Nicholas Denton

Systemic Delivery of SapC-DOPS Has Antiangiogenic and Antitumor Effects Against Glioblastoma

Saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles are a nanotherapeutic which effectively target and destroy cancer cells. Here, we explore the systemic use of SapC-DOPS in several models of brain cancer, including glioblastoma multiforme (GBM), and the molecular mechanism behind its tumor-selective targeting specificity. Using two validated spontaneous brain tumor models, we demonstrate the ability of SapC-DOPS to selectively and effectively cross the blood-brain tumor barrier (BBTB) to target brain tumors in vivo and reveal the targeting to be contingent on the exposure of the anionic phospholipid phosphatidylserine (PtdSer). Increased cell surface expression of PtdSer levels was found to correlate with SapC-DOPS-induced killing efficacy, and tumor targeting in vivo was inhibited by blocking PtdSer exposed on cells. Apart from cancer cell killing, SapC-DOPS also exerted a strong antiangiogenic activity in vitro and in vivo. Interestingly, unlike traditional chemotherapy, hypoxic cells were sensitized to SapC-DOPS-mediated killing. This study emphasizes the importance of PtdSer exposure for SapC-DOPS targeting and supports the further development of SapC-DOPS as a novel antitumor and antiangiogenic agent for brain tumors.

Oncolytic HSV virotherapy in murine sarcomas differentially triggers an antitumor T-cell response in the absence of virus permissivity

Multiple studies have indicated that in addition to direct oncolysis, virotherapy promotes an antitumor cytotoxic T cell response important for efficacy. To study this phenomenon further, we tested three syngeneic murine sarcoma models that displayed varied degrees of permissiveness to oncolytic herpes simplex virus replication and cytotoxicity in vitro, with the most permissive being comparable to some human sarcoma tumor lines. The in vivo antitumor effect ranged from no or modest response to complete tumor regression and protection from tumor rechallenge. The in vitro permissiveness to viral oncolysis was not predictive of the in vivo antitumor effect, as all three tumors showed intact interferon signaling and minimal permissiveness to virus in vivo. Tumor shrinkage was T-cell mediated with a tumor-specific antigen response required for maximal antitumor activity. Further analysis of the innate and adaptive immune microenvironment revealed potential correlates of susceptibility and resistance, including favorable and unfavorable cytokine profiles, differential composition of intratumoral myeloid cells, and baseline differences in tumor cell immunogenicity and tumor-infiltrating T-cell subsets. It is likely that a more complete understanding of the interplay between the immunologic immune microenvironment and virus infection will be necessary to fully leverage the antitumor effects of this therapeutic platform.

Pediatric cancer gone viral. Part I: strategies for utilizing oncolytic herpes simplex virus-1 in children

Progress for improving outcomes in pediatric patients with solid tumors remains slow. In addition, currently available therapies are fraught with numerous side effects, often causing significant life-long morbidity for long-term survivors. The use of viruses to kill tumor cells based on their increased vulnerability to infection is gaining traction, with several viruses moving through early and advanced phase clinical testing. The prospect of increased efficacy and decreased toxicity with these agents is thus attractive for pediatric cancer. In part I of this two-part review, we focus on strategies for utilizing oncolytic engineered herpes simplex virus (HSV) to target pediatric malignancies. We discuss mechanisms of action, routes of delivery, and the role of preexisting immunity on antitumor efficacy. Challenges to maximizing oncolytic HSV in children are examined, and we highlight how these may be overcome through various arming strategies. We review the preclinical and clinical evidence demonstrating safety of a variety of oncolytic HSVs. In Part II, we focus on the antitumor efficacy of oncolytic HSV in pediatric tumor types, pediatric clinical advances made to date, and future prospects for utilizing HSV in pediatric patients with solid tumors.

Tumor-Associated Macrophages in Oncolytic Virotherapy: Friend or Foe?

Cancer therapy remains a challenge due to toxicity limitations of chemotherapy and radiation therapy. Oncolytic viruses that selectively replicate and destroy cancer cells are of increasing interest. In addition to direct cell lysis, these vectors stimulate an anti-tumor immune response. A key regulator of tumor immunity is the tumor-associated macrophage population. Macrophages can either support oncolytic virus therapy through pro-inflammatory stimulation of the anti-tumor response at the cost of hindering direct oncolysis or through immunosuppressive protection of virus replication at the cost of hindering the anti-tumor immune response. Despite similarities in macrophage interaction between adult and pediatric tumors and the abundance of research supporting macrophage modulation in adult tumors, there are few studies investigating macrophage modulation in pediatric cancers or modulation of immunotherapy. We review the current state of knowledge regarding macrophages in cancers and their influence on oncolytic virotherapy.

Pediatric cancer gone viral. Part II: potential clinical application of oncolytic herpes simplex virus-1 in children

Oncolytic engineered herpes simplex viruses (HSVs) possess many biologic and functional attributes that support their use in clinical trials in children with solid tumors. Tumor cells, in an effort to escape regulatory mechanisms that would impair their growth and progression, have removed many mechanisms that would have protected them from virus infection and eventual virus-mediated destruction. Viruses engineered to exploit this weakness, like mutant HSV, can be safely employed as tumor cell killers, since normal cells retain these antiviral strategies. Many preclinical studies and early phase trials in adults demonstrated that oncolytic HSV can be safely used and are highly effective in killing tumor cells that comprise pediatric malignancies, without generating the toxic side effects of nondiscriminatory chemotherapy or radiation therapy. A variety of engineered viruses have been developed and tested in numerous preclinical models of pediatric cancers and initial trials in patients are underway. In Part II of this review series, we examine the preclinical evidence to support the further advancement of oncolytic HSV in the pediatric population. We discuss clinical advances made to date in this emerging era of oncolytic virotherapy.

Abstract 2169: SapC-DOPS induces lethal mitophagy in glioblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The goal of this study is to evaluate SapC-DOPS, a novel cancer nanotherapeutic, for glioblastoma multiforme (GBM). SapC-DOPS delivered intravenously (i.v) was found to specifically target intracranial tumors in mice bearing spontaneous brain tumor, as well in nude mice intracranially implanted with human GBM cells. Treatment of tumor bearing mice with SapC-DOPS (i.v.) significantly increased survival: 25% and 75% long-term survivors in U87ΔEGFR-Luc and X12v2 implanted mice, respectively (P 0.05). In contrast, SapC-DOPS treatment increased levels of an autophagic marker LC3-II via western blot. Autophagosome formation was also confirmed through transmission electron microscopy. Utilizing a stable GBM cell line expressing a GFP-LC3 fusion protein, we observed punctuated GFP expression following treatment, indicative of autophagosome formation. Quantification of GFP punctated cells showed a significant increase in SapC-DOPS treated cells compared to control (P<.001). Analysis of red/green fluorescence following acridine orange staining showed an induction of acidic vesicular organelles indicative of autophagolysosomes. In addition, inhibition of autophagosome formation using 3-methyladeneine or inhibition of auotphagic vacuole maturation with bafilomycin A1 resulted in a significant rescue of SapC-DOPS-induced killing (P<.001). Knockdown of ATG5 using siRNA also resulted in a rescue of SapC-DOPS-induced cell death and autophagy induction (P<.001). Interestingly, we did not observe a decrease in the activation of mTOR as determined by the phosphorylation of 4EBP1 and p70S6K which are typically induced during autophagy. This led us to test the use of rapamycin, a known inhibitor of mTOR and inducer of autophagy, in combination with SapC-DOPS. By using the Chou Talalay analysis, we observed strong synergy for multiple drug combinations in primary GBM neurospheres (combination index < .4). To investigate whether SapC-DOPS induced autophagy could be preferentially targeting mitochondria (mitophagy), we utilized MitoTracker Green (mitochondria) and LysoTracker Red (autophagolysosomes). By using confocal microscopy analysis, we were able to observe a decrease in mitochondrial mass in cells treated with SapC-DOPS as well as co-localization of mitochondria with autophagolysosomes. In addition to this, we observed a significant decrease in ATP levels following SapC-DOPS treatment (P<.01). These findings suggest therapeutic implications for treating GBM by using SapC-DOPS alone and in combination with an AKT/mTOR inhibitor. Citation Format: Jeffrey Wojton, Naduparambil K. Jacob, Nicholas Denton, Nina Dmitrieva, Hiroshi Nakashima, Chang-Hyuk Kwon, Lionel Chow, Ennio A. Chiocca, Arnab Chakravarti, Balveen Kaur, Xiaoyang Qi. SapC-DOPS induces lethal mitophagy in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2169. doi:10.1158/1538-7445.AM2013-2169

Pediatric Cancer Immunotherapy: Opportunities and Challenges

Cancer immunotherapies, widely heralded as transformational for many adult cancer patients, are becoming viable options for selected subsets of pediatric cancer patients. Many therapies are currently being investigated, from immunomodulatory agents to adoptive cell therapy, bispecific T-cell engagers, oncolytic virotherapy, and checkpoint inhibition. One of the most exciting immunotherapies recently FDA approved is the use of CD19 chimeric antigen receptor T cells for pre-B-cell acute lymphoblastic leukemia. With this approval and others, immunotherapy for pediatric cancers is gaining traction. One of the caveats to many of these immunotherapies is the challenge of predictive biomarkers; determining which patients will respond to a given therapy is not yet possible. Much research is being focused on which biomarkers will be predictive and prognostic for these patients. Despite many benefits of immunotherapy, including less long-term side effects, some treatments are fraught with immediate side effects that range from mild to severe, although most are manageable. With few downsides and the potential for disease cures, immunotherapy in the pediatric population has the potential to move to the front-line of therapeutic options.

Clodronate and Trabectedin Enhance Oncolytic Herpes Virotherapy in Preclinical Models of Ewing Sarcoma by Modulating the Tumor Immune Microenvironment

Ewing sarcoma is a highly aggressive cancer that promotes the infiltration and activation of pro-tumor M2-like macrophages. Oncolytic virotherapy that selectively infects and destroys cancer cells is a promising option for treating Ewing sarcoma. The effect of tumor macrophages on oncolytic virus therapy, however, is variable among solid tumors and is unknown in Ewing sarcoma. We tested the effects of macrophage reduction using liposomal clodronate (Clodrosome) and trabectedin on the antitumor efficacy of intratumoral oncolytic herpes simplex virus, rRp450, in two Ewing sarcoma xenograft models. Both agents enhanced antitumor efficacy without increasing virus replication. The most profound effects were in A673 with only a transient effect on response rates in TC71. Interestingly, A673 was more dependent than TC71 on macrophages for its tumorigenesis. We found Clodrosome and virus together induced expression of antitumorigenic genes and reduced expression of protumorigenic genes in both the tumor-associated macrophages and the overall tumor stroma. Trabectedin reduced intratumoral natural killer (NK) cells, myeloid-derived suppressor cells, and M2-like macrophages, and prevented their increase following virotherapy. Our data suggest that a combination of trabectedin and oncolytic herpes virotherapy warrants testing in the clinical setting.

Abstract B66: Tumor-specific differences in virus-induced inflammation may determine therapeutic response to HSV virotherapy in sarcoma models

Ewing9s Sarcoma (EWS) is the second most common childhood bone cancer with high rate of metastasis (25%) and tumor recurrence (30~40%). Despite tremendous advances in the five-year survival rate of localized disease (70~80%), overall survival for patients with relapse or metastasis remains poor ( Citation Format: Chun-Yu Chen, Brian J. Hutzen, Pin-Yi Wang, Jennifer L. Leddon, Mark A. Currier, Nicholas Denton, Timothy Eubank, Timothy P. Cripe. Tumor-specific differences in virus-induced inflammation may determine therapeutic response to HSV virotherapy in sarcoma models. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B66.