Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Neuroblastoma

Despite intensive research efforts and therapeutic advances over the last few decades, the pediatric neural crest tumor, neuroblastoma, continues to be responsible for over 15% of pediatric cancer deaths. Novel therapeutic options are needed for this tumor. Recently, investigators have shown that mice with syngeneic murine gliomas treated with an engineered, neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ₁34.5 gene, enabling replication in tumor cells but precluding infection of normal neural cells. We hypothesized that M002 would also be effective in the neural crest tumor, neuroblastoma. We showed that M002 infected, replicated, and decreased survival in neuroblastoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly decreased tumor growth, and that this effect was augmented with the addition of ionizing radiation. Importantly, survival could be increased by subsequent doses of radiation without re-dosing of the virus. Finally, these studies showed that the primary entry protein for oHSV, CD111 was expressed by numerous neuroblastoma cell lines and was also present in human neuroblastoma specimens. We concluded that M002 effectively targeted neuroblastoma and that this oHSV may have potential for use in children with unresponsive or relapsed neuroblastoma.     

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Pediatric Solid Tumors

Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ₁34.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.     

MicroRNA-27a Contributes to Rhabdomyosarcoma Cell Proliferation by Suppressing RARA and RXRA

Background

Rhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis.

Methodology/Principal Findings

The expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA).

Conclusions

Study results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.     

Detection of FOXO1 break-apart status by fluorescence in situ hybridization in atypical alveolar rhabdomyosarcoma

The morphologies of alveolar rhabdomyosarcoma(ARMS) are various. Some cases entirely lack an alveolar pattern and instead display a histological pattern that overlaps with embryonal rhabdomyosarcoma(ERMS). The method of pathological diagnosis of ARMS and ERMS has been updated in the 4th edition of the World Health Organization's guidelines for classification of skeletal muscle tumors. Under the new guidelines, there is still no molecular test to distinguish between these two subtypes of rhabdomyosarcoma(RMS). In the present study, we applied fluorescent in situ hybridization(FISH) and found that the Forkhead box O1(FOXO1) gene broke apart, amplified, and displayed an aneuploid signal that was related to the RMS pathological subtype.Aside from the fact that FOXO1 break-apart and its amplification were correlated with atypical ARMS, aneuploidies were usually found in atypical ERMS. In conclusion, our results detail a potential biomarker to improve the accuracy of pathological diagnosis by discriminating between atypical ARMS and atypical ERMS.     

Differential expression of Snail1 transcription factor and Snail1-related genes in alveolar and embryonal rhabdomyosarcoma subtypes

Rhabdomyosarcoma (RMS) represents the most common sarcoma of soft tissue among children. Two main RMS subtypes are alveolar (ARMS) and embryonal (ERMS). The major goal of this study was to find differentially expressed genes between RMS subtypes that could explain higher metastatic potential in ARMS and would be useful for the differential diagnosis. Using RQ-PCR analysis we compared expression of Snail1 and Snail-related genes among 7 ARMS and 8 ERMS patients' samples obtained from the primary tumors and among 2 alveolar and 2 embryonal cell lines. Our results show that Snail1 is highly expressed both in ARMS patients' samples and the alveolar cell lines. We also found that the expression of E-Cadherin was downregulated and the expression of Matrix Metalloproteinases 2 and 9 (MMP-2 and MMP-9) was upregulated in ARMS. We assume that, as in many tumors, also in RMS Snail1 acts as a regulator for pathways known for their role in cells' metastasis and that Snail1 activity results in increased MMPs and decreased E-Cadherin expression. Our findings may explain higher ARMS aggressiveness. Moreover, we suggest that further studies should be performed to verify if Snail1 can be considered as a potential target for ARMS therapy.     

Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer that is refractory to current treatment modalities. Oncolytic herpes simplex viruses (HSV) used for gene therapy are genetically engineered, replication-competent viruses that selectively target tumor cells while sparing normal host tissue. The localized nature, the potential accessibility and the relative lack of distant metastasis make MPM a particularly suitable disease for oncolytic viral therapy. METHODS: The infectivity, selective replication, vector spread and cytotoxic ability of three oncolytic HSV: G207, NV1020 and NV1066, were tested against eleven pathological types of MPM cell lines including those that are resistant to radiation therapy, gemcitabine or cisplatin. The therapeutic efficacy and the effect on survival of NV1066 were confirmed in a murine MPM model. RESULTS: All three oncolytic HSV were highly effective against all the MPM cell lines tested. Even at very low concentrations of MOI 0.01 (MOI: multiplicity of viral infection, ratio of viral particles per cancer cell), HSV were highly effective against MPM cells that are resistant to radiation, gemcitabine and cisplatin. NV1066, an oncolytic HSV that expresses green fluorescent protein (GFP), was able to delineate the extent of the disease in a murine model of MPM due to selective infection and expression of GFP in tumor cells. Furthermore, NV1066 was able to reduce the tumor burden and prolong survival even when treatment was at an advanced stage of the disease. CONCLUSION: These findings support the continued investigation of oncolytic HSV as potential therapy for patients with therapy-resistant MPM.     

Amplification of CDK4, MDM2, SAS and GLI genes in leiomyosarcoma, alveolar and embryonal rhabdomyosarcoma

We evaluated amplification and overrepresentation of CDK4, MDM2, GLI and SAS genes of the 12q13-15 region, in a group of soft tissue sarcomas including leiomyosarcomas (LMS), alveolar rhabdomyosarcomas (ARMS) and embryonal (anaplastic and classic variants) rhabdomyosarcomas (ERMS), to ascertain genomic alterations and possible differences within histologic subtypes of rhabdomyosarcoma (RMS). Quantitative real-time PCR was performed on DNA samples from 29 LMS, 9 ARMS, 7 anaplastic ERMS and 6 classic ERMS. Alteration of one or more of the 12q13-15 genes was revealed in 13/29 LMS (45%) and 12/22 RMS (54%) including 5/9 ARMS (56%), 5/7 anaplastic ERMS (71%) and 2/6 classic ERMS (33%). The potential importance of overproduction of protein products in neoplastic development, led us also to study a possible high expression of cdk4, mdm2 and gli proteins in immunohistochemical staining experiments on paraffin-embedded tissue samples of the same cases. Among LMS and RMS most cases with CDK4, MDM2 and GLI gene alterations also showed a simultaneous high expression of the relative protein. In summary, these results indicate that amplification or overerepresentation of genes at 12q13-15 region involve both LMS and RMS. Moreover these genes alterations reveal predominantly in the alveolar and in the anaplastic variant of the embryonal subtype. These two seem to have a more similar behavior than anaplastic and classic embryonal that are classified in the same subtype.     

Induction of strong antitumor immunity by an HSV-2-based oncolytic virus in a murine mammary tumor model

Oncolytic viruses have shown considerable promise for the treatment of solid tumors. In previous studies, we demonstrated that a novel oncolytic virus (FusOn‐H2), constructed by replacing the serine/threonine protein kinase (PK) domain of the ICP10 gene of type 2 herpes simplex virus (HSV‐2) with the gene encoding the green fluorescent protein, can selectively replicate in and thus lyse tumor cells. 4T1 tumor cells are weakly immunogenic and the mammary tumors derived from them aggressively metastasize to different parts of body, thus providing an attractive model for evaluating anticancer agents. We thus tested the antitumor effect of FusOn‐H2 in this tumor model, in comparisons with several other oncolytic HSVs derived from HSV‐1, including a nonfusogenic HSV‐1 (Baco‐1) and a doubly fusogenic virus (Synco‐2D). Our results show that FusOn‐H2 and Synco‐2D have greater oncolytic activity in vitro than Baco‐1. Moreover, FusOn‐H2 induced strong T cell responses against primary and metastatic mammary tumors in vivo, and splenocytes adoptively transferred from FusOn‐H2‐treated mice effectively prevented metastasis in naïve mice bearing implanted mammary tumors. We conclude that the HSV‐2‐based FusOn‐H2 oncolytic virus may be an effective agent for the treatment of both primary and metastatic breast cancer. Copyright © 2007 John Wiley & Sons, Ltd.     

Structural and Functional Studies of FKHR-PAX3, a Reciprocal Fusion Gene of the t(2;13) Chromosomal Translocation in Alveolar Rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer of skeletal muscle. More than 70% of ARMS tumors carry balanced t(2;13) chromosomal translocation that leads to the production of two novel fusion genes, PAX3-FKHR and FKHR-PAX3. While the PAX3-FKHR gene has been intensely studied, the reciprocal FKHR-PAX3 gene has rarely been described. We report here the cloning and functional characterization of the FKHR-PAX3 gene as the first step towards a better understanding of its potential impact on ARMS biology. From RH30 ARMS cells, we detected and isolated three versions of FKHR-PAX3 cDNAs whose C-terminal sequences corresponded to PAX3c, PAX3d, and PAX3e isoforms. Unlike the nuclear-specific localization of PAX3-FKHR, the reciprocal FKHR-PAX3 proteins stayed predominantly in the cytoplasm. FKHR-PAX3 potently inhibited myogenesis in both non-transformed myoblast cells and ARMS cells. We showed that FKHR-PAX3 was not a classic oncogene but could act as a facilitator in oncogenic pathways by stabilizing PAX3-FKHR expression, enhancing cell proliferation, clonogenicity, anchorage-independent growth, and matrix adhesion in vitro, and accelerating the onset of tumor formation in xenograft mouse model in vivo. In addition to these pro-oncogenic behaviors, FKHR-PAX3 also negatively affected cell migration and invasion in vitro and lung metastasis in vivo. Taken together, these functional characteristics suggested that FKHR-PAX3 might have a critical role in the early stage of ARMS development.     

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells¹. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway^2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging^6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy⁸. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection⁹. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.     

Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is the most common soft tissue cancer in children. The prognosis of patients with relapsed or metastatic disease remains poor. ERMS genomes show few recurrent mutations, suggesting that other molecular mechanisms such as epigenetic regulation might play a major role in driving ERMS tumor biology. In this study, we have demonstrated the diverse roles of histone deacetylases (HDACs) in the pathogenesis of ERMS by characterizing effects of HDAC inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; also known as vorinostat) in vitro and in vivo. TSA and SAHA suppress ERMS tumor growth and progression by inducing myogenic differentiation as well as reducing the self-renewal and migratory capacity of ERMS cells. Differential expression profiling and pathway analysis revealed downregulation of key oncogenic pathways upon HDAC inhibitor treatment. By gain-of-function, loss-of-function, and chromatin immunoprecipitation (ChIP) studies, we show that Notch1- and EphrinB1-mediated pathways are regulated by HDACs to inhibit differentiation and enhance migratory capacity of ERMS cells, respectively. Our study demonstrates that aberrant HDAC activity plays a major role in ERMS pathogenesis. Druggable targets in the molecular pathways affected by HDAC inhibitors represent novel therapeutic options for ERMS patients.     

Molecular cytogenetic analysis consistently identifies translocations involving chromosomes 1, 2 and 15 in five embryonal rhabdomyosarcoma cell lines and a PAX-FOXO1A fusion gene negative alveolar rhabdomyosarcoma cell line

Rhabdomyosarcoma in children is a "small round blue cell tumour" that displays skeletal muscle differentiation. Two main histological variants are recognised, alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. Whereas consistent chromosome translocations characteristic of ARMS have been reported, no such cytogenetic abnormality has yet been described in ERMS. We have used multiple colour chromosome painting to obtain composite karyotypes for five ERMS cell lines and one PAX-FOXO1A fusion gene negative ARMS. The cell lines were assessed by spectral karyotyping (SKY), tailored multi-fluorophore fluorescence in situ hybridisation (M-FISH) using series of seven colour paint sets generated to examine specific abnormalities, and comparative genomic hybridisation (CGH). This approach enabled us to obtain karyotypes of the cell lines in greater detail than previously possible. Several recurring cytogenetic abnormalities were demonstrated, including translocations involving chromosomes 1 and 15 and chromosomes 2 and 15, in 4/6 and 2/6 cell lines respectively. All six cell lines demonstrated abnormalities of chromosome 15. Translocations between chromosomes 1 and 15 have previously been recorded in two primary cases of ERMS by conventional cytogenetics. Analysis of the translocation breakpoints may suggest mechanisms of ERMS tumourigenesis and may enable the development of novel approaches to the clinical management of this tumour.     

Isolation of Reovirus T3D Mutants Capable of Infecting Human Tumor Cells Independent of Junction Adhesion Molecule-A

Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.     

Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

Background  

Oncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology.     

The role of cancer metabolism in defining the success of oncolytic viro-immunotherapy

Oncolytic viruses infect, replicate in, and kill cancer cells selectively without harming normal cells. The rapidly expanding clinical development of oncolytic virotherapy is an exciting interdisciplinary field that provides insights into virology, oncology, and immunotherapy. Recent years have seen greater focus on rational design of cancer-selective viruses together with strategies to exploit their immunostimulatory capabilities, ultimately to develop powerful oncolytic cancer vaccines. However, despite great interest in the field, many important experiments are still conducted under optimum conditions in vitro, with many nutrients present in excess and with cellular stress kept to a minimum. Whilst this provides a convenient platform for cell culture, it bears little relation to the typical conditions found within a tumour in vivo, where cells are often subject to a range of metabolic and environmental stresses. Viral infection and cancer will both lead to production of metabolites that are also not present in media in vitro. Understanding how oncolytic viruses interact with cells exposed to more representative metabolic conditions in vitro represents an under-explored area of study that could provide valuable insight into the intelligent design of superior oncolytic viruses and help bridge the gap between bench and bedside. This review summarises the major metabolic pathways altered in cancer cells, during viral infection and highlights possible targets for future studies.