Jeffrey Wojton

NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors

The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1−/− mice and a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy.

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.

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.

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.

Aurora-A inhibition offers a novel therapy effective against intracranial glioblastoma

Glioblastoma remains a devastating disease for which novel therapies are urgently needed. Here, we report that the Aurora-A kinase inhibitor alisertib exhibits potent efficacy against glioblastoma neurosphere tumor stem-like cells in vitro and in vivo. Many glioblastoma neurosphere cells treated with alisertib for short periods undergo apoptosis, although some regain proliferative activity upon drug removal. Extended treatment, however, results in complete and irreversible loss of tumor cell proliferation. Moreover, alisertib caused glioblastoma neurosphere cells to partially differentiate and enter senescence. These effects were also observed in glioma cells treated with the Aurora-A inhibitor TC-A2317 or anti-Aurora-A siRNA. Furthermore, alisertib extended median survival of mice bearing intracranial human glioblastoma neurosphere tumor xenografts. Alisertib exerted similar effects on glioblastoma neurosphere cells in vivo and resulted in markedly reduced activated phosphoThr288Aurora-A and increased abnormal mitoses and cellular ploidy, consistent with on-target activity. Our results offer preclinical proof-of-concept for alisertib as a new therapeutic for glioma treatment.

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.

Bortezomib treatment sensitizes oncolytic HSV-1 treated tumors to NK cell immunotherapy

Purpose: Both the proteasome inhibitor bortezomib and an oncolytic herpes simplex virus-1 (oHSV)–expressing GM-CSF are currently FDA approved. Although proteasome blockade can increase oHSV replication, immunologic consequences, and consequent immunotherapy potential are unknown. In this study, we investigated the impact of bortezomib combined with oHSV on tumor cell death and sensitivity to natural killer (NK) cell immunotherapy. Experimental Design: Western blot, flow cytometry, and caspase 3/7 activity assays were used to evaluate the induction of apoptosis/autophagy and/or necroptotic cell death. Cellular and mitochondrial reactive oxygen species (ROS) production was measured using CellROX and MitoSOX. Inhibitors/shRNA–targeting ROS, JNK and RIP1 kinase (RIPK1) were used to investigate the mechanism of cell killing. The synergistic interaction between oHSV and bortezomib was calculated using a Chou–Talalay analysis. NK cells isolated from normal human blood were co-cultured with tumor cells to evaluate cellular interactions. Q-PCR, ELISA, and FACS analysis were used to evaluate NK cell activation. Intracranial tumor xenografts were used to evaluate antitumor efficacy. Results: Combination treatment with bortezomib- and oHSV-induced necroptotic cell death and increased the production of mitochondrial ROS and JNK phosphorylation. Inhibitors/shRNA of RIPK1 and JNK rescued synergistic cell killing. Combination treatment also significantly enhanced NK cell activation and adjuvant NK cell therapy of mice treated with bortezomib and oHSV improved antitumor efficacy. Conclusions: This study provides a significant rationale for triple combination therapy with bortezomib, oHSV, and NK cells to improve efficacy, in glioblastoma patients. Clin Cancer Res; 22(21); 5265–76. ©2016 AACR. See related commentary by Suryadevara et al., p. 5164

BAI1 Orchestrates Macrophage Inflammatory Response to HSV Infection—Implications for Oncolytic Viral Therapy

Purpose: Brain angiogenesis inhibitor (BAI1) facilitates phagocytosis and bacterial pathogen clearance by macrophages; however, its role in viral infections is unknown. Here, we examined the role of BAI1, and its N-terminal cleavage fragment (Vstat120) in antiviral macrophage responses to oncolytic herpes simplex virus (oHSV). Experimental Design: Changes in infiltration and activation of monocytic and microglial cells after treatment of glioma-bearing mice brains with a control (rHSVQ1) or Vstat120-expressing (RAMBO) oHSV was analyzed using flow cytometry. Co-culture of infected glioma cells with macrophages or microglia was used to examine antiviral signaling. Cytokine array gene expression and Ingenuity Pathway Analysis (IPA) helped evaluate changes in macrophage signaling in response to viral infection. TNFα-blocking antibodies and macrophages derived from Bai1−/− mice were used. Results: RAMBO treatment of mice reduced recruitment and activation of macrophages/microglia in mice with brain tumors, and showed increased virus replication compared with rHSVQ1. Cytokine gene expression array revealed that RAMBO significantly altered the macrophage inflammatory response to infected glioma cells via altered secretion of TNFα. Furthermore, we showed that BAI1 mediated macrophage TNFα induction in response to oHSV therapy. Intracranial inoculation of wild-type/RAMBO virus in Bai1−/− or wild-type non–tumor-bearing mice revealed the safety of this approach. Conclusions: We have uncovered a new role for BAI1 in facilitating macrophage anti-viral responses. We show that arming oHSV with antiangiogenic Vstat120 also shields them from inflammatory macrophage antiviral response, without reducing safety. Clin Cancer Res; 23(7); 1809–19. ©2016 AACR.

Humanized chondroitinase ABC sensitizes glioblastoma cells to temozolomide

Malignant gliomas (glioblastomas; GBMs) are extremely aggressive and have a median survival of approximately 15 months. Current treatment modalities, which include surgical resection, radiation and chemotherapy, have done little to prolong the lives of GBM patients. Chondroitin sulfate proteoglycans (CSPG) are critical for cell–cell and cell–extracellular matrix (ECM) interactions and are implicated in glioma growth and invasion. Chondroitinase (Chase) ABC is a bacterial enzyme that cleaves chondroitin sulfate disaccharide chains from CSPGs in the tumor ECM. Wild‐type Chase ABC has limited stability and/or activity in mammalian cells; therefore, we created a mutant humanized version (Chase M) with enhanced function in mammalian cells.

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