Published OnlineFirst September 16, 2016; DOI: 10.1158/1078-0432.CCR-16-0925 Cancer Therapy: Preclinical Clinical Cancer Research Safe and Effective Treatment of Experimental Neuroblastoma and Glioblastoma Using Systemically Delivered Triple MicroRNA- Detargeted Oncolytic Semliki Forest Virus Mohanraj Ramachandran1,DiYu1, Matheus Dyczynski1, Sathishkumar Baskaran1, Lei Zhang1, Aleksei Lulla2, Valeria Lulla2, Sirle Saul2, Sven Nelander1, Anna Dimberg1, Andres Merits2, Justyna Leja-Jarblad1, and Magnus Essand1 Abstract Background: Glioblastoma multiforme and high-risk neuro- Results: The introduction of miRNA target sequences blastoma are cancers with poor outcome. Immunotherapy in the reduced neurovirulence of SFV4 in terms of attenuated rep- form of neurotropic oncolytic viruses is a promising therapeutic lication in mouse CNS cells and ability to cause encephalitis approach for these malignancies. Here we evaluate the oncolytic when administered intravenously. A single intravenous injec- capacity of the neurovirulent and partly IFNb-resistant Semliki tion of SFV4miRT prolonged survival and cured four of eight Forest virus (SFV)-4 in glioblastoma multiformes and neuroblas- mice (50%) with NXS2 and three of 11 mice (27%) with CT- tomas. To reduce neurovirulence we constructed SFV4miRT, 2A, but not for GL261 tumor-bearing mice. In vivo therapeutic which is attenuated in normal central nervous system (CNS) cells efficacy in different tumor models inversely correlated to through insertion of microRNA target sequences for miR124, secretion of IFNb by respective cells upon SFV4 infection miR125, miR134. in vitro. Similarly, killing efficacy of HGCC lines inversely Methods: Oncolytic activity of SFV4miRT was examined in correlatedtoIFNb response and interferon-a/b receptor-1 mouse neuroblastoma and glioblastoma multiforme cell lines expression. and in patient-derived human glioblastoma cell cultures (HGCC). Conclusions: SFV4miRT has reduced neurovirulence, while In vivo neurovirulence and therapeutic efficacy was evaluated in retaining its oncolytic capacity. SFV4miRT is an excellent two syngeneic orthotopic glioma models (CT-2A, GL261) and a candidate for treatment of glioblastoma multiforme and neu- syngeneic subcutaneous neuroblastoma model (NXS2). The role roblastoma with low IFN-b secretion. Clin Cancer Res; 23(6); of IFNb in inhibiting therapeutic efficacy was investigated. 1519–30. Ó2016 AACR. Introduction curative (2). Glioblastoma multiforme is more common in adults but can also arise in children. Neuroblastoma is the Malignant tumors arising from the central nervous system most common extracranial solid cancer in children arising (CNS) are among the most feared types of cancer. Glioblas- from neural crest cells. Low-risk neuroblastomas are associ- toma multiforme is the most common, malignant form of ated with good outcome but children with high-risk neuro- primary brain tumor with poor prognosis and 1-year survival blastoma often relapse even after multimodal therapy (3). rate of only 35.7% (1). Surgical resection combined with Conditionally replicating oncolytic viruses are an attractive radiotherapy and adjuvant temozolomide is the current stan- option for treatment of cancers, as viruses have a natural dard of care therapy, which extends survival but is not ability to replicate inside dividing tumor cells and kill them and simultaneously induce an immune response against the tumor. Herpes simplex virus, adenovirus, and Newcastle 1Department of Immunology, Genetics and Pathology, Science for Life Labora- tory, Uppsala University, Uppsala, Sweden. 2Institute of Technology, University disease virus have been evaluated in phases I and II clinical of Tartu, Tartu, Estonia. trials for treatment of glioblastoma multiforme. They show Note: Supplementary data for this article are available at Clinical Cancer excellent safety data but have so far not resulted in successful Research Online (http://clincancerres.aacrjournals.org/). tumor shrinkage and prolonged survival (4). Adenovirus has been used to treat children with neuroblastoma with encour- M. Ramachandran, D. Yu, J. Leja-Jarblad, and M. Essand have contributed equally to this article. aging results (5, 6). Semliki Forest virus (SFV) is an Alphavirus belonging to the Current affiliation for M. Dyczynski: Department of Oncology and Pathology, Togaviridae family (7). It is an enveloped, positive strand RNA CCK, Karolinska Institute, Stockholm, Sweden. virus, having high replication efficacy and ability to kill a variety Corresponding Author: Magnus Essand, Uppsala University, SE-75185 Uppsala, of tumor cells (7–10). It has a natural neurotropism (11), Sweden. Phone: 46 18 471 4535; E-mail: [email protected] making it an attractive candidate for use as an oncolytic doi: 10.1158/1078-0432.CCR-16-0925 immunotherapy agent to treat neuroblastoma and glioblasto- Ó2016 American Association for Cancer Research. ma multiforme. Several strains of SFV have been described, www.aacrjournals.org 1519 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 16, 2016; DOI: 10.1158/1078-0432.CCR-16-0925 Ramachandran et al. Translational Relevance Materials and Methods Quantitative real-time PCR With Talimogene laherparepvec, the first oncolytic virus Total RNA from cell lines was isolated for miRNA detection approved for treatment of cancer, oncolytic virus immuno- using miRNeasy Mini Kit (Qiagen). To isolate RNA from unin- therapy has a very potential future. Glioblastoma multiforme fected mouse tissues: brain, spinal cord, and dorsal root ganglion is a rapidly growing neoplasm that is very difficult to cure with (DRG) were excised, dissected, immersed in RNAlater (Ambion, the current standard therapies. Oncolytic viruses have been Thermo Fisher Scientific) and homogenized before total RNA was previously tested in the clinic, but without much therapeutic prepared by TRizol extraction (Invitrogen). cDNA was synthe- benefit. We have engineered a safe oncolytic Semliki Forest sized using QuantiMir RT Kit (System Biosciences). miRNAs virus (SFV) detargeted using three central nervous system were quantified from cDNA by SYBR green based real-time PCR (CNS)-related microRNAs (SFV4miRT) that has potential to using the forward primers miR124.F: 50-TAAGGCACGCGGTGA- kill malignant gliomas and neuroblastomas in preclinical ATGCC-30, miR125.F: 50-TCCCTGAGACCCTTTAACCTGTGA-30, murine models. Because SFV4 is partly type-I IFN insensitive, miR134.F: 50-TGTGACTGGTTGACCAGAGGGG-30, and the uni- the SFV4miRT virus could kill also tumors cells with moderate versal reverse primer provided in the QuantiMir RT Kit. Human secretion of type-I IFNs. By using the newly established human (hU6.F: 50-CGCAAGGATGACACGCAATTC-30) and mouse glioblastoma cell culture (HGCC) resource (www.hgcc.se), we (mU6.F: 50-TGGCCCCTGCGCAAGGATG-30) U6 snRNA was demonstrate that SFV4miRT could kill human glioblastoma used to normalize expression levels of miRNAs of interest. cell cultures of all molecular subtypes (proneural, classical, Total RNA from 15 mg mouse brain tissue infected with SFV4 or mesenchymal, neural), which indicates the potential for its SFV4miRT was extracted using RNeasy-Mini Kit (Qiagen). RNA clinical translational. samples (1 mL) at the concentration of 100 ng/mL were used directly to determine viral genome copies, IFNa and IFNb mRNA by real-time PCR analysis using iScript One-Step RT-PCR Kit including the neurovirulent strain SFV4 and the avirulent strain (BioRad). Viral genomes were determined by using primers annealing to nsP1 encoding region: nsP1.F 50-CGCCAAAA- SFV-A(7)74 (referred to as A7/74 from now; ref. 7). The 0 0 neurovirulence of SFV4 is mainly due to initial replication in GATTTTGTTCCA-3 ; nsP1.R, 5 -CCATCGTGGGTGGTTAATCT- 0 a a 0 neurons and oligodendrocytes, and subsequent spread to the 3 . Primers used for murine IFN detection were mIFN .F: 5 - 0 a 0 rest of the brain resulting in encephalitis (12, 13). A7/74 is AGGACAGGAAGGATTTTGGA-3 , mIFN .R: 5 -GCTGCTGATG- GAGGTCATT-30, for murine IFNb detection were mIFNb.F: 50- usually preferred for use as an oncolytic agent due to its natural 0 0 a b a b CACAGCCCTCTCCATCAACT-3 , mIFN-b.R: 5 -GCATCTTCTCC- avirulence in adult rodents. However, type-I IFN / (IFN / ) 0 responses elicited by the host in response to virus infection GTCATCTCC-3 , and for house-keeping murine hypoxanthine- impede A7/74 efficacy (14, 15). The nonstructural proteins guanine phosphoribosyltransferase (HPRT1) detection were HPRT.F: 50-CATAACCTGGTTCATCATCGC-30, HPRT.R: 50- GG- nsP3-nsP4 of SFV4 are involved in reducing STAT1 Tyr701 0 phosphorylation (P-STAT1) upon stimulation of virus infected AGCGGTAGCACCTCCT-3 . All primers were from Sigma- a b cells with exogenous IFNb, and it confers its ability to partly Aldrich. Relative expression of murine IFN and murine IFN was calculated relative to HPRT1 expression levels. Data were resist type-I antiviral defense (16). Thus, SFV4 can be a prom- ÀDDCT ising alternative to treat IFNa/b responsive tumors if its neu- evaluated using the 2 method (23). rovirulence can be attenuated. miRNA are small noncoding RNAs involved in posttranscrip- In vitro cell killing assay using GL261, CT-2A, NXS2, and HGCC tional regulation of gene expression. miRNA expression is cell- cells type restricted, which has been taken advantage of when Mouse glioma cell lines GL261, CT-2A, and mouse neuroblas- designing oncolytic virus to prevent their replication in healthy toma cell