide 3-kinase/Akt Pathway Inhibitors to Target Glioblastoma Stem Cells Ryuichi Kanai, Hiroaki Wakimoto, Robert L. Martuza, and Samuel D. Rabkin Abstract Purpose: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC), an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. Experimental Design: MG18L, containing a US3 deletion and an inactivating LacZ insertion in UL39, was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV- susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs, alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002, triciribine, GDC-0941, and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou–Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. Results: MG18L was severely neuroattenuated in mice, replicated well in GSCs, and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs, whereas their combination with MG18L synergized in killing GSCs and glioma cell lines, but not human astrocytes, through enhanced induction of apoptosis. Importantly, synergy was independent of inhibitor sensitivity. In vivo, the combination of MG18L and LY294002 significantly prolonged survival of mice, as compared with either agent alone, achieving 50% long-term survival in GBM-bearing mice. Conclusions: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM, being especially effective when combined with PI3K/Akt pathway–targeted agents. Clin Cancer Res; 17(11); 3686–96. Ó2011 AACR. Introduction molecular analysis of GBMs has shown that genetic altera- tions in the phosphoinositide-3-kinase (PI3K)/Akt path- Glioblastoma (GBM) is the most common and deadly way, including in PTEN and PIK3CA, occur in about 80% primary brain tumor in adults (1). Despite advances in of tumors (4, 5). These molecular changes confer GBMs drug development, overall survival has not substantially with proliferative and survival advantages and may play a improved. Recently, cancer stem or tumor-initiating cells role in regulating GSCs (6). There has been rapid growth in have been isolated from solid tumors, including GBM, that the development and clinical testing of small molecule are tumorigenic with characteristics of adult stem cells inhibitors of the PI3K/Akt pathway including for glioma including self-renewal and differentiation into multiple (7). It has been reported that GSCs are sensitive to Akt lineages (2). Glioblastoma stem cells (GSC) are thought inhibitors (8, 9). Unfortunately, the combination of to be important in GBM progression, heterogeneity, recur- genetic alterations and the number of cross-regulatory rence, and resistance to therapy and provide an important feedback loops in the PI3K/Akt pathway raise questions target for the development of new therapies (3). Detailed about the likely clinical efficacy of inhibitors alone (7, 10). Oncolytic viruses are a distinct class of targeted antic- ancer agents with unique mechanisms of action compared Authors' Affiliation: Brain Tumor Research Center, Department of Neu- with other cancer therapies; they selectively replicate in and rosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts kill cancer cells (oncolysis), amplifying themselves and spreading but sparing normal tissue. The safety of oncolytic Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). herpes simplex virus (oHSV) therapy has been shown in Corresponding Author: Samuel D. Rabkin, Brain Tumor Research Center, clinical trials for GBM; however, efficacy remains anecdotal MGH, 185 Cambridge St., CPZN-3800, Boston, MA 02114. Phone: 617- and needs improvement (11, 12). Combining oHSV with 726-6817; Fax: 617-643-3422; E-mail: [email protected] chemotherapeutic drugs is an effective strategy to improve doi: 10.1158/1078-0432.CCR-10-3142 overall efficacy and depending on the specific oHSV muta- Ó2011 American Association for Cancer Research. tions and drug, synergistic cancer cell killing can be 3686 Clin Cancer Res; 17(11) June 1, 2011 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst April 19, 2011; DOI: 10.1158/1078-0432.CCR-10-3142 oHSV and PI3K/Akt Inhibitors Synergize in Glioblastoma Translational Relevance Type Culture Collection and used at low-passage number. Human astrocytes were obtained from ScienCell. Cells Glioblastoma (GBM) is an invariably lethal brain were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (DMEM-FCS) at tumor. Glioblastoma stem cells (GSC) are thought to be important in disease progression, recurrence, and 37 C and 5% CO2. Human GSCs were isolated as pre- resistance to therapy, providing an important target for viously described (15) and cultured in EF20 medium the development of new therapies. Oncolytic herpes composed of Neurobasal medium (Invitrogen) supple-  simplex viruses (oHSV) hold promise for GBM treat- mented with 3 mmol/L L-Glutamine (Mediatech), 1  ment; however, evidence for clinical efficacy remains B27 supplement (Invitrogen), 0.5 N2 supplement (Invi- trogen), 2 mg/mL heparin (Sigma), 20 ng/mL human EGF elusive. One strategy to enhance the efficacy of US3- deleted oHSV is combination with phosphoinositide-3- (epidermal growth factor; R&D systems), 20 ng/mL human  kinase (PI3K)/Akt pathway inhibitors. This study shows FGF-2 (fibroblast growth factor 2; Peprotech) and 0.5 that oHSV-MG18L replicates well in GSCs and is effi- penicillin G/streptomycin sulfate/amphotericin B complex cacious in mice bearing GSC-derived brain tumors, yet (Mediatech). The stem cell features of GBM4, GBM8, and nonneuropathogenic in mice. Notably, MG18L syner- BT74 have been previously described (15). Spheres were gizes with PI3K/Akt inhibitors in killing GSCs and their dissociated using NeuroCult Chemical Dissociation kit intracerebral tumors. This study is the first to show (StemCell Technologies). Passaged cells were confirmed effective targeting of cancer stem cells by the combina- to be mycoplasma free. LY294002 (LC Laboratories), tri- tion of oHSV and small molecule inhibitors. Synergy ciribine (Akt inhibitor V; Santa Cruz Biotechnology), GDC- with MG18L may be utilized to increase efficacy of 0941 (Chemdea), BEZ235 (Chemdea), and Z-VAD-FMK PI3K/Akt inhibitors. These data provide support for (Tocris Bioscience) were dissolved in dimethyl sulfoxide translation of this novel strategy in GBM patients. (Sigma-Aldrich). Viruses observed (13). All oHSV vectors developed for clinical use in All viruses were constructed on a HSV-1 strain F back- À the brain contain deletions of the g34.5 gene, the major viral ground. G207 (g34.5D, ICP6 , LacZ), G47D (g34.5D, À À neurovirulence factor, which is associated with attenuated ICP6 , ICP47/ US11proD, LacZ), and FD6 (ICP6 , LacZ) replication, even in cancer cells, and an inability to replicate have been previously described (14, 15, 19). R7041 (US3 in GSCs (14, 15). To overcome these obstacles and target deleted) was provided by Dr. B. Roizman (University of different tumorigenic pathways, we have developed a new Chicago, Chicago, IL; ref. 20). Viruses were grown, purified, class of oHSV vectors for the treatment of tumors in the and titered on Vero cells (19). brain that contain a deletion in the HSV-1 US3 gene. US3 encodes a serine-threonine kinase that among its many Construction of MG18L activities includes inhibition of virus-induced apoptosis Construction and characterization of MG18L was as and Akt activation (16–18). As apoptotic pathways are described (19). Briefly, the 5.3-kb fragment of pKX2-bG3 frequently dysfunctional in tumor cells, US3 mutants (from S.K. Weller, University of Connecticut Health Center, should engender tumor selectivity based on enhanced apop- Farmington, CT), containing the Escherichia coli lacZ tosis in normal cells, preventing further replication, as was sequence inserted in-frame in UL39, was cotransfected with À found with R7041 (US3 ) infected human umbilical vein R7041 viral DNA into Vero cells using Lipofectamine endothelial cell (HUVEC; ref. 18). We previously reported (Invitrogen). Recombinant viruses, isolated by limiting that R7041 (i) was efficacious in vitro against a number of dilution and identified as plaques staining blue after X- cancer cell lines, including glioma U87 and T98G, and in gal histochemistry (Supplementary Fig. S2A), were plaque vivo against U87 subcutaneous tumors; (ii) synergized with purified 3 times in Vero cells. The genomic structure of PI3K/Akt inhibitors; and (iii) was safe after systemic delivery MG18L was confirmed by restriction endonuclease diges- in the periphery (18). To extend these findings to human tion and
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