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author:("Kaur, baleen")
1.  VSTAT120 REDUCES THE INNATE INFLAMMATORY RESPONSE TO INFECTION AND ENHANCES ONCOLYTIC VIRAL THERAPY FOR GLIOBLASTOMA 
Neuro-Oncology  2014;16(Suppl 3):iii42.
BACKGROUND: A major barrier to oncolytic virus (OV) efficacy for the treatment of Glioblastoma is the innate immune response; this response limits virus replication and reduces tumor cell destruction. Our lab developed a novel OV, RAMBO, which expresses the anti-angiogenic fragment Vasculostatin (Vstat120). In addition to its anti-angiogenic effects, we observe the RAMBO virus replicates more efficiently and persists longer in vivo than a control virus (rHSVQ1). The primary objective of this study is to investigate the effects RAMBO directed Vstat120 expression on microglia/macrophage activation and OV efficacy. METHODS: Mice bearing intracranial tumors were treated with RAMBO, rHSVQ1, or PBS, and flow cytometry utilized to examine monocyte/microglia activation/recruitment. Coculture of i9nfected glioma cells with microglia were utilized in in vitro experiemnts. Cytokines were analyzed via qPCR and ELISA. Virus replication was determined by plaque forming unit (PFU) assays. RESULTS: Treatment of intracranial tumors with OV resulted in significant monocyte infiltration into the tumor. Surprisingly, this infiltration was reduced 4.5 fold in tumors treated with the RAMBO virus as compared to rHSVQ1 (p < 0.05). Infiltrating monocytes from RAMBO treated tumors had significantly less MHCII, Ly6C, and CD86 than rHSVQ1 treated tumors (p < 0.05). The microglia of RAMBO treated tumors had significantly less MHCII and CD206 expression than those treated with rHSVQ1 (p< 0.05). To further examine the interaction of Vstat120 with microglia/monocytes we developed a coculture system with murine microglia and infected human glioma cells. Glioma cells were treated with PBS, rHSVQ1, or RAMBO and cocultured with microglia. We observed a 75-fold induction of TNF-α expression in microglia cultured with rHSVQ1 infected glioma cells compared to untreated microglia (p < 0.05). Interestingly, we observed a 3.4-fold reduction in microglia TNF-α expression in RAMBO treated cocultures compared to rHSVQ1 cocultures (p < 0.01). Consistent with this ELISA confirmed a 6.9 fold decrease in microglia TNF-α secretion (p < 0.01), and virus titers revealed a 2.6 fold increase in infectious virus in RAMBO Vs eHSVQ1 treated cocultures (p < 0.01). Cocultures treated with a microglia (murine) specific TNF-α blocking antibody rescued the differences in viral replication between rHSVQ1 and RAMBO virus titers. There was no difference in virus replication of glioma cells or microglia alone. These results suggest Vstat120 reduces inflammation and enhances OV replication in part through the suppression of TNF-α expression/secretion on microglia/monocytes. This transient suppression of the microglia/monocyte inflammatory response enhances OV replication and promotes anti-tumor efficacy. CONCLUSIONS: Vasculostatin affects macrophage recruitment and activity to improve viral replication and efficacy. SECONDARY CATEGORY: Immunobiology & Immunotherapy.
doi:10.1093/neuonc/nou209.2
PMCID: PMC4144633
2.  TGF-β treatment enhances glioblastoma virotherapy by inhibiting the innate immune response 
Cancer research  2015;75(24):5273-5282.
Oncolytic viruses including oncolytic herpes simplex virus (oHSV) have produced provocative therapeutic responses in patients with glioblastoma (GB), the most aggressive brain tumor. Paradoxically, innate immune responses mediated by natural killer (NK) cells and macrophages/microglia appear to limit oHSV efficacy. Therefore, we investigated whether pretreatment with an immunosuppressive cytokine, TGF-β, might reverse these effects and thereby potentiate oHSV efficacy. TGF-β treatment of NK cells rendered them less cytolytic against oHSV-infected GB cells and stem-like cells in vitro. Further, TGF-β treatment of NK cells, macrophages or microglia increased viral titers of oHSV in co-cultures with GB cells. In a syngeneic mouse model of GB, administering TGF-β prior to oHSV injection inhibited intracranial infiltration and activation of NK cells and macrophages. Notably, a single administration of TGF-β prior to oHSV therapy was sufficient to phenocopy NK cell depletion and suppress tumor growth and prolong survival in both orthograft and syngeneic models of GB. Collectively, our findings show how administering a single dose of TGF-β prior to oncolytic virus treatment of GB can transiently inhibit innate immune cells that limit efficacy, thereby improving therapeutic responses and survival outcomes.
doi:10.1158/0008-5472.CAN-15-0894
PMCID: PMC4681611  PMID: 26631269
TGF-β; glioblastoma; NK cells; oncolytic viruses; innate immune response; Oncolytic Herpes Simplex Virus
3.  Glucose-mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth 
Cancer cell  2015;28(5):569-581.
SUMMARY
Tumorigenesis is associated with increased glucose consumption and lipogenesis, but how these pathways are interlinked is unclear. Here, we delineate a pathway in which EGFR signaling, by increasing glucose uptake, promotes N-glycosylation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and consequent activation of SREBP-1, an endoplasmic reticulum-bound transcription factor with central roles in lipid metabolism. Glycosylation stabilizes SCAP and reduces its association with Insig-1, allowing movement of SCAP/SREBP to the Golgi and consequent proteolytic activation of SREBP. Xenograft studies reveal that blocking SCAP N-glycosylation ameliorates EGFRvIII-driven glioblastoma growth. Thus, SCAP acts as key glucose-responsive protein linking oncogenic signaling and fuel availability to SREBP-dependent lipogenesis. Targeting SCAP N-glycosylation may provide a promising means of treating malignancies and metabolic diseases.
Graphical Abstract
doi:10.1016/j.ccell.2015.09.021
PMCID: PMC4643405  PMID: 26555173
SCAP; N-glycosylation; SREBP-1; EGFR signaling; glioblastoma; Insig-1
7.  Medroxyprogesterone acetate and levonorgestrel increase genital mucosal permeability and enhance susceptibility to genital herpes simplex virus type 2 infection 
Mucosal immunology  2016;9(6):1571-1583.
Depot-medroxyprogesterone acetate (DMPA) is a hormonal contraceptive especially popular in areas with high prevalence of HIV and other sexually transmitted infections (STI). While observational studies identify DMPA as an important STI risk factor, mechanisms underlying this connection are undefined. Levonorgestrel (LNG) is another progestin used for hormonal contraception, but its effect on STI susceptibility is much less explored. Using a mouse model of genital HSV-2 infection, we herein found DMPA and LNG similarly reduced genital expression of the desmosomal cadherin desmoglein-1α (DSG1α), enhanced access of inflammatory cells to genital tissue by increasing mucosal epithelial permeability, and increased susceptibility to viral infection. Additional studies with uninfected mice revealed DMPA-mediated increases in mucosal permeability promoted tissue inflammation by facilitating endogenous vaginal microbiota invasion. Conversely, concomitant treatment of mice with DMPA and intravaginal estrogen restored mucosal barrier function and prevented HSV-2 infection. Evaluating ectocervical biopsy tissue from women before and 1 month after initiating DMPA remarkably revealed inflammation and barrier protection were altered by treatment identically to changes seen in progestin-treated mice. Together, our work reveals DMPA and LNG diminish the genital mucosal barrier; a first-line defense against all STI, but may offer foundation for new contraceptive strategies less compromising of barrier protection.
doi:10.1038/mi.2016.22
PMCID: PMC5035233  PMID: 27007679
genital mucosal barrier protection; medroxyprogesterone; levonorgestrel; estrogen
9.  Feedback Loop Regulation of SCAP/SREBP-1 by miR-29 Modulates EGFR Signaling-driven Glioblastoma Growth 
Cell reports  2016;16(6):1527-1535.
Summary
Dysregulated lipid metabolism is a characteristic of malignancies. Sterol regulatory element-binding protein-1 (SREBP-1), a transcription factor playing a central role in lipid metabolism, is highly activated in malignancies. Here, we unraveled a previously unappreciated link between miR-29 and the SCAP/SREBP-1 pathway in glioblastoma (GBM) growth. EGFR signaling enhances miR-29 expression in GBM cells via upregulation of SCAP/SREBP-1, and SREBP-1 activates miR-29 expression via binding to specific sites in its promoter. In turn, miR-29 inhibits SCAP and SREBP-1 expression by interacting with their 3′-untranslated regions. miR-29 transfection suppressed lipid synthesis and GBM cell growth, which were rescued by addition of fatty acids or N-terminal SREBP-1 expression. Xenograft studies showed that miR-29 mimics significantly inhibit GBM growth and prolong the survival of GBM-bearing mice. Our study reveals a previously unrecognized negative feedback loop in SCAP/SREBP-1 signaling mediated by miR-29, and suggests that miR-29 treatment may represent an effective means to target GBM.
eTOC Blurb
In this study, Peng et al. unravel a negative feedback loop mediated by miR-29 in SCAP/SREBP-1 signaling, advancing our understanding of lipid metabolism. The study also indicates that miR-29-mediated inhibition of SCAP/SREBP-1 may be a promising approach for targeting glioblastoma.
doi:10.1016/j.celrep.2016.07.017
PMCID: PMC4981543  PMID: 27477273
miR-29; SCAP; SREBP-1; EGFR; lipid metabolism; Glioblastoma
10.  The impact of macrophage and microglia secreted TNFα on oncolytic HSV-1 therapy in the glioblastoma tumor microenvironment 
Purpose
Oncolytic herpes simplex viruses [oHSV] represent a promising therapy for glioblastoma [GB], but their clinical success has been limited. Early innate immune responses to viral infection reduce oHSV replication, tumor destruction, and efficacy. Here, we characterized the antiviral effects of macrophages and microglia on viral therapy for GB.
Experimental Design
Quantitative flow cytometry of mice with intracranial gliomas [± oHSV] was utilized to examine macrophage/microglia infiltration and activation. In vitro co-culture assays of infected glioma cells with microglia/macrophages were utilized to test their impact on oHSV replication. Macrophages from TNFα knockout mice and blocking antibodies were used to evaluate the biological effects of TNFα on virus replication. TNFα blocking antibodies were utilized to evaluate the impact of TNFα on oHSV therapy in vivo.
Results
Flow cytometry analysis revealed a 7.9 fold increase in macrophage infiltration after virus treatment. Tumor infiltrating macrophages/microglia were polarized towards a M1, pro-inflammatory phenotype and they expressed high levels of CD86, MHCII, and Ly6C. Macrophages/microglia produced significant amounts of TNFα in response to infected glioma cells in vitro and in vivo. Utilizing TNFα blocking antibodies and macrophages derived from TNFα knockout mice we discovered TNFα induced apoptosis in infected tumor cells and inhibited virus replication. Finally, we demonstrated the transient blockade of TNFα from the tumor microenvironment with TNFα blocking antibodies significantly enhanced virus replication and survival in GB intracranial tumors.
Conclusions
The results of these studies suggest FDA approved TNFα inhibitors may significantly improve the efficacy of oncolytic virus therapy.
doi:10.1158/1078-0432.CCR-14-3118
PMCID: PMC4780415  PMID: 25829396
TNFα; macrophage; microglia; innate immune responses; oncolytic virus; apoptosis
11.  The integrin inhibitor cilengitide enhances the anti-glioma efficacy of vasculostatin-expressing oncolytic virus 
Cancer gene therapy  2013;20(8):437-444.
Oncolytic viral (OV) therapy has been considered as a promising treatment modality for brain tumors. Vasculostatin, the fragment of brain-specific angiogenesis inhibitor-1, shows anti-angiogenic activity against malignant gliomas. Previously, a vasculostatin-expressing oncolytic HSV-1, Rapid Antiangiogenesis Mediated By Oncolytic virus (RAMBO), was reported to have a potent antitumor effect. Here, we investigated the therapeutic efficacy of RAMBO and cilengitide, an integrin inhibitor, combination therapy for malignant glioma. In vitro, tube formation was significantly decreased in RAMBO and cilengitide combination treatment compared to RAMBO or cilengitide monotherapy. Moreover, combination treatment induced a synergistic suppressive effect on endothelial cell migration compared to the control virus. RAMBO, combined with cilengitide, induced synergistic cytotoxicity on glioma cells. In the caspase-8 and -9 assays, the relative absorption of U87ΔEGFR cell clusters treated with cilengitide and with RAMBO was significantly higher than of those treated with control. In addition, the activity of caspase 3/7 was significantly increased with combination therapy. In vivo, there was a significant increase in the survival of mice treated with combination therapy compared to RAMBO or cilengitide monotherapy. These results indicate that cilengitide enhanced vasculostatin-expressing OV therapy for malignant glioma and provide a rationale for designing future clinical trials combining these two agents.
doi:10.1038/cgt.2013.38
PMCID: PMC4862362  PMID: 23827879
oncolytic viral therapy; cilengitide; glioma
12.  Design, synthesis, and evaluation of cisplatin-containing EGFR targeting bioconjugates as potential therapeutic agents for brain tumors 
OncoTargets and therapy  2016;9:2769-2781.
The aim of this study was to evaluate four different platinated bioconjugates containing a cisplatin (cis-diamminedichloroplatinum [cis-DDP]) fragment and epidermal growth factor receptor (EGFR)-targeting moieties as potential therapeutic agents for the treatment of brain tumors using a human EGFR-expressing transfectant of the F98 rat glioma (F98EGFR) to assess their efficacy. The first two bioconjugates employed the monoclonal antibody cetuximab (C225 or Erbitux®) as the targeting moiety, and the second two used genetically engineered EGF peptides. C225-G5-Pt was produced by reacting cis-DDP with a fifth-generation polyamidoamine dendrimer (G5) and then linking it to C225 by means of two heterobifunctional reagents. The second bioconjugate (C225-PG-Pt) employed the same methodology except that polyglutamic acid was used as the carrier. The third and fourth bioconjugates used two different EGF peptides, PEP382 and PEP455, with direct coordination to the Pt center of the cis-DDP fragment. In vivo studies with C225-G5-Pt failed to demonstrate therapeutic activity following intracerebral (ic) convection-enhanced delivery (CED) to F98EGFR glioma-bearing rats. The second bioconjugate, C225-PG-Pt, failed to show in vitro cytotoxicity. Furthermore, because of its high molecular weight, we decided that lower molecular weight peptides might provide better targeting and microdistribution within the tumor. Both PEP382-Pt and PEP455-Pt bioconjugates were cytotoxic in vitro and, based on this, a pilot study was initiated using PEP455-Pt. The end point for this study was tumor size at 6 weeks following tumor cell implantation and 4 weeks following ic CED of PEP455-Pt to F98 glioma-bearing rats. Neuropathologic examination revealed that five of seven rats were either tumor-free or only had microscopic tumors at 42 days following tumor implantation compared to a mean survival time of 20.5 and 26.3 days for untreated controls. In conclusion, we have succeeded in reformatting the toxicity profile of cis-DDP and demonstrated the therapeutic efficacy of the PEP455-Pt bioconjugate in F98 glioma-bearing rats.
doi:10.2147/OTT.S99242
PMCID: PMC4869632  PMID: 27274273
cisplatin; F98EGFR rat glioma; molecular targets; peptides; monoclonal antibodies
13.  A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases 
Oncotarget  2016;7(19):27764-27777.
Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs.
doi:10.18632/oncotarget.8526
PMCID: PMC5053686  PMID: 27050072
breast cancer brain metastases; chimeric antigen receptor; natural killer cells; EGFR; oncolytic virus
14.  Bimodal anti-glioma mechanisms of cilengitide demonstrated by novel invasive glioma models 
Background
Integrins are expressed in tumor cells and tumor endothelial cells, and likely play important roles in glioma angiogenesis and invasion.
Objective
We investigated the anti-glioma mechanisms of cilengitide (EMD121974), an αvβ3 integrin inhibitor, utilizing the novel invasive glioma models, J3T-1and J3T-2.
Methods and results
Immunohistochemical staining of cells in culture and brain tumors in rats revealed positive αvβ3 integrin expression in J3T-2 cells and tumor endothelial cells, but not in J3T-1 cells. Established J3T-1 and J3T-2 orthotopic gliomas in athymic rats were treated with cilengitide or solvent. J3T-1 gliomas showed perivascular tumor cluster formation and angiogenesis, while J3T-2 gliomas showed diffuse single-cell infiltration without obvious angiogenesis. Cilengitide treatment resulted in a significantly decreased diameter of the J3T-1 tumor vessel clusters and its core vessels when compared with controls, while an anti-invasive effect was shown in the J3T-2 glioma with a significant reduction of diffuse cell infiltration around the tumor center. The survival of cilengitide-treated mice harboring J3T-1 tumors was significantly longer than that of control animals (median survival; 57.5 days and 31.8 days, respectively, P < 0.005), while cilengitide had no effect on the survival of mice with J3T-2 tumors (median survival; 48.9 days and 48.5, P = 0.69).
Conclusion
Our results indicate that cilengitide exerts a phenotypic anti-tumor effect by inhibiting angiogenesis and glioma cell invasion. These two mechanisms are clearly shown by the experimental treatment of two different animal invasive glioma models.
doi:10.1111/j.1440-1789.2012.01344.x
PMCID: PMC4861321  PMID: 22989076
angiogenesis; animal model; glioma; integrin; invasion
15.  Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity 
Disease Models & Mechanisms  2016;9(2):199-210.
ABSTRACT
Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for drug screening.
Summary: This zebrafish xenotransplant model of glioblastoma enables in vivo imaging of tumor cells and rapid screening for anti-glioma agents. It provides standardization of a model that is easily replicated across laboratories.
doi:10.1242/dmm.022921
PMCID: PMC4770147  PMID: 26659251
GBM9 neurospheres; Glial fibrillary acidic protein; Glioblastoma; Sox2; X12 cells; Temozolomide
16.  Changes in BAI1 and Nestin expression are prognostic indicators for survival and metastases in breast cancer and provide opportunities for dual targeted therapies 
Molecular cancer therapeutics  2014;14(1):307-314.
The 2 year survival rate of patients with breast cancer [BC] brain metastases is less than 2%. Treatment options for BC brain metastases are limited and there is an unmet need to identify novel therapies for this disease. Brain angiogenesis inhibitor 1 [BAI1] is a GPCR involved in tumor angiogenesis, invasion, phagocytosis, and synaptogenesis. For the first time, we identify BAI1 expression is significantly reduced in BC and higher expression is associated with better patient survival. Nestin is an intermediate filament whose expression is up-regulated in several cancers. We found higher Nestin expression significantly correlated with BC lung and brain metastases, suggesting both BAI1 and Nestin can be therapeutic targets for this disease. Here, we demonstrate the ability of an oncolytic virus, 34.5ENVE, to target and kill high nestin expressing cells and deliver Vstat120 [extracellular fragment of BAI1]. Finally, we created two orthotopic immune competent murine models of BC brain metastases and demonstrated 34.5ENVE extended the survival of immune competent mice bearing intracranial breast-cancer tumors.
doi:10.1158/1535-7163.MCT-14-0659
PMCID: PMC4297221  PMID: 25376607
Breast Cancer Brain Metastases; Brain Angiogenesis Inhibitor 1; Nestin; Oncolytic Virus
17.  Doxorubicin synergizes with 34.5 ENVE to enhance antitumor efficacy against metastatic ovarian cancer 
Purpose
Novel therapeutic regimens are needed to improve dismal outcomes associated with late-stage ovarian cancer. Oncolytic viruses are currently being tested in patients with ovarian cancer. Here we tested the therapeutic efficacy of combining doxorubicin with 34.5ENVE, an oncolytic herpes simplex virus transcriptionally driven by a modified stem cell-specific nestin promoter, and encoding for anti-angiogenic Vasculostatin-120 (VStat120) for use against progressive ovarian cancer.
Experimental Design
Anti-tumor efficacy of 34.5ENVE was assessed in ovarian cancer cell lines, mouse ascites-derived tumor cells, and primary patient ascites-derived tumor cells by standard MTT assay. The ability of conditioned medium derived from 34.5ENVE-infected ovarian cancer cells to inhibit endothelial cell migration was measured by a transwell chamber assay. Scope of cytotoxic interactions between 34.5ENVE and doxorubicin were evaluated using Chou-Talalay synergy analysis. Viral replication, HSV receptor expression, and apoptosis were evaluated. Efficacy of oncolytic viral therapy in combination with doxorubicin was evaluated in vivo in the murine xenograft model of human ovarian cancer.
Results
Treatment with 34.5ENVE reduced cell viability of ovarian cancer cell lines, and mouse ascites-derived and patient ascites-derived ovarian tumor cells. Conditioned media from tumor cells infected with 34.5ENVE reduced endothelial cell migration. When combined with doxorubicin, 34.5ENVE killed synergistically with a significant increase in caspase-3/7 activation, and an increase in sub-G1 population of cells. The combination of doxorubicin and 34.5ENVE significantly prolonged survival in nude mice bearing intraperitoneal ovarian cancer tumors.
Conclusions
This study indicates significant antitumor efficacy of 34.5ENVE alone, and in combination with doxorubicin against disseminated peritoneal ovarian cancer.
doi:10.1158/1078-0432.CCR-14-0463
PMCID: PMC4268177  PMID: 25294909
18.  ET-34HIGHLY SELECTIVE HSV VIROTHERAPY FOR TREATMENT OF GLIOBLASTOMA 
Neuro-Oncology  2014;16(Suppl 5):v87.
Glioblastoma Multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSV) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals. Although proven safe for treatment of GBM in patients, oHSVs efficacy has been limited, a consequence of poor intra-tumoral virus replication and spread. To counter these limitations, we have developed oHSVs whose selective replication in GBM cells does not rely on defective genes. This was accomplished by (i) full retargeting of oHSV to utilize the epidermal growth factor receptor (EGFR) for infection of human GBM tumor cells and (ii) further vector engineering to modify the essential HSV immediate early gene (ICP4) for sensitivity to repression by the microRNA mir-124. Mir-124 is highly expressed in neurons but virtually absent in GBM and highly conserved among different species. The mir-124-regulated vector was unable to replicate in nude mice following intracranial inoculation supporting vector safety and was shown to be effective (50-60% long term survival) in the treatment of human GBM in nude mice. To enhance vector intra-tumor vector spread, our EGFR retargeted-mir-124 controlled vector was further modified by vector arming with the matrix metalloproteinase gene encoding MMP9. MMP9 degrades collagen type IV, a major component of the extracellular matrix (ECM) and basement membranes of glioblastomas but absent in normal brain tissue. Studies are ongoing to determine whether MMP9 expression enhances vector spread in GBM neurospheres and as a therapeutic agent for enhanced treatment of human GBM in animals.
doi:10.1093/neuonc/nou255.34
PMCID: PMC4218131
19.  ET-40ONCOLYTIC VIRUS INDUCED HMGB1 SECRETION AND ITS IMPACT ON TUMOR ANGIOGENESIS 
Neuro-Oncology  2014;16(Suppl 5):v88.
Glioblastoma multiforme (GBM) is a difficult-to-treat, and the most aggressive of primary brain tumors. Survival is less than 2 years despite aggressive therapy. Oncolytic virus (OV) therapy has shown tremendous benefits in the treatment of GBM and other tumors. This benefit is limited by host antiviral immune responses that enhance OV clearance and tumor progression. In this study, using both in-vivo and in-vitro models of GBM, we have tested the role of High mobility group box 1 (HMGB1) in OV therapy. HMGB1 is a nuclear protein that binds and regulates transcription factors. Extracellular HMGB1 functions as damage associated molecular pattern (DAMP), it mediates the inflammatory response upon binding to inflammation mediators, such as receptor of advanced glycated end product (RAGE), toll-like receptors (TLRs) 2, 4, and 9. Here, we report that; 1) HSV1716 infection induced significant secretion of HMGB1 in human glioma cell lines and glioma neurosphere cells alone or when co-cultured with microglia. 2) Measurement of HMGB1 in serum of mice with intracranial tumors revealed an increase in serum HMGB1 levels after virus treatment. 3) HSV1716 infection enhanced human brain microvascular endothelial cells (HBMEC) migration towards infected cells, which was blocked by HMGB1 blocking antibodies. 4) HMGB1 both directly enhanced HBMEC migration and induced production of VEGF by microglia cells co-cultured with glioma cells. 5) Pharmacologic inhibitor of HMGB1 (Garbexate (100uM)), and anti-HMGB1 polyclonal blocking antibody blocked HSV1716 induced HMGB1 secretion of VEGF in vitro and in vivo. Our findings suggest that OV therapy with HSV1716 activates proangiogenic pathway by induction of HMGB1, and that tumor microenvironment contributes to it proangiogenic property. Thus we conclude that OV therapy that target HMGB1 secretion and angiogenic axis may improve virotherapy of HSV1716, and other oncolytic herpes simplex virus in tumors.
doi:10.1093/neuonc/nou255.40
PMCID: PMC4218138
20.  ET-70COMBINATION OF oHSV WITH INTEGRIN β1 NEUTRALIZING ANTIBODY (OS2966) AUGMENTS oHSV THERAPY 
Neuro-Oncology  2014;16(Suppl 5):v94.
Oncolytic HSV (oHSV) is a biologic therapy currently being tested in patients. We have previously shown that oHSV-induced Cysteine-rich 61 protein (CCN1) binds with integrin α6β1, activating a type 1 IFN-antiviral defense response. oHSV-induced CCN1 induction also increases macrophage infiltration and activation, resulting in increased virus clearance in glioblastoma cells. Integrin β1 is cell surface molecule involved in multiple functions like cellular proliferation, invasion and inflammation. Inhibition of integrin β1 has shown anti-tumor efficacy in preclinical modles of glioma. Furthermore, incorporating the Rembrandt database of the National Cancer Institute, we found integrin β1 expression is negatively associated with glioblastoma patient survival (p value < 0.0001). Interestingly, oHSV infection strongly increased integrin β1 expression (western blot). Based on this, we hypothesized that the combination of oHSV with integrin β1neutralizing antibody (OS2966) will augment oHSV therapy. Combination treatment with OS2966 increased oHSV replication and cell killing. Increased viral replication and cell killing was more obvious in the laminin-coated cells, consistent with integrin β1 signaling pathway. OS2966 treatment strongly inhibited oHSV-induced raw246.7 macrophage cell migration towards infected glioblastoma cells as dose dependent manner (migration assay). Inhibition of integrin β1in glioma cell lines and patient derived primary GSCs significantly increased markers of sensencsence such as enhanced β-gal activity, along with induction of p21, and reduction of phosphorylation of AKT and Rb, indicating that integrin β1 activation is involved in self-renewal, and proliferation. In conclusion, oHSV-induced integrin β1 expression sensitize to OS2966 effect, resulting in enhanced viral replication and inhibition of macrophage migration. Our findings reveal how integrin β1 limits oHSV replication. Our findings also reveal how OS2966, an integrin β1 blocking antibody which has been humanized in anticipation of potential clinical trials, inhibits the innate immune response elicited upon oHSV therapy. The results will lead to the development of a novel oHSV therapeutic strategy.
doi:10.1093/neuonc/nou255.67
PMCID: PMC4218167
21.  NT-12IMPACT OF HUMANIZED CHONDROITINASE ABC ON OV THERAPY FOR INTRACRANIAL TUMORS 
Neuro-Oncology  2014;16(Suppl 5):v161.
Breast cancer is a leading cause of cancer death and source of brain metastases (BM) among women in the US. With a dismal two year survival of 2%, novel treatment modalities are essential. Chondroitin sulfate proteoglycan (CSPG) expression has been directly associated with metastatic potential in breast cancer. The Chondroitinase ABC (Chase) enzyme is able to digest CSPGs, thereby disrupting critical extracellular matrix interactions. We recently generated an oncolytic virus expressing Chase which resulted in some efficacy in mice bearing intracranial gliomas. Here, we developed a second generation Chase mutant (ChaseM) vector, which results in optimal enzymatic expression and function in mammalian cells. ChaseM decreases neurosphere formation in vitro, akin to the phenotype observed with pharmacologic blockade of CSPG assembly. In nude mice bearing glioma xenografts, OV-ChaseM led to a significant increase in median survival over control virus treated animals (p = 0.005). CSPGs in the CNS are formed as an integral part of the glial scar and implicated in the sequestration of inflammation at the site of insult. Thus, we hypothesize that the dissolution of CSPGs by OV-ChaseM will facilitate the influx of immune cells into the tumor stroma, alleviate microglia suppression and elevate T-cell responses following viral therapy, resulting in long term responses. We generated a syngeneic model of BM to test the ability of OV-ChaseM to facilitate these effects. Intracranial implantation of DB7 breast cancer cells results in 100% tumor development and pathology akin to human BM disease. In this model, a single dose of OV-ChaseM resulted in a significant survival advantage compared to controls (p = 0.02). Ongoing studies are evaluating the contribution of host anti-tumor immune activation towards this survival efficacy. With this data, we aim to broaden the scope of treatment for brain cancer patients and provide therapeutic evidence for regiments to be implemented in the clinic.
doi:10.1093/neuonc/nou265.12
PMCID: PMC4218421
22.  RM-01RADIATION INDUCED PRMT5 EXPRESSION IS REGULATED BY miRNA-96 IN GLIOBLASTOMA 
Neuro-Oncology  2014;16(Suppl 5):v186.
BACKGROUND: Glioblastoma (GB) represent the most common and aggressive histological subtype among malignant gliomas and are associated with poor outcomes. It is composed of multiple types of tumor cells displaying multiple genetic and epigenetic defects affecting tumor suppressor gene expression, regulation of growth and apoptosis. Recurrence is the major concern with the current standard therapy. Protein arginine methyltransferase enzyme 5 (PRMT5), that regulates many cellular processes through its methylation-dependent and independent activity is overexpressed in this disease. We hypothesize that PRMT5 contributes to radioresistance in GB. Using patient derived GB neurosphere models we are probing the role of PRMT5 in inducing radioresistance in GB. METHODS: GB neurospheres dispersed into single cell, were treated with radiation or transfected with specified siRNAs. At appropriate time, cells were collected and probed for mRNA expression by qPCR and protein level by immunblotting. RESULTS: We found that PRMT5 associates with Hsp90 in GB neurospheres and that this binding is essential for PRMT5 stability. In addition to higher levels of baseline expression, radiation treatment increases PRMT5 mRNA level by 2 fold and protein expression by 60%. This increase is independent of its post-translational stability conferred by its association with Hsp90. However radiation treatment decreases miRNA-96 level significantly (p ≤ 0.01). Overxpression of miRNA-96 down regulates the expression of PRMT5 protein. Additionally we found that PRMT5 is important for regulation of HIF1alpha, a transcription factor induced post radiation and known to be involved in radiation resistance. We are currently investigating the implications of PRMT5 inhibition in conjunction with radiation in vivo. CONCLUSIONS: Based on our findings we conclude that radiation treatment induced suppression of miRNA-96 causes increased expression of PRMT5. This suggests that increased expression of PRMT5 may contribute for HIF-mediated radioresistance in GB.
doi:10.1093/neuonc/nou273.1
PMCID: PMC4218557
23.  HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide 
Oncotarget  2015;6(31):31134-31150.
Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9–5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.
PMCID: PMC4741593  PMID: 26429859
myeloma; CD44; miR-9–5p; IGF2BP3; lenalidomide
24.  Piperlongumine treatment inactivates peroxiredoxin 4, exacerbates endoplasmic reticulum stress, and preferentially kills high-grade glioma cells 
Neuro-Oncology  2014;16(10):1354-1364.
Backgrounds
Piperlongumine, a natural plant product, kills multiple cancer types with little effect on normal cells. Piperlongumine raises intracellular levels of reactive oxygen species (ROS), a phenomenon that may underlie the cancer-cell killing. Although these findings suggest that piperlongumine could be useful for treating cancers, the mechanism by which the drug selectively kills cancer cells remains unknown.
Methods
We treated multiple high-grade glioma (HGG) sphere cultures with piperlongumine and assessed its effects on ROS and cell-growth levels as well as changes in downstream signaling. We also examined the levels of putative piperlongumine targets and their roles in HGG cell growth.
Results
Piperlongumine treatment increased ROS levels and preferentially killed HGG cells with little effect in normal brain cells. Piperlongumine reportedly increases ROS levels after interactions with several redox regulators. We found that HGG cells expressed higher levels of the putative piperlongumine targets than did normal neural stem cells (NSCs). Furthermore, piperlongumine treatment in HGG cells, but not in normal NSCs, increased oxidative inactivation of peroxiredoxin 4 (PRDX4), an ROS-reducing enzyme that is overexpressed in HGGs and facilitates proper protein folding in the endoplasmic reticulum (ER). Moreover, piperlongumine exacerbated intracellular ER stress, an effect that was mimicked by suppressing PRDX4 expression.
Conclusions
Our results reveal that the mechanism by which piperlongumine preferentially kills HGG cells involves PRDX4 inactivation, thereby inducing ER stress. Therefore, piperlongumine treatment could be considered as a novel therapeutic option for HGG treatment.
doi:10.1093/neuonc/nou088
PMCID: PMC4165421  PMID: 24879047
Endoplasmic reticulum stress; high-grade glioma; piperlongumine; peroxiredoxin 4; reactive oxygen species
25.  Aurora-A inhibition offers a novel therapy effective against intracranial glioblastoma 
Cancer research  2014;74(19):5364-5370.
Glioblastoma (GB) 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 GB neurosphere tumor stem-like cells in vitro and in vivo. Many GB 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 GB 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 GB neurosphere tumor xenografts. Alisertib exerted similar effects on GB neurosphere cells in vivo based on the presence of activated phosphoThr288Aurora-A, abnormal mitoses and increased cellular ploidy, consistent with on-target activity. Our results offer preclinical proof-of-concept for alisertib as a new therapeutic for glioma treatment.
doi:10.1158/0008-5472.CAN-14-0386
PMCID: PMC4528677  PMID: 25106428
Aurora-A; alisertib; glioblastoma; neurospheres; orthotopic xenograft model

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