Amy Haseley

Genetic Validation of the Protein Arginine Methyltransferase PRMT5 as a Candidate Therapeutic Target in Glioblastoma

Glioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cell survival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease.

Effective Treatment of an Orthotopic Xenograft Model of Human Glioblastoma Using an EGFR-retargeted Oncolytic Herpes Simplex Virus

Glioblastoma multiforme (GBM) remains an untreatable human brain malignancy. Despite promising preclinical studies using oncolytic herpes simplex virus (oHSV) vectors, efficacy in patients has been limited by inefficient virus replication in tumor cells. This disappointing outcome can be attributed in part to attenuating mutations engineered into these viruses to prevent replication in normal cells. Alternatively, retargeting of fully replication-competent HSV to tumor-associated receptors has the potential to achieve tumor specificity without impairment of oncolytic activity. Here, we report the establishment of an HSV retargeting system that relies on the combination of two engineered viral glycoproteins, gD and gB, to mediate highly efficient HSV infection exclusively through recognition of the abundantly expressed epidermal growth factor receptor (EGFR) on glioblastoma cells. We demonstrate efficacy in vitro and in a heterotopic tumor model in mice. Evidence for systemically administered virus homing to the tumor mass is presented. Treatment of orthotopic primary human GBM xenografts demonstrated prolonged survival with up to 73% of animals showing a complete response as confirmed by magnetic resonance imaging. Our study describes an approach to HSV retargeting that is effective in a glioma model and may be applicable to the treatment of a broad range of tumor types.

Advances in oncolytic virus therapy for glioma.

The World Health Organization grossly classifies the various types of astrocytomas using a grade system with grade IV gliomas having the worst prognosis. Oncolytic virus therapy is a novel treatment option for GBM patients. Several patents describe various oncolytic viruses used in preclinical and clinical trials to evaluate safety and efficacy. These viruses are natural or genetically engineered from different viruses such as HSV-1, Adenovirus, Reovirus, and New Castle Disease Virus. While several anecdotal studies have indicated therapeutic advantage, recent clinical trials have revealed the safety of their usage, but demonstration of significant efficacy remains to be established. Oncolytic viruses are being redesigned with an interest in combating the tumor microenvironment in addition to defeating the cancerous cells. Several patents describe the inclusion of tumor microenvironment modulating genes within the viral backbone and in particular those which attack the tumor angiotome. The very innovative approaches being used to improve therapeutic efficacy include: design of viruses which can express cytokines to activate a systemic antitumor immune response, inclusion of angiostatic genes to combat tumor vasculature, and also enzymes capable of digesting tumor extra cellular matrix (ECM) to enhance viral spread through solid tumors. As increasingly more novel viruses are being tested and patented, the future battle against glioma looks promising.

Antitumor efficacy of 34.5ENVE: a transcriptionally retargeted and "Vstat120"-expressing oncolytic virus.

Here, we describe the construction and testing of a novel herpes simplex virus type 1 (HSV-1) derived oncolytic virus (OV): 34.5ENVE (viral ICP34.5 Expressed by Nestin promotor and Vstat120 Expressing), for the treatment of cancer. This virus showed significant glioma-specific killing and antiangiogenic effects in vitro and in vivo. Treatment of subcutaneous and intracranial glioma-bearing mice with 34.5ENVE showed a significant increase in median survival of mice in four different glioma models. Histology and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) revealed reduced microvessel density (MVD) and increased tumoral necrosis in 34.5ENVE-treated tumor tissue compared to control OV-treated tumor tissue. Collectively, these results describe the construction, efficacy, and impact on tumor microenvironment of a transcriptionally driven OV armed with Vstat120 gene expression. These preclinical results will facilitate future clinical testing of 34.5ENVE.

Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. Cysteine rich 61 (CCN1) is an extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. In this study, we examined a hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Strikingly, we found that exogenous CCN1 in glioma ECM orchestrated a cellular antiviral response that reduced viral replication and limited cytolytic efficacy. Gene expression profiling and real-time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1 exposure. This induction was accompanied by activation of the Jak/Stat signaling pathway, consistent with induction of an innate antiviral cellular response. Both effects were mediated by the binding of CCN1 to the cell surface integrin α6β1, activating its signaling and leading to rapid secretion of interferon-α, which was essential for the innate antiviral effect. Together, our findings reveal how an integrin signaling pathway mediates activation of a type-I antiviral interferon response that can limit the efficacy of oncolytic viral therapy. Furthermore, they suggest therapeutic interventions to inhibit CCN1-integrin α6 interactions to sensitize gliomas to viral oncolysis.

Copper Chelation Enhances Antitumor Efficacy and Systemic Delivery of Oncolytic HSV

Purpose: Copper in serum supports angiogenesis and inhibits replication of wild-type HSV-1. Copper chelation is currently being investigated as an antiangiogenic and antineoplastic agent in patients diagnosed with cancer. Herpes simplex virus–derived oncolytic viruses (oHSV) are being evaluated for safety and efficacy in patients, but several host barriers limit their efficacy. Here, we tested whether copper inhibits oHSV infection and replication and whether copper chelation would augment therapeutic efficacy of oHSV. Experimental Design: Subcutaneous and intracranial tumor-bearing mice were treated with oHSV ± ATN-224 to evaluate tumor burden and survival. Virus replication and cell killing was measured in the presence or absence of the copper chelating agent ATN-224 and in the presence or absence of copper in vitro. Microvessel density and changes in perfusion were evaluated by immunohistochemistry and dynamic contrast enhanced MRI (DCE-MRI). Serum stability of oHSV was measured in mice fed with ATN-224. Tumor-bearing mice were injected intravenously with oHSV; tumor burden and amount of virus in tumor tissue were evaluated. Results: Combination of systemic ATN-224 and oHSV significantly reduced tumor growth and prolonged animal survival. Immunohistochemistry and DCE-MRI imaging confirmed that ATN-224 reduced oHSV-induced blood vessel density and vascular leakage. Copper at physiologically relevant concentrations inhibited oHSV replication and glioma cell killing, and this effect was rescued by ATN-224. ATN-224 increased serum stability of oHSV and enhanced the efficacy of systemic delivery. Conclusion: This study shows that combining ATN-224 with oHSV significantly increased serum stability of oHSV and greatly enhanced its replication and antitumor efficacy. Clin Cancer Res; 18(18); 4931–41. ©2012 AACR.

VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

Understanding the host response to oncolytic viruses is important to maximize their antitumor efficacy. Despite robust cytotoxicity and high virus production of an oncolytic herpes simplex virus (oHSV) in cultured human sarcoma cells, intratumoral (ITu) virus injection resulted in only mild antitumor effects in some xenograft models, prompting us to characterize the host inflammatory response. Virotherapy induced an acute neutrophilic infiltrate, a relative decrease of ITu macrophages, and a myeloid cell-dependent upregulation of host-derived vascular endothelial growth factor (VEGF). Anti-VEGF antibodies, bevacizumab and r84, the latter of which binds VEGF and selectively inhibits binding to VEGF receptor-2 (VEGFR2) but not VEGFR1, enhanced the antitumor effects of virotherapy, in part due to decreased angiogenesis but not increased virus production. Neither antibody affected neutrophilic infiltration but both partially mitigated virus-induced depletion of macrophages. Enhancement of virotherapy-mediated antitumor effects by anti-VEGF antibodies could largely be recapitulated by systemic depletion of CD11b(+) cells. These data suggest the combined effect of oHSV virotherapy and anti-VEGF antibodies is in part due to modulation of a host inflammatory reaction to virus. Our data provide strong preclinical support for combined oHSV and anti-VEGF antibody therapy and suggest that understanding and counteracting the innate host response may help enable the full antitumor potential of oncolytic virotherapy.

STAT3 Activation Promotes Oncolytic HSV1 Replication in Glioma Cells

Recent studies report that STAT3 signaling is a master regulator of mesenchymal transformation of gliomas and that STAT3 modulated genes are highly expressed in the mesenchymal transcriptome of gliomas. A currently studied experimental treatment for gliomas consists of intratumoral injection of oncolytic viruses (OV), such as oncolytic herpes simplex virus type 1 (oHSV). We have described one particular oHSV (rQNestin34.5) that exhibits potent anti-glioma activity in animal models. Here, we hypothesized that alterations in STAT3 signaling in glioma cells may affect the replicative ability of rQNestin34.5. In fact, human U251 glioma cells engineered to either over-express STAT3 or with genetic down-regulation of STAT3 supported oHSV replication to a significantly higher or lesser degree, respectively, when compared to controls. Administration of pharmacologic agents that increase STAT3 phosphorylation/activation (Valproic Acid) or increase STAT3 levels (Interleukin 6) also significantly enhanced oHSV replication. Instead, administration of inhibitors of STAT3 phosphorylation/activation (LLL12) significantly reduced oHSV replication. STAT3 led to a reduction in interferon signaling in oHSV infected cells and inhibition of interferon signaling abolished the effect of STAT3 on oHSV replication. These data thus indicate that STAT3 signaling in malignant gliomas enhances oHSV replication, likely by inhibiting the interferon response in infected glioma cells, thus suggesting avenues for possible potentiation of oncolytic virotherapy.

DNA Demethylating Agents Synergize with Oncolytic HSV1 against Malignant Gliomas

Purpose: Oncolytic viruses (OV) based on herpes simplex virus type 1 (HSV1) are being used in clinical trials for a variety of cancers. The OV, rQNestin34.5, uses a nestin promoter/enhancer to selectively drive robust viral replication in malignant glioma cells. We have discovered that this promoter becomes extensively methylated in infected glioma cells, reducing OV efficacy. Experimental Design: We used demethylating drugs [5-azacytidine (5-Aza)], decitabine, or valproic acid (VPA) in both in vitro and in vivo malignant glioma models to determine if they improved the efficacy of rQNestin34.5 therapy. Results: The use of demethylating agents, such as 5-Aza, improved OV replication and tumor cell lysis in vitro and, in fact, synergized pharmacologically on Chou–Talalay analysis. In vivo, the combination of the demethylating agents, 5-Aza or decitabine, with rQNestin34.5 significantly prolonged the survivorship of athymic mice harboring intracranial human glioma xenografts over single agent alone. Conclusion: These results, thus, provide further justification for the exploration of demethylating agents when combined with the OV, rQNestin34.5, in preclinical therapeutics and, possibly, clinical trials for malignant glioma. Clin Cancer Res; 19(21); 5952–9. ©2013 AACR.

Abstract 1505: The effect of Cyr61 on oncolytic HSV-1 therapy for glioblastoma multiforme

Gliomas are the most prevalent primary brain tumor and categorized by WHO into four grades. Glioblastoma multiforme (GBM), the grade IV gliomas, have the worst prognosis, wherein despite standard radiation and chemotherapy, patients have a median survival of less than fifteen months. Hence there is a need for radical therapeutic options such as oncolytic viruses (OVs). OVs are viruses that are either natural occurring or genetically modified to selectively replicate and kill cancer cells. Clinical trials have shown the safety of OV therapy, however evidence of significant efficacy remains to be established. We have recently reported a significant induction of the secreted Cyr61 protein in glioma cells infected with an oncolytic HSV-1 (oHSV). Cyr61 is a secreted heparin-binding protein encoded by a growth factor immediate early gene. It has been associated with accelerated tumor growth, invasion, and vascularization in athymic nude mice. Cyr61 is known to promote cell proliferation, adhesion, and migration of various cell lines by its interaction with integrin receptors and heparan sulfate proteoglycans (HSPGs). To confirm the role of Cyr61 in gliomagenesis we performed a meta analysis of gene expression in all the studies available on Oncomine database on Cyr61 gene expression in normal brain to GBM. Our results showed a significant increase in Cyr61 expression in GBM compared to normal brain expression in each study. Additionally we have observed a negative correlation between levels of endogenous Cyr61 in a panel of glioma cell lines and their ability to be infected. Thus, we hypothesized that apart from its pro-angiogenic function, increased levels of Cyr61 inhibits oHSV entry into the cell and may contribute to glioma onco-resistance. We have found that transient transfection of two different glioma cells (Gli36, U251) with a plasmid expressing Cyr61 showed a reduction in viral presence following oHSV infection (fold=1.67, p=0.0118 for gli36 cells; fold=15.19, p=0.0043. for U251T2 cells). To confirm this result we have created tetracycline (tet) inducible glioma cells which express Cyr61 under a tet promotor. Western blot analysis confirmed rapid and dose-dependent induction of secreted Cyr61 in these cells. Furthermore, overexpression of Cyr61 inhibited OV infection/replication at 6 and 24 hours after infection (fold=1.47, p=0.0032; fold=3.79, p=0.0399). Consistent with this, a statistically significant reduction in viral propagation as measured by viral titration was also found in cells overexpressing Cyr61 (fold=3.74;p=0.0202). Thus we have found that Cyr61 has a negative impact on OV therapy. The results from this study will lead to a better understanding of the impact of the tumor microenvironment on OV therapy. Ultimately we hope to use the knowledge gained from these studies to create a more efficient OV, improving this therapeutic modality. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1505.