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BackgroundOncolytic viruses (OVs) show significant potential for treating tumors alongside immunotherapies.1 VV1 is an OV derived from the innocuous vesicular stomatitis virus (VSV). VV1 has been engineered to expresses human interferon (IFN) β and thyroidal sodium iodide symporter (NIS).2 VV1-infected cells produce IFNβ, which protects non-cancer cells from VV1 and allows VV1 to spread more efficiently in cancerous tissue.3 4 NIS expression on cells imports 99mTc pertechnetate, which facilitates in vivo imaging of virus infection.2 This three-part, phase 1–2 study was designed to determine the safety and tolerability of VV1 in patients with advanced unresectable and metastatic solid tumors. Here we report on the second part of this study: selection of recommended phase 2 regimen (RP2D), comprising further assessment of both duration and dose.MethodsPatients (n=29) were enrolled to receive a single IV infusion of VVI monotherapy. 23 patients received IV VV1 1.7 x1010 TCID50 over 15, 30, 60 or 180 min. Six patients received 1.0 x1011 TCID50 over 30 min with aggressive premedication and fluid support overnight. Patients were monitored for dose limiting toxicities over 21 days with efficacy assessments after 6 weeks and then every 3 months for survival. The primary objective was to establish the safety and tolerability of IV VV1. Secondary objectives included preliminary efficacy, pharmacokinetics and pharmacodynamics.ResultsIn this study VV1, demonstrated an acceptable safety profile. No deaths or Grade 4 infusion-related reactions (IRR) were reported. VV1 shedding by buccal swabs was negative at all study visits. Peak IFNβ serum levels and preliminary efficacy signals (2 PRs) were associated with 30 min infusion duration and higher dose, with RECIST data pending for 1 x 1011(table 1).Abstract 505 Table 1ConclusionsIn this study, the absence of viral shedding demonstrates that VV1 is safe for patient and caregiver with little/no environmental impact. There was no difference in safety between the lower and the higher dose infusions. In this patient population acceptable tolerability was observed at the higher dose with 30 min duration, thus the RP2D is 1x 1011 over 30 mins.Trial RegistrationNCT02923466ReferencesHemminki O, Dos Santos JM, Hemminki A. Oncolytic viruses for cancer immunotherapy. J Hematol Oncol 2020;13(1):84.Naik S, Nace R, Federspiel MJ, Barber GN, Peng KW, Russell SJ. Curative one-shot systemic virotherapy in murine myeloma. Leukemia 2012;26(8):1870–1878.Barber GN. Vesicular stomatitis virus as an oncolytic vector. Viral Immunol 2004;17(4):516–527.Lichty BD, Power AT, Stojdl DF, Bell JC. Vesicular stomatitis virus: re-inventing the bullet. Trends Mol Med 2004;10(5):210–216.Ethics ApprovalEthics approval was granted by WCG IRB. IRB tracking number: 20163005. Voluntary written informed consent was obtained from every patient prior to participation.

The use of mutant strains of oncolytic herpes simplex virus (oHSV) in early-phase human clinical trials for the treatment of glioblastoma multiforme (GBM) has proven safe, but limited efficacy suggests that more potent vector designs are required for effective GBM therapy. Inadequate vector performance may derive from poor intratumoral vector replication and limited spread to uninfected cells. Vector replication may be impaired by mutagenesis strategies to achieve vector safety, and intratumoral virus spread may be hampered by vector entrapment in the tumor-specific extracellular matrix (ECM) that in GBM is composed primarily of type IV collagen. In this report, we armed our previously described epidermal growth factor receptor (EGFR)vIII-targeted, neuronal microRNA-sensitive oHSV with a matrix metalloproteinase (MMP9) to improve intratumoral vector distribution. We show that vector-expressed MMP9 enhanced therapeutic efficacy and long-term animal survival in a GBM xenograft model. Keywords: matrix metalloproteinase 9 = MMP9, extracellular matrix = ECM, Oncolytic vectors = OVs, Glioblastoma multiforme = GBM, OVs derived from herpes simplex virus = oHSV

Summary Osteosarcoma remains one of the deadliest cancers in pediatrics and young adults. We administered two types of immunotherapies, oncolytic virotherapy and immune checkpoint inhibition, to two murine osteosarcoma models and observed divergent results. Mice bearing F420 showed no response, whereas those with K7M2 showed prolonged survival in response to combination therapy. K7M2 had higher expression of immune-related genes and higher baseline immune cell infiltrates, but there were no significant differences in tumor mutational burden or predicted MHC class I binding of nonsynonymous mutations. Instead, we found several mouse endogenous retrovirus sequences highly expressed in K7M2 compared with F420. T cell tetramer staining for one of them, gp70, was detected in mice with K7M2 but not F420, suggesting that endogenous retrovirus proteins are targets for the anti-tumor immune reaction. Given prior observations of endogenous retrovirus expression in human osteosarcomas, our findings may be translatable to human disease., Graphical abstract, Highlights • K7M2 but not F420 responds to viroimmunotherapy • K7M2 and F420 exhibit similar tumor mutational burdens • K7M2 shows much higher expression of endogenous retrovirus genes including gp70 • Tetramer staining suggest gp70 is the immune target in K7M2, Cancer; Immunology

CARMIL2 deficiency is a rare combined immunodeficiency (CID) characterized by defective CD28-mediated T cell co-stimulation, altered cytoskeletal dynamics, and susceptibility to Epstein Barr Virus smooth muscle tumors (EBV-SMTs). Case reports associated with EBV-SMTs are limited. We describe herein a novel homozygous CARMIL2 variant (c.1364_1393del) in two Saudi Arabian male siblings born to consanguineous parents who developed EBV-SMTs. CARMIL2 protein expression was significantly reduced in CD4+ T cells and CD8+ T cells. T cell proliferation on stimulation with soluble (s) anti-CD3 or (s) anti-CD3 plus anti-CD28 antibodies was close to absent in the proband, confirming altered CD28-mediated co-signaling. CD28 expression was substantially reduced in the proband's T cells, and was diminished to a lesser degree in the T cells of the younger sibling, who has a milder clinical phenotype. Defects in both T and B cell compartments were observed, including absent central memory CD8+ T cells, and decreased frequencies of total and class-switched memory B cells. FOXP3+ regulatory T cells (Treg) were also quantitatively decreased, and furthermore CD25 expression within the Treg subset was substantially reduced. These data confirm the pathogenicity of this novel loss-of-function (LOF) variant in CARMIL2 and expand the genotypic and phenotypic spectrum of CIDs associated with EBV-SMTs.

Oncolytic viruses are lytic for many types of cancers but are attenuated or replication-defective in normal tissues. Aside from tumor lysis, oncolytic viruses can induce host immune responses against cancer cells and may thus be viewed as a form of immunotherapy. Although recent successes with checkpoint inhibitors have shown that enhancing antitumor immunity can be effective, the dynamic nature of the immunosuppressive tumor microenvironment presents significant hurdles to the broader application of these therapies. Targeting one immune-suppressive pathway may not be sufficient to eliminate tumors. Here we focus on the development of the combination of oncolytic virotherapy with checkpoint inhibitors designed to target the programmed cell death protein 1 and programmed cell death ligand 1 signaling axis. We also discuss future directions for the clinical application of this novel combination therapy.

Herpes simplex virus (HSV) is one of the many viruses that have been modified or adapted for oncolytic purposes. There are two serotypes of HSV, HSV-1 and HSV-2. The majority of oncolytic HSVs, including T-VEC which has recently been approved by the US Food and Drug Administration (FDA) for clinical use in treating late stage melanoma patients, are derived from HSV-1. Recently, we and others have developed several HSV-2 based oncolytic viruses. During our in vitro characterization of oncolytic viruses developed from both serotypes (Baco-1 from HSV-1 and FusOn-H2 from HSV-2), we noticed there is a subpopulation of cancer cells in which both viruses could infect but only FusOn-H2 could spread from cell to cell on monolayers. This observation prompted us to investigate the virus receptor expression profiles in these and other tumor cells. Our data show the following: 1) This subpopulation of tumor cells only express nectin-2, not the other two major receptors (HVEM or nectin-1). 2) Baco-1 grows to a higher titer than FusOn-H2 in this subpopulation of tumor cells, but the latter kills these tumor cells more efficiently than the former. 3) FusOn-H2 is effective at treating tumors formed from these tumor cells while Baco-1 is completely ineffective. Our results suggest that this subpopulation of tumor cells may be intrinsically resistant to the therapeutic effect of a HSV-1 based oncolytic virus but they remain sensitive to a HSV-2 based virotherapy.

Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo. Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2–M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo. This combination warrants further study. Mol Cancer Ther; 16(8); 1555–65. ©2017 AACR.

Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)–dynein–dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. Implications: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.

Ewing sarcoma (EWS) is the second most common and aggressive type of metastatic bone tumor in adolescents and young adults. There is unmet medical need to develop and test novel pharmacological targets and novel therapies to treat EWS. Here, we found that EWS expresses high levels of a p53 isoform, delta133p53. We further determined that aberrant expression of delta133p53 induced HGF secretion resulting in tumor growth and metastasis. Thereafter, we evaluated targeting EWS tumors with HGF receptor neutralizing antibody (AMG102) in preclinical studies. Surprisingly, we found that targeting EWS tumors with HGF receptor neutralizing antibody (AMG102) in combination with GD2-specific, CAR-reengineered T-cell therapy synergistically inhibited primary tumor growth and establishment of metastatic disease in preclinical models. Furthermore, our data suggested that AMG102 treatment alone might increase leukocyte infiltration including efficient CAR-T access into tumor mass and thereby improves its antitumor activity. Together, our findings warrant the development of novel CAR-T-cell therapies that incorporate HGF receptor neutralizing antibody to improve therapeutic potency, not only in EWS but also in tumors with aberrant activation of the HGF/c-MET pathway.

3090 Background: VV1 (Voyager V1) is derived from VSV, an RNA virus with low human seroprevalence, engineered to replicate selectively in and kill human cancer cells. In Part 1 of this study, we demonstrated the safety of intratumoral VV1 and dose-response, using serum IFNβ as a biomarker; we observed viral replication in tumor and concomitant lymphocyte/neutrophil trafficking (SITC 2018). 2 other studies suggested greater efficacy and higher IFNβ levels with IV administration. Longer infusion durations were reported to mitigate infusion reactions (IRRs) for another oncolytic. Methods: We studied 3 different infusion durations of VV1 monotherapy at the recommended phase 2 IV dose (1.7 x 1010 TCID50) in patients with advanced solid tumors. Endpoints included safety, preliminary anti-tumor activity, viral titers, IFNβ PD and shedding. Patients received IV VV1 once on D1 and were monitored for DLT over 21 days with efficacy assessments every 6 weeks. IRRs were classified using Lee 2014 criteria for CRS as either constitutional symptoms only (G1) or involving hypotension (G2). Results: 18 patients were treated at 30, 60 and 180-minute durations (n = 7, 5 and 6, respectively). No DLTs, deaths or G3-4 related IRR AEs were observed. Most pts were female (67%), white (100%), with ECOG PS 0 (61%) and median 4 lines of prior systemic therapy (range 1-14) for colorectal (CRC; 56%), squamous cell carcinoma (11%), pheochromocytoma (11%), sarcoma (11%) or other (11%) cancers. The table shows results (number of patients) by infusion duration. Conclusions: There was no difference in safety between the 3 infusion durations, while efficacy and PD markers suggested better anti-tumor effect with 30-minute infusion. VV1 is safe for caregivers, with no viral shedding. Part 3 of this study will now treat CRC patients with VV1 in combination with a checkpoint inhibitor (avelumab). A 5-arm phase 2 basket study in combination with cemiplimab is proceeding with 30-minute infusions. Clinical trial information: NCT02923466 . [Table: see text]

High Mobility Group Box 1 (HMGB1) is a multifunctional protein that plays various roles in the processes of inflammation, cancer, and other diseases. Many reports document abundant HMGB1 release following infection with oncolytic viruses (OVs). Further, other groups including previous reports from our laboratory highlight the synergistic effects of OVs with chemotherapy drugs. Here, we show that virus-free supernatants have varying cytotoxic potential, and HMGB1 is actively secreted by two established fibroblast cell lines (NIH 3T3 and 3T6-Swiss albino) following HSV1716 infection in vitro. Further, pharmacologic inhibition or genetic knock-down of HMGB1 reveals a role for HMGB1 in viral restriction, the ability to modulate bystander cell proliferation, and drug sensitivity in 3T6 cells. These data further support the multifactorial role of HMGB1, and suggest it could be a target for modulating the efficacy of oncolytic virus therapies alone or in combination with other frontline cancer treatments.

Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3′UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.

Oncolytic virotherapy is an effective immunotherapeutic approach for cancer treatment via a multistep process including direct tumor cell lysis, induction of cytotoxic or apoptosis-sensitizing cytokines and promotion of antitumor T cell responses. Solid tumors limit the effectiveness of immunotherapeutics in diverse ways such as secretion of immunosuppressive cytokines and expression of immune inhibitory ligands to inhibit antitumor T cell function. Blocking programmed cell death protein (PD)-1 signaling, which mediates T cell suppression via engagement of its inhibitory ligands, PD-L1 or PD-L2, is of particular interest due to recent successes in many types of cancer. In syngeneic murine rhabdomyosarcoma models, we found that M3-9-M (MHC I high) but not 76-9 (MHC I low) tumors respond to oncolytic herpes simplex virus-1 (oHSV-1) and PD-1 blockade combination therapy. In addition, the therapeutic outcomes in M3-9-M tumor models correlated with the increased incidence of CD4+ and CD8+ T cells but not with the CD4+CD25+Foxp3+ regulatory T cell populations in the tumor. Overall, our data suggest the combination of PD-1 blockade and oHSV-1 may be an effective treatment strategy for childhood soft tissue sarcoma.

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 antiangiogenic Vasculostatin-120 (VStat120) for use against progressive ovarian cancer. Experimental Design: Antitumor 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, herpes simplex virus 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. Clin Cancer Res; 20(24); 6479–94. ©2014 AACR.

Neuroblastoma is unique amongst common pediatric cancers for its expression of the norepinephrine transporter (NET), enabling tumor-selective imaging and therapy with radioactive analogues of norepinephrine. The majority of neuroblastoma tumors are avid for (123)I-metaiodobenzaguanidine (mIBG) on imaging, yet the therapeutic response to (131) I-mIBG is only 30% in clinical trials, and off-target effects cause short- and long-term morbidity. We review the contemporary understanding of the tumor-selective uptake, retention, and efflux of meta-iodobenzylguanidine (mIBG) and strategies currently in development for improving its efficacy. Combination treatment strategies aimed at enhancing NET are likely necessary to reach the full potential of (131)I-mIBG therapy.

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene are common in cancer and can cause resistance to therapy. Using transcriptome analysis we identified MAF as an NF1- regulated transcription factor and verified MAF regulation through RAS/MAPK/AP-1 signaling in malignant peripheral nerve sheath tumor (MPNST) cell lines. MAF was also downregulated in human MPNST. Acute re-expression of MAF promoted expression of glial differentiation markers in MPNST cells in vitro, decreased self-renewal of embryonic precursors and transiently affected tumor cell phenotypes in vitro by increasing MPNST cell death and reducing metabolic activity and anchorage-independent growth. Paradoxically, chronic MAF overexpression enhanced MPNST cell tumor growth in vivo, correlating with elevated pS6 in vitro and in vivo. RAD001 blocked MAF-mediated tumor growth, and MAF regulated the mTOR pathway through DEPTOR. MAPK inhibition with NF1 loss of function is predicted to show limited efficacy due to reactivation of mTOR signaling via MAF.

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers. Cancer Res; 73(16); 5169–82. ©2013 AACR.

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.

Oncolytic viruses are an emerging class of cancer therapeutics that couple cytotoxicity with the induction of an anti-tumor immune response. Host-virus interactions are complex and modulated by a tumor microenvironment whose immunosuppressive activities can limit the effectiveness of cancer immunotherapies. In an effort to improve this aspect of oncolytic virotherapy, we combined the oncolytic herpes virus HSV1716 with the transforming growth factor beta receptor 1 (TGF-βR1) inhibitor A8301 to treat syngeneic models of murine rhabdomyosarcoma. Mice that received HSV1716 or A8301 alone showed little to no benefit in efficacy and survival over controls. Conversely, mice given combination therapy exhibited tumor stabilization throughout the treatment regimen, which was reflected in significantly prolonged survival times including some complete responses. In vitro cell viability and virus replication assays showed that the rhabdomyosarcoma cell lines were generally insensitive to HSV1716 and A8301. Likewise, in vivo virus replication assays showed that HSV1716 titers moderately decreased in the presence of A8301. The enhanced efficacy instead appears to be dependent on the generation of an improved anti-tumor T cell response as determined by its loss in athymic nude mice and following in vivo depletion of either CD4+ or CD8+ cells. These data suggest TGF-β inhibition can augment the immunotherapeutic efficacy of oncolytic herpes virotherapy., Graphical Abstract

Malignant peripheral nerve sheath tumors (MPNSTs) develop sporadically or in the context of neurofibromatosis type 1. Epidermal growth factor receptor (EGFR) overexpression has been implicated in MPNST formation, but its precise role and relevant signaling pathways remain unknown. We found that EGFR overexpression promotes mouse neurofibroma transformation to aggressive MPNST (GEM-PNST). Immunohistochemistry demonstrated phosphorylated STAT3 (Tyr705) in both human MPNST and mouse GEM-PNST. A specific JAK2/STAT3 inhibitor FLLL32 delayed MPNST formation in an MPNST xenograft nude mouse model. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR-STAT3 pathway is necessary for MPNST transformation and establishment of MPNST xenografts growth but not for tumor maintenance. Efficacy of the FLLL32 pharmacological inhibitor in delaying MPNST growth suggests that combination therapies targeting JAK/STAT3 might be useful therapeutics.

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.

Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1(fl/fl);Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.

Item does not contain fulltext Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disease (LPD) after hematopoietic stem cell or solid organ transplantation remains a life-threatening complication. Expression of the virus-encoded gene product, EBER, has been shown to prevent apoptosis via blockade of PKR activation. As PKR is a major cellular defense against Herpes simplex virus (HSV), and oncolytic HSV-1 (oHSV) mutants have shown promising antitumor efficacy in preclinical models, we sought to determine whether EBV-LPD cells are susceptible to infection by oHSVs. We tested three primary EBV-infected lymphocyte cell cultures from neuroblastoma (NB) patients as models of naturally acquired EBV-LPD. NB12 was the most susceptible, NB122R was intermediate and NB88R2 was essentially resistant. Despite EBER expression, PKR was activated by oHSV infection. Susceptibility to oHSV correlated with the expression of the HSV receptor, nectin-1. The resistance of NB88R2 was reversed by exogenous nectin-1 expression, whereas downregulation of nectin-1 on NB12 decreased viral entry. Xenografts derived from the EBV-LPDs exhibited only mild (NB12) or no (NB88R2) response to oHSV injection, compared with a NB cell line that showed a significant response. We conclude that EBV-LPDs are relatively resistant to oHSV virotherapy, in some cases, due to low virus receptor expression but also due to intact antiviral PKR signaling.

Purpose: Patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to use comprehensive gene expression analysis to identify novel therapeutic targets. Experimental Design: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST because of the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase overexpression in MPNST in vitro and in vivo using Aurora kinase short hairpin RNAs (shRNA) and compounds that inhibit Aurora kinase. Results: We identified 2,000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically overexpressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. Conclusion: Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST. Clin Cancer Res; 18(18); 5020–30. ©2012 AACR.

Background Expression of epidermal growth factor receptor (EGFR), a potent regulator of cellular homeostasis, is associated with aggressive tumor behavior. The mechanism by which EGFR inhibition functions is unclear, with controversial results demonstrating an effect on the tumor cells, endothelial cells, or pericytes. EGFR activation has been linked to the expression of vascular endothelial growth factor (VEGF), a known mitogen of angiogenesis, but the relationship between these factors and their effect on tumor vessel development is vague. We hypothesized that using an EGFR inhibitor on a human Ewing's sarcoma model would inhibit tumor growth by suppressing vessel proliferation. Methods A cell proliferation assay was performed on the Ewing's sarcoma (SK-NEP-1) cell line. Tumor cells were implanted intrarenally in athymic mice. Animals received daily gavage with vehicle or gefitinib 1 wk following implantation. Mice ( n = 12/cohort) were euthanized 6 wk following implantation. Remaining mice were maintained without treatment for 2 wk. Vascular changes were assessed by angiography and immunohistochemically. EGFR and vascular endothelial growth factor (VEGF) expression were quantified using quantitative polymerase chain reaction (qPCR). Results Gefitinib suppressed in vitro cell growth with an IC 50 = 1.36 μM. Minimal tumor growth suppression was noted at 6 wk (6.01 ± 1.2 g in control versus 4.61 ± 0.9 g treated, P = 0.36). After cessation of gefitinib, tumor growth was increased in both groups (7.37 ± 1.62 g versus 6.77 ± 1.53 g, P = 0.79). Microvessel density was unchanged despite EGFR inhibition (161,000 ± 16,000 pixels versus 135,000 ± 18,000 pixels, P = 0.31). At 6 wk, the vascular maturity index was similar in both groups (3.63 ± 1.12 versus 4.09 ± 1.71, P = 0.83). A downward trend in EGFR expression (49% of control) and an upward trend in VEGF levels (50% of control) occurred in the treated group. Conclusions EGFR expression was suppressed in cultured cells and xenograft tumors. Despite a cytotoxic effect on cell lines, gefitinib had little effect on tumor growth. No effects on the tumor vasculature were noted in the setting of EGFR suppression, suggesting that angiogenesis induced by SK-NEP-1 cells is refractory to EGFR inhibition. Interestingly, the resulting increase in VEGF expression following EGFR blockade, provides an alternative pro-angiogenic pathway promoting tumor survival.

Background Expression profile analyses of midkine (MDK), a multifunctional protein important in development but repressed postnataly, indicate that it is highly expressed in approximately 80% of adult carcinomas and many childhood cancers including malignant peripheral nerve sheath tumors (MPNST). In the present study, we sought to leverage its selective expression to develop a novel oncolytic herpes simplex virus (oHSV) capable of targeting developmentally primitive cancers that express MDK. Methods We sought to increase the oncolytic efficacy of the virus by fusing the human MDK promoter to the HSV type 1 neurovirulence gene, γ134.5, whose protein product increases viral replication. Results Tissue-specific MDK promoter activity in human tumor cells and transgene biological activity was confirmed in human MPNST tumor cells. In vitro replication and cytotoxicity in human fibroblasts and MPNST cells by plaque and MTT assays showed that oHSV-MDK-34.5 increased replication and cytotoxicity compared to oHSV-MDK-Luc. By contrast, no significant difference in cytotoxicity was detected between these viruses in normal human fibroblasts. oHSV-MDK-34.5 impaired in vivo tumor growth and increased median survival of MPNST tumor-bearing nude mice. Conclusions The transcriptional targeting of HSV lytic infection to MDK-expressing tumor cells is feasible. oHSV-MDK-34.5 shows enhanced anti-tumor effects both in vitro and in vivo. Further studies are warranted and may lead to its use in clinical trials. Copyright © 2010 John Wiley & Sons, Ltd.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas without effective therapeutics. Bioinformatics was used to identify potential therapeutic targets. Paired Box (PAX), Eyes Absent (EYA), Dachsund (DACH) and Sine Oculis (SIX) genes, which form a regulatory interactive network in Drosophila, were found to be dysregulated in human MPNST cell lines and solid tumors. We identified a decrease in DACH1 expression, and increases in the expressions of PAX6, EYA1, EYA2, EYA4, and SIX1-4 genes. Consistent with the observation that half of MPNSTs develop in neurofibromatosis type 1 (NF1) patients, subsequent to NF1 mutation, we found that exogenous expression of the NF1-GTPase activating protein-related domain normalized DACH1 expression. EYA4 mRNA was elevated more than 100-fold as estimated by quantitative real-time PCR in most MPNST cell lines. In vitro, suppression of EYA4 expression using short hairpin RNA reduced cell adhesion and migration and caused cellular necrosis without affecting cell proliferation or apoptotic cell death. MPNST cells expressing shEYA4 either failed to form tumors in nude mice or formed very small tumors, with extensive necrosis but similar levels of proliferation and apoptosis as control cells. Our findings identify a role of EYA4 and possibly interacting SIX and DACH proteins in MPNSTs and suggest the EYA4 pathway as a rational therapeutic target.

Oncolytic viral therapy is a promising biological therapy for the treatment of cancer. Recent advances in genetic engineering have facilitated the construction of custom-built oncolytic viruses that can be exquisitely targeted to tumors by exploiting each cancer's unique biology and their efficacy can be further enhanced by "arming" them with additional therapeutic genes. Such an approach allows the virus to unload its "therapeutic cargo" at the tumor site, thereby enhancing its anti-neoplastic properties. While several clever strategies have been recently described using genes that can induce cellular apoptosis/suicide and/or facilitate tumor/virus imaging, viruses armed with genes that also affect the tumor microenvironment present an exciting and promising approach to therapy. In this review we discuss recently developed oncolytic viruses armed with genes encoding for angiostatic factors, inflammatory cytokines, or proteases that modulate the extracellular matrix to regulate tumor vascularization, anti-tumor immune responses and viral spread throughout the solid tumor.

Oncolytic herpes simplex viruses (oHSVs) are promising anticancer therapeutics. We sought to characterize the functional genomic response of human cancer cells to oHSV infection using G207, an oHSV previously evaluated in a phase I trial. Five human malignant peripheral nerve sheath tumor cell lines, with differing sensitivity to oHSV, were infected with G207 for 6 h. Functional genomic analysis of virus-infected cells demonstrated large clusters of downregulated cellular mRNAs and smaller clusters of those upregulated, including 21 genes commonly upregulated in all five lines. Of these, 7 are known to be HSV-1 induced and 14 represent novel virus-regulated genes. Gene ontology analysis revealed that a majority of G207-upregulated genes are involved in Janus kinase/signal transducer and activator of transcription signaling, transcriptional regulation, nucleic acid metabolism, protein synthesis and apoptosis. Ingenuity networks highlighted nodes for AP-1 subunits and interferon signaling via STAT1, suppressor of cytokine signaling-1 (SOCS1), SOCS3 and RANTES. As biological confirmation, we found that virus-mediated upregulation of SOCS1 correlated with sensitivity to G207 and that depletion of SOCS1 impaired virus replication by >10-fold. Further characterization of roles provided by oHSV-induced cellular genes during virus replication may be utilized to predict oncolytic efficacy and to provide rational strategies for designing next-generation oncolytic viruses.

Malignant peripheral nerve sheath tumors (MPNST) are chemoresistant sarcomas with poor 5-year survival that arise in patients with neurofibromatosis type 1 (NF1) or sporadically. We tested three drugs for single and combinatorial effects on collected MPNST cell lines and in MPNST xenografts. The mammalian target of rapamycin complex 1 inhibitor RAD001 (Everolimus) decreased growth 19% to 60% after 4 days of treatment in NF1 and sporadic-derived MPNST cell lines. Treatment of subcutaneous sporadic MPNST cell xenografts with RAD001 significantly, but transiently, delayed tumor growth, and decreased vessel permeability within xenografts. RAD001 combined with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib caused additional inhibitory effects on growth and apoptosis in vitro, and a small but significant additional inhibitory effect on MPNST growth in vivo that were larger than the effects of RAD001 with doxorubicin. RAD001 plus erlotinib, in vitro and in vivo, reduced phosphorylation of AKT and total AKT levels, possibly accounting for their additive effect. The results support the consideration of RAD001 therapy in NF1 patient and sporadic MPNST. The preclinical tests described allow rapid screening strata for drugs that block MPNST growth, prior to tests in more complex models, and should be useful to identify drugs that synergize with RAD001. [Mol Cancer Ther 2008;7(5):1237–45]

Malignant solid tumors remain a significant clinical challenge, necessitating innovative therapeutic approaches. Oncolytic viral therapy is a nonmutagenic, biological anticancer therapeutic shown to be effective against human cancer in early studies. Because matrix metalloproteinases (MMP) play important roles in the pathogenesis and progression of cancer, we sought to determine if “arming” an oncolytic herpes simplex virus (oHSV) with an MMP-antagonizing transgene would increase virus-mediated antitumor efficacy. We generated oHSVs that express human tissue inhibitor of metalloproteinases 3 (TIMP3) or firefly luciferase and designated them rQT3 and rQLuc, respectively. We evaluated the antitumor efficacy of these viruses against neuroblastoma and malignant peripheral nerve sheath tumor (MPNST) xenografts. Relative to rQLuc, rQT3-infected primary human MPNST and neuroblastoma cells exhibited equivalent virus replication but increased cytotoxicity and reduced MMP activity. In vivo, rQT3-treated tumors showed delayed tumor growth, increased peak levels of infectious virus, immature collagen extracellular matrix, and reduced tumor vascular density. Remarkably, rQT3 treatment reduced circulating endothelial progenitors, suggesting virus-mediated antivasculogenesis. We conclude that rQT3 enhanced antitumor efficacy through multiple mechanisms, including direct cytotoxicity, elevated virus titer, and reduced tumor neovascularization. These findings support the further development of combined TIMP-3 and oncolytic virotherapy for cancer. [Cancer Res 2008;68(4):1170–9]

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Malignant peripheral nerve sheath tumors (MPNSTs), driven in part by hyperactive Ras and epidermal growth factor receptor (EGFR) signaling, are often incurable. Testing of therapeutics for MPNST has been hampered by lack of adequate xenograft models. We previously documented that human MPNST cells are permissive for lytic infection by oncolytic herpes simplex viruses (oHSV). Herein we developed and characterized a xenograft model of human MPNST and evaluated the antitumor effects of oHSV mutants (G207 and hrR3) and the EGFR inhibitor, erlotinib. Additive cytotoxicity of these agents was found in human MPNST cell lines, suggesting that EGFR signaling is not critical for virus replication. Mice bearing human MPNST tumors treated with G207 or hrR3 by intraperitoneal or intratumoral injection showed tumor-selective virus biodistribution, virus replication, and reduced tumor burden. oHSV injection demonstrated more dramatic antitumor activity than erlotinib. Combination therapies showed a trend toward an increased antiproliferative effect. Both oHSV and erlotinib were antiangiogenic as measured by proangiogenic gene expression, effect on endothelial cells and xenograft vessel density. Overall, oHSVs showed highly potent antitumor effects against MPNST xenografts, an effect not diminished by EGFR inhibition. Our data suggest that inclusion of MPNSTs in clinical trials of oHSV is warranted.

Oncolytic viral (OV) therapy, which uses genetically engineered tumor-targeting viruses, is being increasingly used in cancer clinical trials due to the direct cytolytic effects of this treatment that appear to provoke a robust immune response against the tumor. As OVs enter tumor cells, intrinsic host defenses have the potential to hinder viral replication and spread within the tumor mass. In this report, we show that histone deacetylase 6 (HDAC6) in tumor cells appears to alter the trafficking of post-entry OVs from the nucleus toward lysosomes. In glioma cell lines and glioma-stem-like cells, HDAC6 inhibition (HDAC6i) by either pharmacologic or genetic means substantially increased replication of oncolytic herpes simplex virus type 1 (oHSV). Moreover, HDAC6i increased shuttling of post-entry oHSV to the nucleus. In addition, electron microscopic analysis revealed that post-entry oHSVs are preferentially taken up into glioma cells through the endosomal pathway rather than via fusion at the cell surface. Together, these findings illustrate a mechanism of glioma cell defense against an incoming infection by oHSV and identify possible approaches to enhance oHSV replication and subsequent lysis of tumor cells.

Background Malignant peripheral nerve sheath tumors (MPNSTs) occur most frequently in patients with neurofibromatosis type 1 and are often fatal. Current therapy relies upon radical surgical resection, which often fails to completely remove the tumor. To address the need for novel treatment approaches for this disease, we sought to determine if human MPNST-derived cell lines are sensitive to oncolytic Herpes simplex virus (oHSV) infection. Activation of the Ras pathway and its inhibitory effects on protein kinase R (PKR) activation have been shown to dictate cellular permissivity to oHSV mutants. Because NF-1-associated MPNSTs possess inherent hyperactive Ras, we hypothesized these tumors would be ideal therapeutic targets for oHSVs. Procedure Human MPNST-derived cell lines were examined for sensitivity to oHSV-mediated gene transduction, virus replication, cytotoxicity, and apoptosis. These parameters were correlated with PKR activation following oHSV infection and compared with normal human Schwann cells (NHSCs) without hyperactive Ras. Results MPNST-derived cell lines were efficiently transduced, supported virus replication and were killed by the oncolytic HSV mutants, including sporadic MPNSTs without hyperactive Ras. In contrast to the highly sensitive MPNST cell lines, NHSCs did not support mutant virus replication. Conclusions MPNSTs are susceptible to lysis by oncolytic HSV mutants, regardless of Ras status. Tumor-selective virus replication in MPNST cells appears to be mediated by both cellular expression of ribonucleotide reductase and prevention of eIF2α phosphorylation. Virus-induced cytotoxicity of MPNST cell lines was caused by both direct lysis and apoptosis. Our data suggest the use of oncolytic HSV mutants may represent a novel treatment approach for patients with MPNSTs. © 2005 Wiley-Liss, Inc.

Background Inflammatory myofibroblastic tumors (IMTs) are neoplasms that are highly vascularized, have an intermediate prognosis, and are associated with infiltration, obstruction, local recurrence, and rare metastasis. Resection of large IMTs can lead to substantial morbidity and even mortality. Anecdotal experience suggests that nonsteroidal anti-inflammatory drugs may eradicate large IMTs or shrink them to a more readily resectable size and configuration. To support the hypothesis that nonsteroidal anti-inflammatory drugs are antiangiogenic for IMTs by interfering with vascular endothelial growth factor (VEGF) signaling via cyclooxygenase 2 (COX-2) inhibition, IMT specimens were immunohistochemically examined for expression of COX-2 enzyme and VEGF. Methods The diagnosis of IMT was confirmed in all 18 cases comprising the study. Intensity of COX-2 and VEGF staining was graded, and staining uniformity was examined. ALK-1 protein expression, found in up to two thirds of IMTs, was also determined. Results COX-2 and VEGF expression were identified in all tissue examined, with staining intensity varying independently. ALK-1 protein expression was identified in 33% of specimens. Its presence was not related to the intensity of COX-2 or VEGF staining. Conclusions Our data suggest that the mediators of angiogenesis, VEGF and COX-2, are present and may play an important role in the growth of IMTs.

Background High-risk neuroblastoma (Nb) is incurable using current treatment regimens in the majority of patients. Oncolytic virotherapy is a novel approach being tested for several types of adult cancers. Objectives To compare the susceptibility of Nb tumor models to oncolytic adenovirus and HSV mutants and delineate the mechanisms of resistance or sensitivity. Methods Human Nb cell lines were used to determine susceptibility to adenovirus type 5 wild-type and HSV1 mutant (NV1066) infection, adenovirus receptor expression, support of NV1066 replication, and induction of apoptosis. Human xenograft tumors in immunodeficient mice were evaluated for histological effects and tumor response to intratumoral injection of an oncolytic HSV mutant. Results All eight Nb cell lines tested in culture were relatively resistant to infection with wild type and attenuated adenoviruses. Cells expressed the cocksackie-adenovirus attachment receptor (CAR) but had low or absent expression of the internalization receptors (αvβ3, αvβ5 integrins). In contrast, all cells were uniformly sensitive to infection with the attenuated HSV mutant, NV1066. Productive virus replication and induction of apoptosis were observed in HSV-infected cells. CHLA-20 and LAN-5 xenograft tumors injected with a single dose of NV1066 showed a significant antitumor response, and the animals had a prolonged survival post infection in comparison to the PBS-treated control group. HSV injected tumors showed extensive areas of necrosis and morphologic evidence of apoptosis. Conclusions Nb tumor models are resistant to adenovirus mediated oncolysis but highly sensitive to HSV mediated oncolysis. Further studies of HSV virotherapy as a novel treatment for Nb are warranted. © 2004 Wiley-Liss, Inc.

Novel methods of local control for sarcomas are needed. We investigated the antitumor effect of two related herpes simplex virus (HSV) mutants, NV1020 and NV1066, on human rhabdomyosracoma cells and xenografts. Cell death correlated with virus replication and apoptosis in cultured cells and tumors. Complete regression was seen in all tumors250 mm(3) following a single injection, yet only half of tumors250 mm(3) showed a complete response. Fractionation of the virus dose into five injection sites did not increase transduction efficiency, transgene expression, or virus production, but did yield more widespread intratumoral distribution. Despite the same total dose of virus, improved control of large tumors was seen using fractionated injections as all large tumors (500-700 mm(3)) had durable, complete regression. Our data suggest that oncolytic HSVs may be useful for local control of bulky rhabdomyosarcoma tumors and that fractionated virus administration results in a more widespread virus infection and better tumor control. Therefore, strategies to maximize intratumoral virus distribution at initial delivery should be sought.

Aurora A Kinase (AURKA) inhibition with the investigational agent alisertib results in abnormal mitotic progression and the induction of apoptosis in multiple tumor cell lines. Cell cycle arrest is a strategy employed by a variety of viruses, including herpes simplex virus (HSV), to bolster viral reproduction. We previously demonstrated the anti-tumor efficacy of oncolytic HSV and alisertib as monotherapies in models of malignant peripheral nerve sheath tumor (MPNST). We then proceeded to test alisertib and the oncolytic HSV Seprehvir in combination, finding a synergistic anti-tumor effect between the two therapies. Considering this, we hypothesized that alisertib potentiates Seprehvir infection, leading to more rapid and robust viral proliferation with concomitant tumor regression. However, our efforts to quantify enhanced viral infection with combination therapy have uncovered no significant differences in Seprehvir viral growth kinetics witih or without alisertib therapy. Therefore, the synergistic tumor response of Seprehvir combined with alisertib in vitro or in vivo is not due to augmented viral infection. We then hypothesized that Seprehvir infection potentiates the anti-tumor effect of alisertib. In line with this hypothesis, there is a signficant decrease in tumor cell proliferation in alisertib-treated tumor cells that are exposed to soluble factors released by Seprehvir-infected cells. Furthermore, by UV inactivation of Seprehvir conditioned media, we demonstrate that these soluble death signals are not replication competent virus released by infected cells. Taken together, our results suggest a novel mechanism through which Seprehvir-infected cells release a paracrine signal to surrounding cells promoting cell death and increasing the anti-tumor activity of alisertib therapy.

Purpose: Attenuated viruses derived from herpes simplex virus (HSV) type 1 that kill tumor cells (oncolysis) are currently in clinical trials for selected cancers, primarily carcinomas and gliomas. The authors sought to determine if pediatric sarcoma cells are also sensitive to HSV-mediated oncolysis. Materials and Methods: The authors tested a panel of ten cell lines derived from rhabdomyosarcoma, osteosarcoma, Ewing sarcoma, and a secondary malignant fibrous histiocytoma for survival after exposure to attenuated HSV vectors. The viruses used included NV1020, haploid for the neurovirulence gene ICP34.5, and G207, deleted for both ICP34.5 and ribonucleotide reductase but expressing the β-galactosidase reporter gene. G207 transduction was determined by measuring β-galactosidase expression. Results: Sarcoma cells differed in their sensitivity to viral oncolysis but were relatively consistent by histologic type. Rhabdomyosarcoma and malignant fibrous histiocytoma cells were most sensitive while osteosarcoma cells were intermediately sensitive to oncolysis by both HSV recombinants. Although Ewing sarcoma cells showed efficient viral entry and gene transfer, these cells were the least susceptible to oncolysis by HSV. Conclusions: Conditionally replication-competent HSV-derived vectors may be useful for the treatment of rhabdomyosarcoma and osteosarcoma, but may not be as efficacious for treating Ewing sarcoma until the mechanism of resistance is defined and circumvented.

Glioblastoma (GBM) is a World Health Organization grade IV astrocytoma with poor patient survival, characterized by extreme heterogeneity and invasiveness, and by resistance to radiation and chemotherapeutic drugs. Brain metastases are 2-3x more frequent than GBM, with breast cancer among the top 3 brain metastasizing tumor types. The tumor suppressor phosphatase and tensin homolog on chromosome ten (PTEN) is mutated or deleted in ~50% of all high grade gliomas and is lost in 33-49% of all breast cancers. We propose to target the dysregulated PTEN pathway in brain tumors to enhance patient survival. Oncolytic viruses are engineered to destroy tumor cells while sparing non-neoplastic cells, and can be designed to incorporate payloads that enhance their oncolytic effects. PTEN-L is a recently discovered, longer form of PTEN which is secreted and membrane permeable, and retains its phosphatase activity after secretion and re-entry into cells. We have generated a novel oncolytic herpesvirus (RAPTOR) that secretes PTEN-L from infected cells for the treatment of GBM and breast cancer brain metastases (BCBM). In vitro assays to evaluate virally mediated tumor cell killing, virus replication, transgene expression, and flow cytometry to evaluate intracellular pAkt and cell surface programmed death ligand 1 (PD-L1) were performed. In vivo studies using mice bearing orthotopic human GBM (nude mice) or murine BCBM (nude and FVB/N) were used to evaluate brain immune infiltrates and survival. Western blot analyses of infected cell lysates and conditioned media show that RAPTOR produces functional secreted PTEN-L, as confirmed by intracellular pAkt FACS. Tumor cell killing and replication were equivalent between RAPTOR and control HSVQ. In vivo, treatment of mice bearing established orthotopic BCBM revealed improved anti-tumor efficacy of RAPTOR. Significantly, 9/10 RAPTOR treated mice were long term survivors in immunocompetent FVB/N mice vs. 4/10 HSVQ treated mice (p=0.044). Treatment of nude mice bearing the same tumors did not produce long term survivors, implicating the development of antitumor immunity in RAPTOR treated animals. Flow cytometry revealed significant down regulation of the T-cell repressor PD-L1 on RAPTOR vs. HSVQ treated cells. Consistent with this, FACS of virus treated tumor bearing brain hemispheres revealed RAPTOR induced a significant influx in antigen presenting, NK, and CD8 T cells 7 days post treatment. Our findings demonstrate that RAPTOR enhances survival of brain tumor bearing mice using a two-pronged approach: lytic tumor cell death and immune cell education and activation against the brain tumor. RAPTOR inhibits virally induced PD-L1 expression on tumor cells, and could pave the way for a paradigm shift in immune therapy where immune checkpoint inhibition occurs locally within the tumor rather than globally, overriding toxicity of neutralizing antibodies. Citation Format: Luke Russell, Alexander Sprague, Alena Cristina Jaime-Ramirez, Chelsea Bolyard, Yeshavanth Banasavadi-Siddegowda, JiYoung Yoo, Pin Yi Wang, Timothy Cripe, Balveen Kaur. Oncolytic and immune effects of RAPTOR: a novel oncolytic Herpes Simplex Virus 1 expressing PTEN-L for brain tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 601. doi:10.1158/1538-7445.AM2017-601

High-grade gliomas remain clinically challenging and under current therapies the prognosis is generally dismal. Intratumoral heterogeneity, therapy resistance, and tumor recurrence likely caused by glioma stem cells (GSCs) still represent major challenges, which need to be addresses by novel, multimodal therapies. We attempted to combine rQnestin34.5, an oncolytic herpes simplex virus expressing its ICP34.5 virulence gene under the glioma-specific nestin promoter, with specific inhibition of histone deacetylase 6 (HDAC6). We have previously reported that pan-HDAC inhibition using valproic acid enhances rQnestin34.5's antitumor activity and our data obtained in established glioma cell lines indicate that sole inhibition of HDAC6 can at least in part be accounted for this effect. We now analyzed the influence of the specific HDAC6 inhibitor Tubacin on rQnestin34.5 replication in GSC lines originating from proneural, classic, and mesenchymal glioma subtypes. Time-lapse microscopy revealed that plaque formation and spread were accelerated within the first 48 hours after infection when cells were pre-treated with Tubacin, translating into significantly increased cytotoxicity in the respective time frame. Viral DNA replication was enhanced after combination treatment as increased viral copy numbers per cell were detected by qPCR. Furthermore, HDAC6 inhibition increased mRNA expression levels of immediate early and late genes. Importantly, the positive effect of Tubacin on rQnestin34.5 replication and spread in GSCs was critically dependent on drug pre-treatment regimens and very low infectious titers. We also noted that the benefit of rQnestin34.5 oncolysis from HDAC6 inhibition varied quantitatively between GSCs. While responsiveness was not determined by glioma subtype or HDAC6 expression levels, we are currently investigating whether the choice of the exact viral entry mechanism can be used as a predictive marker. Future studies will also aim at establishing a therapeutic regimen demonstrating the potential benefit of HDAC6 inhibition in addition to herpes simplex oncolysis in a GSC-derived orthotopic glioma model.

After decades of unfulfilled promise, immunotherapies for cancer have finally reached a tipping point, with several FDA approved products now on the market and many more showing promise in both adult and pediatric clinical trials. Tumor cell expression of MHC Class I has emerged as a potential determinant of the therapeutic success of many immunotherapy approaches. Here we review current knowledge regarding MHC Class I expression in pediatric cancers including a discussion of prognostic significance, the opposing influence of MHC on T-cell versus NK-mediated therapies, and strategies to reverse or circumvent MHC down-regulation.

Background: Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic herpes simplex virus-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for antitumor efficacy. Experimental Design: The synergistic interaction between oHSV and bortezomib was calculated using Chou–Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western blot assays were used to evaluate induction of estrogen receptor (ER) stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Antitumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan–Meier curves and two-sided log-rank test. Results: Combination treatment with bortezomib and oHSV (34.5ENVE), displayed strong synergistic interaction in ovarian cancer, head and neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK, and IRE1α (Western blot analysis) and the UPR (induction of hsp40, 70, and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (P < 0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced antitumor efficacy in multiple different tumor models in vivo. Conclusions: The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib-induced UPR and warrants future clinical testing in patients. Clin Cancer Res; 20(14); 3787–98. ©2014 AACR.

The addition of replication-defective recombinant adenovirus to plasmid transfection (termed here "adenofection") has been shown to increase plasmid transgene expression in limited studies. Similarly, the addition of cationic liposomes to adenovirus increases adenovirus-mediated gene transduction (termed here "lipoduction"). Here we demonstrate that adenofection was effective at enhancing transgene expression when used in conjunction with a variety of different transfection reagents, including a monocationic liposome, a polycationic liposome, an activated dendrimer, a large multilamellar liposomal vesicle, and a protein/amphipathic polyamine complex. The effect was seen regardless of the cellular expression of the adenovirus receptor, CAR, in three different human cancer cell lines derived from rhabdomyosarcomas (Rh18 and RD, CAR-) and cervical carcinoma (HeLa, CAR+). The protein/amphipathic polyamine complex showed an adenofection effect but did not show a lipoduction effect, consistent with different mechanisms of action for adenofection and lipoduction. Using dual-color flow cytometric analysis of cells transfected with a plasmid expressing the enhanced blue fluorescent protein (pEBFP) and a recombinant adenovirus expressing the green fluorescent protein (Ad5-GFP), we demonstrate that adenofection works primarily by increasing gene expression within a cell, whereas lipoduction increases the percentage of cells expressing the transgene. In addition, these studies show that both adenofection and lipoduction can occur simultaneously, further increasing gene transfer. The combination of lipofection and adenovirus transduction also prolonged the duration of transient gene expression and was generally no more toxic than lipofection alone. The enhancement of gene transfer was also seen after injection of complexes directly into subcutaneous human xenograft tumors. Therefore, more effective gene transfer in vitro and in vivo of either plasmid DNA, adenovirus DNA, or both can be achieved by combining liposomal transfection with adenoviral transduction.

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was ≈250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3–6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3–6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.

Ras superfamily proteins participate in TGF-β—mediated developmental pathways that promote either tumor suppression or progression. However, the specific Ras proteins, which integrate in vivo with TGF-β signaling pathways, are unknown. As a general approach to this question, we activated all Ras proteins in vivo by genetic deletion of the RasGAP protein Nf1 and examined mice doubly deficient in a Ras protein to determine its requirement in formation of TGF-β—dependent neurofibromas that arise in Nf1-deficient mice. Animals lacking Nf1 and the Ras-related protein R-Ras2/TC21 displayed a delay in formation of neurofibromas but an acceleration in formation of brain tumors and sarcomas. Loss of R-Ras2 was associated with elevated expression of TGF-β in Nf1-deficient Schwann cell precursors, blockade of a Nf1/TGFβRII/AKT-dependent autocrine survival loop in tumor precursor cells, and decreased precursor cell numbers. Furthermore, the increase in size of sarcomas from xenografts doubly deficient in these genes was also found to be TGF-β—dependent, in this case resulting from cell nonautonomous effects on endothelial cells and myofibroblasts. Extending these findings in clinical specimens, we documented an increase in TGF-β ligands and an absence of TGF-β receptor II in malignant peripheral nerve sheath tumors, which correspond to tumors in the Nf1-deficient mouse model. Together, our findings reveal R-Ras2 as a critical regulator of TGF-β signaling in vivo. Cancer Res; 72(20); 5317–27. ©2012 AACR.

Purpose: The inhibitory role of secreted chondroitin sulfate proteoglycans on oncolytic viral (OV) therapy was examined. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove chondroitin sulfate glycosaminoglycans from proteoglycans without any deleterious effects in vivo. We examined the effect of Chase-ABC on OV spread and efficacy. Experimental Design: Three-dimensional glioma spheroids placed on cultured brain slices were utilized to evaluate OV spread. Replication-conditional OV-expressing Chase-ABC (OV-Chase) was engineered using HSQuik technology and tested for spread and efficacy in glioma spheroids. Subcutaneous and intracranial glioma xenografts were utilized to compare antitumor efficacy of OV-Chase, rHsvQ (control), and PBS. Titration of viral particles was performed from OV-treated subcutaneous tumors. Glioma invasion was assessed in collagen-embedded glioma spheroids in vitro and in intracranial tumors. All statistical tests were two sided. Results: Treatment with Chase-ABC in cultured glioma cells significantly enhanced OV spread in glioma spheroids grown on brain slices (P < 0.0001). Inoculation of subcutaneous glioma xenografts with Chase-expressing OV significantly increased viral titer (>10 times, P = 0.0008), inhibited tumor growth, and significantly increased overall animal survival (P < 0.006) compared with treatment with parental rHsvQ virus. Single OV-Chase administration in intracranial xenografts also resulted in longer median survival of animals than rHsvQ treatment (32 vs. 21 days, P < 0.018). Glioma cell migration and invasion were not increased by OV-Chase treatment. Conclusions: We conclude that degradation of glioma extracellular matrix with OV-expressing bacterial Chase-ABC enhanced OV spread and antitumor efficacy. Clin Cancer Res; 17(6); 1362–72. ©2010 AACR.

Nanovesicles composed of the phospholipid dioleylphosphatidylserine (DOPS) and a fusogenic protein, saposin C (SapC), selectively target and induce apoptotic cell death in a variety of human cancer cells in vitro and in vivo. We tested whether such tumor-homing nanovesicles are capable of delivering fluorescent probes and magnetic resonance (MR) contrast agents to cancerous tissue to aid in earlier detection and improve visualization.SapC-DOPS nanovesicles labeled with either a far-red fluorescent probe (CellVue® Maroon, CVM) or conjugated with a dextran coated MR contrast agent, ultrasmall superparamagnetic iron oxide (USPIO), were systemically administrated into xenografts for tumor detection using optical and MR imaging systems.SapC-DOPS nanovesicles were effectively detected in vivo in tumor-bearing animals using both optical and MR imaging techniques, thereby demonstrating the cancer-selective properties of these nanovesicles.SapC-DOPS nanovesicles offer promise as a new and robust theranostic agent for broad cancer-selective detection, visualization, and potential therapy.