Oncolytic herpes simplex virus expressing IL-2 controls glioblastoma growth and improves survival.

BACKGROUND: Glioblastoma (GBM), a highly immunosuppressive and often fatal primary brain tumor, lacks effective treatment options. GBMs contain a subpopulation of GBM stem-like cells (GSCs) that play a central role in tumor initiation, progression, and treatment resistance. Oncolytic viruses, especially oncolytic herpes simplex virus (oHSV), replicate selectively in cancer cells and trigger antitumor immunity-a phenomenon termed the "in situ vaccine" effect. Although talimogene laherparepvec (T-VEC), an oHSV armed with granulocyte macrophage-colony stimulating factor (GM-CSF), is Food and Drug Administration (FDA)-approved for melanoma, its use in patients with GBM has not been reported. Interleukin 2 (IL-2) is another established immunotherapy that stimulates T cell growth and orchestrates antitumor responses. IL-2 is FDA-approved for melanoma and renal cell carcinoma but has not been widely evaluated in GBM, and IL-2 treatment is limited by its short half-life, minimal tumor accumulation, and significant systemic toxicity. We hypothesize that local intratumoral expression of IL-2 by an oHSV would avoid the systemic IL-2-related therapeutic drawbacks while simultaneously producing beneficial antitumor immunity. METHODS: We developed G47Δ-mIL2 (an oHSV expressing IL-2) using the flip-flop HSV BAC system to deliver IL-2 locally within the tumor microenvironment (TME). We then tested its efficacy in orthotopic mouse GBM models (005 GSC, CT-2A, and GL261) and evaluated immune profiles in the treated tumors and spleens by flow cytometry and immunohistochemistry. RESULTS: G47Δ-mIL2 significantly prolonged median survival without any observable systemic IL-2-related toxicity in the 005 and CT-2A models but not in the GL261 model due to the non-permissive nature of GL261 cells to HSV infection. The therapeutic activity of G47Δ-mIL2 in the 005 GBM model was associated with increased intratumoral infiltration of CD8+ T cells, critically dependent on the release of IL-2 within the TME, and CD4+ T cells as their depletion completely abrogated therapeutic efficacy. The use of anti-PD-1 immune checkpoint blockade did not improve the therapeutic outcome of G47Δ-mIL2. CONCLUSIONS: Our findings illustrate that G47Δ-mIL2 is efficacious, stimulates antitumor immunity against orthotopic GBM, and may also target GSC. OHSV expressing IL-2 may represent an agent that merits further exploration in patients with GBM.

Photodynamic augmentation of oncolytic virus therapy for central nervous system malignancies.

Oncolytic viruses (OVs) have emerged as a clinical therapeutic modality potentially effective for cancers that evade conventional therapies, including central nervous system malignancies. Rationally designed combinatorial strategies can augment the efficacy of OVs by boosting tumor-selective cytotoxicity and modulating the tumor microenvironment (TME). Photodynamic therapy (PDT) of cancer not only mediates direct neoplastic cell death but also primes the TME to sensitize the tumor to secondary therapies, allowing for the combination of two potentially synergistic therapies with broader targets. Here, we created G47Δ-KR, clinical oncolytic herpes simplex virus G47Δ that expresses photosensitizer protein KillerRed (KR). Optical properties and cytotoxic effects of G47Δ-KR infection followed by amber LED illumination (peak wavelength: 585-595 nm) were examined in human glioblastoma (GBM) and malignant meningioma (MM) models in vitro. G47Δ-KR infection of tumor cells mediated KR expression that was activated by LED and produced reactive oxygen species, leading to cell death that was more robust than G47Δ-KR without light. In vivo, we tested photodynamic-oncolytic virus (PD-OV) therapy employing intratumoral injection of G47Δ-KR followed by laser light tumor irradiation (wavelength: 585 nm) in GBM and MM xenografts. PD-OV therapy was feasible in these models and resulted in potent anti-tumor effects that were superior to G47Δ-KR alone (without laser light) or laser light alone. RNA sequencing analysis of post-treatment tumor samples revealed PD-OV therapy-induced increases in TME infiltration of variable immune cell types. This study thus demonstrated the proof-of-concept that G47Δ-KR enables PD-OV therapy for neuro-oncological malignancies and warrants further research to advance potential clinical translation.

Histone deacetylase inhibitors enhance oncolytic herpes simplex virus therapy for malignant meningioma.

Approximately 20% of meningiomas are not benign (higher grade) and tend to relapse after surgery and radiation therapy. Malignant (anaplastic) meningioma (MM) is a minor subset of high-grade meningioma that is lethal with no effective treatment options currently. Oncolytic herpes simplex virus (oHSV) is a powerful anti-cancer modality that induces both direct cell death and anti-tumor immunity, and has shown activity in preclinical models of MM. However, clinically meaningful efficacy will likely entail rational mechanistic combination approaches. We here show that epigenome modulator histone deacetylase inhibitors (HDACi) increase anti-cancer effects of oHSV in human MM models, IOMM-Lee (NF2 wild-type) and CH157 (NF2 mutant). Minimally toxic, sub-micromolar concentrations of pan-HDACi, Trichostatin A and Panobinostat, substantively increased the infectability and spread of oHSV G47Δ within MM cells in vitro, resulting in enhanced oHSV-mediated killing of target cells when infected at low multiplicity of infection (MOI). Transcriptomics analysis identified selective alteration of mRNA processing and splicing modules that might underlie the potent anti-MM effects of combining HDACi and oHSV. In vivo, HDACi treatment increased intratumoral oHSV replication and boosted the capacity of oHSV to control the growth of human MM xenografts. Thus, our work supports further translational development of the combination approach employing HDACi and oHSV for the treatment of MM.

In Situ Cancer Vaccination and Immunovirotherapy Using Oncolytic HSV.

Herpes simplex virus (HSV) can be genetically altered to acquire oncolytic properties so that oncolytic HSV (oHSV) preferentially replicates in and kills cancer cells, while sparing normal cells, and inducing anti-tumor immune responses. Over the last three decades, a better understanding of HSV genes and functions, and improved genetic-engineering techniques led to the development of oHSV as a novel immunovirotherapy. The concept of in situ cancer vaccination (ISCV) was first introduced when oHSV was found to induce a specific systemic anti-tumor immune response with an abscopal effect on non-injected tumors, in the process of directly killing tumor cells. Thus, the use of oHSV for tumor vaccination in situ is antigen-agnostic. The research and development of oHSVs have moved rapidly, with the field of oncolytic viruses invigorated by the FDA/EMA approval of oHSV talimogene laherparepvec in 2015 for the treatment of advanced melanoma. Immunovirotherapy can be enhanced by arming oHSV with immunomodulatory transgenes and/or using them in combination with other chemotherapeutic and immunotherapeutic agents. This review offers an overview of the development of oHSV as an agent for ISCV against solid tumors, describing the multitude of different oHSVs and their efficacy in immunocompetent mouse models and in clinical trials.

Modification of Extracellular Matrix Enhances Oncolytic Adenovirus Immunotherapy in Glioblastoma.

PURPOSE: Extracellular matrix (ECM) component hyaluronan (HA) facilitates malignant phenotypes of glioblastoma (GBM), however, whether HA impacts response to GBM immunotherapies is not known. Herein, we investigated whether degradation of HA enhances oncolytic virus immunotherapy for GBM. EXPERIMENTAL DESIGN: Presence of HA was examined in patient and murine GBM. Hyaluronidase-expressing oncolytic adenovirus, ICOVIR17, and its parental virus, ICOVIR15, without transgene, were tested to determine if they increased animal survival and modulated the immune tumor microenvironment (TME) in orthotopic GBM. HA regulation of NF-κB signaling was examined in virus-infected murine macrophages. We combined ICOVIR17 with PD-1 checkpoint blockade and assessed efficacy and determined mechanistic contributions of tumor-infiltrating myeloid and T cells. RESULTS: Treatment of murine orthotopic GBM with ICOVIR17 increased tumor-infiltrating CD8+ T cells and macrophages, and upregulated PD-L1 on GBM cells and macrophages, leading to prolonged animal survival, compared with control virus ICOVIR15. High molecular weight HA inhibits adenovirus-induced NF-κB signaling in macrophages in vitro, linking HA degradation to macrophage activation. Combining ICOVIR17 with anti-PD-1 antibody further extended the survival of GBM-bearing mice, achieving long-term remission in some animals. Mechanistically, CD4+ T cells, CD8+ T cells, and macrophages all contributed to the combination therapy that induced tumor-associated proinflammatory macrophages and tumor-specific T-cell cytotoxicity locally and systemically. CONCLUSIONS: Our studies are the first to show that immune modulatory ICOVIR17 has a dual role of mediating degradation of HA within GBM ECM and subsequently modifying the immune landscape of the TME, and offers a mechanistic combination immunotherapy with PD-L1/PD-1 blockade that remodels innate and adaptive immune cells.

Adjuvant Radiation Therapy Versus Surveillance After Surgical Resection of Atypical Meningiomas.

PURPOSE: The optimal timing of adjuvant radiation therapy (RT) in the management of atypical meningiomas remains controversial. We compared the outcomes of atypical meningiomas managed with upfront adjuvant RT versus postoperative surveillance. METHODS AND MATERIALS: Patients with intracranial atypical meningiomas who underwent resection between 2000 and 2015 at a single institution were identified. Patients receiving adjuvant RT (n = 51), defined as RT within the first year of surgery before tumor progression/recurrence (P/R), were compared with those undergoing initial surveillance (n = 179). The primary endpoints were radiographic evidence of P/R and time to P/R from surgery. RESULTS: A total of 230 patients were identified. Fifty-one (22%) patients received upfront adjuvant RT, and 179 (78%) underwent surveillance. Compared with the surveillance group, patients who received adjuvant RT had larger tumors (5.2 cm vs 4.6 cm; P = .04), were more likely to have undergone subtotal resection (65% vs 26%; P < . 01), and more often had bone invasion (18% vs 7%; P = .02). On multivariable analysis, receipt of adjuvant RT was associated with a lower risk of P/R compared with surveillance (hazard ratio, 0.21; 95% confidence interval, 0.11-0.41; P < .01). Patients who initially underwent surveillance and then received salvage RT at time of P/R had a shorter median time to local progression after RT compared with patients who developed local P/R after upfront adjuvant RT (19 vs 64 months, respectively; P < . 01). CONCLUSION: Upfront adjuvant RT was associated with improved local control in atypical meningiomas irrespective of extent of initial resection compared with surveillance. Early adjuvant RT should be strongly considered after gross total resection of atypical meningiomas.

Characterization and oncolytic virus targeting of FAP-expressing tumor-associated pericytes in glioblastoma.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts constituting the major stromal components in many types of cancer. CAFs contribute to hallmarks of cancer such as proliferation, invasion and immunosuppressive tumor microenvironment, and are associated with poor prognosis of patients with cancer. However, in glioblastoma (GBM), the most common and aggressive primary malignant brain tumor, our knowledge about CAFs or CAF-like stromal cells is limited. Here, using commonly accepted CAF markers, we characterized CAF-like cell populations in clinical glioma specimens and datasets along with mouse models of GBM. We found that tumor-associated pericytes marked by co-expression of fibroblast activation protein α (FAP) and PDGFRß represent major stromal cell subsets in both human GBM and mouse GBM models, while a fraction of mesenchymal neoplastic cells also express FAP in patient tumors. Since oncolytic viruses can kill cancer cells and simultaneously modulate the tumor microenvironment by impacting non-neoplastic populations such as immune cells and tumor vasculature, we further investigated the ability of oncolytic viruses to target GBM-associated stromal cells. An oncolytic adenovirus, ICOVIR15, carrying ∆24-E1A and an RGD-fiber, infects and depletes FAP+ pericytes as well as GBM cells in murine GBM. Our study thus identifies FAP+/PDGFRß+ pericytes as a major CAF-like stromal cell population in GBM, and highlights the unique property of this oncolytic adenovirus to target both GBM cells and GBM-associated stromal FAP+ cells.

Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma.

BACKGROUND: Temozolomide (TMZ) chemotherapy is a current standard of care for glioblastoma (GBM), however it has only extended overall survival by a few months. Because it also modulates the immune system, both beneficially and negatively, understanding how TMZ interacts with immunotherapeutics is important. Oncolytic herpes simplex virus (oHSV) is a new class of cancer therapeutic with both cytotoxic and immunostimulatory activities. Here, we examine the combination of TMZ and an oHSV encoding murine interleukin 12, G47Δ-mIL12, in a mouse immunocompetent GBM model generated from non-immunogenic 005 GBM stem-like cells (GSCs. METHODS: We first investigated the cytotoxic effects of TMZ and/or G47Δ-IL12 treatments in vitro, and then the antitumor effects of combination therapy in vivo in orthotopically implanted 005 GSC-derived brain tumors. To improve TMZ sensitivity, O6-methylguanine DNA methyltransferase (MGMT) was inhibited. The effects of TMZ on immune cells were evaluated by flow cytometery and immunohistochemistry. RESULTS: The combination of TMZ+G47Δ-IL12 kills 005 GSCs in vitro better than single treatments. However, TMZ does not improve the survival of orthotopic tumor-bearing mice treated with G47Δ-IL12, but rather can abrogate the beneficial effects of G47Δ-IL12 when the two are given concurrently. TMZ negatively affects intratumor T cells and macrophages and splenocytes. Addition of MGMT inhibitor O6-benzylguanine (O6-BG), an inactivating pseudosubstrate of MGMT, to TMZ improved survival, but the combination with G47Δ-IL12 did not overcome the antagonistic effects of TMZ treatment on oHSV therapy. CONCLUSIONS: These results illustrate that chemotherapy can adversely affect oHSV immunovirotherapy. As TMZ is the standard of care for GBM, the timing of these combined therapies should be taken into consideration when planning oHSV clinical trials with chemotherapy for GBM.

Atypical Histopathological Features and the Risk of Treatment Failure in Nonmalignant Meningiomas: A Multi-Institutional Analysis.

BACKGROUND: Histopathological grading of meningiomas is insufficient for optimal risk stratification. The purpose of the present study was to determine the prognostic value of atypical histopathological features across all nonmalignant meningiomas (World Health Organization [WHO] grade I-II). METHODS: The data from 334 patients with WHO grade I (n = 275) and grade II (n = 59) meningiomas who had undergone surgical resection from 2001 to 2015 at 2 academic centers were pooled. Progression/recurrence (P/R) was determined radiographically and measured from the date of surgery. RESULTS: The median follow-up was 52 months. The patients were stratified by the number of atypical features: 0 (n = 151), 1 (n = 71), 2 (n = 66), 3 (n = 22), and 4 or 5 (n = 24). The risk of P/R increased with an increasing number of atypical features (log-rank test, P = 0.001). The 5-year actuarial rates of P/R stratified by the number of atypical features were as follows: 0, 16.3% (95% confidence interval [CI], 10.7-24.4); 1, 21.7% (95% CI, 12.8-35.2); 2, 28.2% (95% CI, 18.4-41.7); 3, 30.4% (95% CI, 13.8-58.7); and 4 or 5, 51.4% (95% CI, 31.7-74.5). On univariate analysis, the presence of high nuclear/cytoplasmic ratio (P = 0.007), prominent nucleoli (P = 0.007), and necrosis (P < 0.00005) were associated with an increased risk of P/R. On multivariate analysis, the number of atypical features (hazard ratio [HR], 1.30; 95% CI, 1.03-1.63; P = 0.03), ≥4 mitoses per high-power fields (HR, 2.45; 95% CI, 1.17-5.15; P = 0.02), subtotal resection (HR, 3.9; 95% CI, 2.5-6.3; P < 0.0005), and the lack of adjuvant radiotherapy (HR, 2.40; 95% CI, 1.19-4.80; P = 0.01) were associated with an increased risk of P/R. CONCLUSIONS: An increased number of atypical features, ≥4 mitoses per 10 high-power fields, subtotal resection, and the lack of adjuvant radiotherapy were independently associated with P/R of WHO grade I-II meningiomas. Patients with these features might benefit from intensified therapy.

Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma.

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.

A Monoclonal Antibody Against ß1 Integrin Inhibits Proliferation and Increases Survival in an Orthotopic Model of High-Grade Meningioma.

BACKGROUND: High-grade meningiomas (HGMs; World Health Organization [WHO] classification grade II and III) have high relapse rates and poor clinical outcomes despite surgery and radiation treatments. No effective medical therapy currently exists for HGMs, and developing novel therapeutic strategies depends on the identification of molecular drivers. In cancer, ß1 integrin enhances malignant characteristics, including proliferation, invasion, and drug resistance. OBJECTIVE: We conducted this study to investigate whether ß1 integrin could be a therapeutic target in HGMs. PATIENTS AND METHODS: Expression of ß1 integrin was examined in gene array datasets, with proteomics of clinical meningioma specimens, and in patient-derived HGM xenografts. Anti-tumor activity of OS2966, a first-in-class humanized antagonizing monoclonal antibody against ß1 integrin, was tested in vitro and in vivo using an orthotopic mouse model of patient-derived malignant meningioma. RESULTS: ß1 integrin was expressed in meningiomas of all WHO grades and two xenografts tested. In vitro, OS2966 suppressed the viability of NF2-deficient MN3 sphere cells and NF2-wild-type IOMM-Lee malignant meningioma cells only when plated on laminin-coated plastic. While OS2966 decreased phosphorylation of ERK1/2 in both MN3 cells and laminin-grown IOMM-Lee cells, OS2966 only affected the phosphorylation of FAK (Tyr397) in MN3, and of Akt (Ser473) in IOMM-Lee cells, respectively, indicating differential pathway inhibition. Systemic administration of OS2966 in mice bearing orthotopic MN3 HGMs inhibited HGM cell proliferation and significantly extended overall survival of the treated mice. CONCLUSIONS: ß1 Integrin may be a therapeutic target in HGMs, and further preclinical and clinical development of OS2966 for HGM therapy is warranted.

Oncolytic Herpes Simplex Virus and PI3K Inhibitor BKM120 Synergize to Promote Killing of Prostate Cancer Stem-like Cells.

Novel therapies to override chemo-radiation resistance in prostate cancer (PCa) are needed. Prostate cancer sphere-forming cells (PCSCs) (also termed prostate cancer stem-like cells) likely participate in tumor progression and recurrence, and they are important therapeutic targets. We established PCSC-enriched spheres by culturing human (DU145) and murine (TRAMP-C2) PCa cells in growth factor-defined serum-free medium, and we characterized stem-like properties of clonogenicity and tumorigenicity. The efficacy of two different oncolytic herpes simplex viruses (oHSVs) (G47Δ and MG18L) in PCSCs was tested alone and in combination with radiation; chemotherapy; and inhibitors of phosphoinositide 3-kinase (PI3K), Wnt, and NOTCH in vitro; and, G47Δ was tested with the PI3K inhibitor BKM120 in a PCSC-derived tumor model in vivo. PCSCs were more tumorigenic than serum-cultured parental cells. Human and murine PCSCs were sensitive to oHSV and BKM120 killing in vitro, while the combination was synergistic. oHSV combined with radiation, docetaxel, Wnt, or NOTCH inhibitors was not. In athymic mice bearing DU145 PCSC-derived tumors, the combination of intra-tumoral G47Δ and systemic BKM120 induced complete regression of tumors in 2 of 7 animals, and it exhibited superior anti-tumor activity compared to either monotherapy alone, with no detectable toxicity. oHSV synergizes with BKM120 in killing PCSCs in vitro, and the combination markedly inhibits tumor growth, even inducing regression in vivo.

Genetically distinct glioma stem-like cell xenografts established from paired glioblastoma samples harvested before and after molecularly targeted therapy.

Intratumoural heterogeneity underlies tumour escape from molecularly targeted therapy in glioblastoma. A cell-based model preserving the evolving molecular profiles of a tumour during treatment is key to understanding the recurrence mechanisms and development of strategies to overcome resistance. In this study, we established a matched pair of glioblastoma stem-like cell (GSC) cultures from patient glioblastoma samples before and after epidermal growth factor receptor (EGFR)-targeted therapy. A patient with recurrent glioblastoma (MGG70R) harboring focal, high-level EGFR amplification received the irreversible EGFR tyrosine kinase inhibitor dacomitinib. The tumour that subsequently recurred (MGG70RR) showed diploid EGFR, suggesting inhibitor-mediated elimination of EGFR-amplified tumour cells and propagation of EGFR non-amplified cell subpopulations. The MGG70R-GSC line established from MGG70R formed xenografts retaining EGFR amplification and EGFR overexpression, while MGG70RR-GSC established from MGG70RR generated tumours that lacked EGFR amplification and EGFR overexpression. MGG70R-GSC-derived intracranial xenografts were more proliferative than MGG70RR-GSC xenografts, which had upregulated mesenchymal markers, mirroring the pathological observation in the corresponding patient tumours. In vitro MGG70R-GSC was more sensitive to EGFR inhibitors than MGG70RR-GSC. Thus, these molecularly distinct GSC lines recapitulated the subpopulation alteration that occurred during glioblastoma evasion of targeted therapy, and offer a valuable model facilitating therapeutic development for recurrent glioblastoma.

Author Correction: Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors.

In the version of this article originally published, the figure callout in this sentence was incorrect: "Furthermore, in S1P1-KI mice themselves, whereas PD-1 blockade was ineffectual as monotherapy, the effects of 4-1BB agonism and checkpoint blockade proved additive, with the combination prolonging median survival and producing a 50% long-term survival rate (Fig. 6f)." The callout should have been to Supplementary Fig. 6b. The error has been corrected in the PDF and HTML versions of the article.

Performance of a Hospital Pathway for Patients With a New Single Brain Mass.

PURPOSE:: To reduce care variation and improve the management of patients with newly identified single brain masses and no history of cancer, we implemented a dedicated admission protocol. METHODS:: We reviewed records of 206 patients who presented to our emergency department between January 2010 and May 2016 with a new single brain mass but no history of cancer. Patients admitted before the protocol implementation were designated the pre-implementation group (PRE), and those admitted after implementation were designated the post-implementation group (POST). RESULTS:: Ninety-six patients were in the PRE group and 110 in the POST group. Length of stay for POST patients was significantly shorter than for PRE patients (6 v 7 days, respectively; P = .042), and this effect was more robust after excluding the 66 patients who were discharged to rehabilitation, skilled nursing, or hospice facilities (5 v 7 days, respectively; P = .001). Additional comparison of POST with PRE patients showed that time to surgery was significantly reduced (2.7 v 3.5 days, respectively; P = .006) and that computed tomography scans of the chest, abdomen, and pelvis were reduced (12% v 47%, respectively; P < .001). No difference was found in the 30-day readmission rates. For patients with GBM, there also was no significant difference in time to initiation of chemoradiation or in median overall survival. CONCLUSION:: Implementation of a specialized admission pathway for patients with a new single brain mass decreased average length of hospital stay and time to surgery and reduced unnecessary diagnostic imaging tests in patients with primary brain tumors.

Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors.

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell-deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell-activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.

Oncolytic herpes simplex virus immunovirotherapy in combination with immune checkpoint blockade to treat glioblastoma.

Oncolytic viruses, such as oncolytic herpes simplex virus (oHSV), are a new class of cancer therapeutic, which selectively replicate and kill cancer cells, while inducing an inflammatory microenvironment, immunovirotherapy. Recently, an oHSV (talimogene laherparepvec) has been approved for the treatment of advanced melanoma. Glioblastoma (GBM) is an almost always lethal primary tumor in the brain that is highly immunosuppressive, and posited to contain GBM stem-like cells (GSCs). Immune checkpoint blockade has revolutionized therapy for some cancers, but not GBM. We have used a syngeneic GSC-derived orthotopic GBM model (005) to develop immunotherapeutic strategies. Curative therapy required oHSV expressing IL-12 in combination with two checkpoint inhibitors, anti-PD-1 and anti-CTLA-4. This response required CD4+ and CD8+ T cells, and macrophages in a complex interplay.

Restriction of Replication of Oncolytic Herpes Simplex Virus with a Deletion of γ34.5 in Glioblastoma Stem-Like Cells.

Oncolytic viruses, including herpes simplex viruses (HSVs), are a new class of cancer therapeutic engineered to infect and kill cancer cells while sparing normal tissue. To ensure that oncolytic HSV (oHSV) is safe in the brain, all oHSVs in clinical trial for glioma lack the γ34.5 genes responsible for neurovirulence. However, loss of γ34.5 attenuates growth in cancer cells. Glioblastoma (GBM) is a lethal brain tumor that is heterogeneous and contains a subpopulation of cancer stem cells, termed GBM stem-like cells (GSCs), that likely promote tumor progression and recurrence. GSCs and matched serum-cultured GBM cells (ScGCs), representative of bulk or differentiated tumor cells, were isolated from the same patient tumor specimens. ScGCs are permissive to replication and cell killing by oHSV with deletion of the γ34.5 genes (γ34.5- oHSV), while patient-matched GSCs were not, implying an underlying biological difference between stem and bulk cancer cells. GSCs specifically restrict the synthesis of HSV-1 true late (TL) proteins, without affecting viral DNA replication or transcription of TL genes. A global shutoff of cellular protein synthesis also occurs late after γ34.5- oHSV infection of GSCs but does not affect the synthesis of early and leaky late viral proteins. Levels of phosphorylated eIF2α and eIF4E do not correlate with cell permissivity. Expression of Us11 in GSCs rescues replication of γ34.5- oHSV. The difference in degrees of permissivity between GSCs and ScGCs to γ34.5- oHSV illustrates a selective translational regulatory pathway in GSCs that may be operative in other stem-like cells and has implications for creating oHSVs.IMPORTANCE Herpes simplex virus (HSV) can be genetically engineered to endow cancer-selective replication and oncolytic activity. γ34.5, a key neurovirulence gene, has been deleted in all oncolytic HSVs in clinical trial for glioma. Glioblastoma stem-like cells (GSCs) are a subpopulation of tumor cells thought to drive tumor heterogeneity and therapeutic resistance. GSCs are nonpermissive for γ34.5- HSV, while non-stem-like cancer cells from the same patient tumors are permissive. GSCs restrict true late protein synthesis, despite normal viral DNA replication and transcription of all kinetic classes. This is specific for true late translation as early and leaky late transcripts are translated late in infection, notwithstanding shutoff of cellular protein synthesis. Expression of Us11 in GSCs rescues the replication of γ34.5- HSV. We have identified a cell type-specific innate response to HSV-1 that limits oncolytic activity in glioblastoma.

Combinatorial Effects of VEGFR Kinase Inhibitor Axitinib and Oncolytic Virotherapy in Mouse and Human Glioblastoma Stem-Like Cell Models.

Purpose: Glioblastoma (GBM), a fatal brain cancer, contains a subpopulation of GBM stem-like cells (GSCs) that contribute to resistance to current therapy. Angiogenesis also plays a key role in GBM progression. Therefore, we developed a strategy to target the complex GBM microenvironment, including GSCs and tumor vasculature.Experimental Design: We evaluated the cytotoxic effects of VEFGR tyrosine kinase inhibitor (TKI) axitinib in vitro and then tested antitumor efficacy of axitinib in combination with oncolytic herpes simplex virus (oHSV) expressing antiangiogenic cytokine murine IL12 (G47Δ-mIL12) in two orthotopic GSC-derived GBM models: patient-derived recurrent MGG123 GSCs, forming vascular xenografts in immunodeficient mice; and mouse 005 GSCs, forming syngeneic tumors in immunocompetent mice.Results: GSCs form endothelial-like tubes and were sensitive to axitinib. G47Δ-mIL12 significantly improved survival, as did axitinib, while dual combinations further extended survival significantly compared with single therapies alone in both models. In MGG123 tumors, axitinib was effective only at high doses (50 mg/kg), alone and in combination with G47Δ-mIL12, and this was associated with greatly decreased vascularity, increased macrophage infiltration, extensive tumor necrosis, and PDGFR/ERK pathway inhibition. In the mouse 005 model, antiglioma activity, after single and combination therapy, was only observed in immunocompetent mice and not the T-cell-deficient athymic mice. Interestingly, immune checkpoint inhibition did not improve efficacy.Conclusions: Systemic TKI (axitinib) beneficially combines with G47Δ-mIL12 to enhance antitumor efficacy in both immunodeficient and immunocompetent orthotopic GBM models. Our results support further investigation of TKIs in combination with oHSV for GBM treatment. Clin Cancer Res; 24(14); 3409-22. ©2018 AACR.

Histopathological prognostic factors of recurrence following definitive therapy for atypical and malignant meningiomas.

OBJECTIVE Patients with atypical and malignant (WHO Grade II and III) meningiomas have a worse prognosis than patients with benign (WHO Grade I) meningiomas. However, there is limited understanding of the pathological risk factors that affect long-term tumor control following combined treatment with surgery and radiation therapy. Here, the authors identify clinical and histopathological risk factors for the progression and/or recurrence (P/R) of high-grade meningiomas based on the largest series of patients with atypical and malignant meningiomas, as defined by the 2007 WHO classification. METHODS Patients diagnosed with WHO Grade II and III meningiomas between 2007 and 2014 per the WHO 2007 criteria and treated with both surgery and external beam radiation therapy were retrospectively reviewed for clinical and histopathological factors at the time of diagnosis and assessed for P/R outcomes at the last available follow-up. RESULTS A total of 76 patients met the inclusion criteria (66 Grade II meningiomas, 10 Grade III meningiomas). Median follow-up from the time of pathological diagnosis was 52.6 months. Three factors were found to predict P/R: Grade III histology, brain and/or bone invasion, and a Ki-67 proliferation rate at or above 3%. The crude P/R rate was 80% for patients with Grade III histology, 40% for those with brain and/or bone involvement (regardless of WHO tumor grade), and 20% for those with a proliferative index ≥ 3% (regardless of WHO tumor grade). The median proliferation index was significantly different between patients in whom treatment failed and those in whom it did not fail (11% and 1%, respectively). CONCLUSIONS In patients with atypical or malignant meningiomas, the presence of Grade III histology, brain and/or bone involvement, and a high mitotic index significantly predicted an increased risk of treatment failure despite combination therapy. These patients can be stratified into risk groups predicting P/R. Patients with high-risk features may benefit from more treatment and counseling than is typically offered currently.