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MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409

We previously showed that derivatives of the Chinese traditional medicine indirubin promote survival in murine glioblastoma models (Williams et al. Cancer Research 2011). However, poor drug solubility hampered further development of this approach. Here we introduce PPRX-1701, a 6’-bromoindirubin acetoxime (BiA) containing drug/polymer nanoparticle formulation which can be injected intravenously at relatively high concentrations. Mechanistically, BiA is thought to act as a broadly selective serine-threonine protein kinase inhibitor, with activity against Src family kinases, GSK-3 and JAK2. Our preliminary data show that intravenous administration of PPRX-1701 is well-tolerated and can reach intracranial murine glioblastoma as assessed by luminescent reporter assays. PPRX-1701 administration leads to improved survival in the GL261 glioblastoma mouse model (median survival 30 days (control), 47 days (treated), p < 0.0001). Treatment with PPRX-1701 was associated with alterations in the tumor immune microenvironment, with reduced Tregs and pro-tumor macrophages, and increased CD8+ T cells. Further preliminary mechanistic studies have shown that PPRX-1701/BiA blocks the expression multiple immunosuppressive molecules in GBM downstream of interferon-g (IFNg) including PD-L1 and indoleamine 2,3-dioxygenase 1 (IDO1) - a key enzyme in the tryptophan–kynurenine–aryl hydrocarbon receptor (Trp–Kyn–AhR) immunosuppressive pathway. BiA promoted more effective T-cell mediated tumor cell killing using patient derived glioblastoma ex vivo co-culture models. This data supports further development of PPRX-1701 is a candidate immunotherapeutic agent for glioblastoma treatment. Ongoing pre-clinical studies are investigating combination with other relevant therapies.

One of the cancer hallmarks is immune evasion mediated by the tumour microenvironment (TME). Oncolytic virotherapy is a form of immunotherapy based on the application of oncolytic viruses (OVs) that selectively replicate in and induce the death of tumour cells. Virotherapy confers reciprocal interaction with the host’s immune system. The aim of this review is to explore the role of macrophage-mediated responses in oncolytic virotherapy efficacy. The approach was to study current scientific literature in this field in order to give a comprehensive overview of the interactions of OVs and macrophages and their effects on the TME. The innate immune system has a central influence on the TME; tumour-associated macrophages (TAMs) generally have immunosuppressive, tumour-supportive properties. In the context of oncolytic virotherapy, macrophages were initially thought to predominantly contribute to anti-viral responses, impeding viral spread. However, macrophages have now also been found to mediate transport of OV particles and, after TME infiltration, to be subjected to a phenotypic shift that renders them pro-inflammatory and tumour-suppressive. These TAMs can present tumour antigens leading to a systemic, durable, adaptive anti-tumour immune response. After phagocytosis, they can recirculate carrying tissue-derived proteins, which potentially enables the monitoring of OV replication in the TME. Their role in therapeutic efficacy is therefore multifaceted, but based on research applying relevant, immunocompetent tumour models, macrophages are considered to have a central function in anti-cancer activity. These novel insights hold important clinical implications. When optimised, oncolytic virotherapy, mediating multifactorial inhibition of cancer immune evasion, could contribute to improved patient survival.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV(+) mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV(+) mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV(+) mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV(+) mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.

The authors wish to make the following corrections to this paper [1] [...]

Purpose: Oncolytic herpes simplex virus-1 (oHSV) infection of brain tumors activates NOTCH, however the consequences of NOTCH on oHSV-induced immunotherapy is largely unknown. Here we evaluated the impact of NOTCH blockade on virus-induced immunotherapy. Experimental Design: RNA sequencing (RNA-seq), TCGA data analysis, flow cytometry, Luminex- and ELISA-based assays, brain tumor animal models, and serum analysis of patients with recurrent glioblastoma (GBM) treated with oHSV was used to evaluate the effect of NOTCH signaling on virus-induced immunotherapy. Results: TCGA data analysis of patients with grade IV glioma and oHSV treatment of experimental brain tumors in mice showed that NOTCH signaling significantly correlated with a higher myeloid cell infiltration. Immunofluorescence staining and RNA-seq uncovered a significant induction of Jag1 (NOTCH ligand) expression in infiltrating myeloid cells upon oHSV infection. Jag1-expressing macrophages further spread NOTCH activation in the tumor microenvironment (TME). NOTCH-activated macrophages increased the secretion of CCL2, which further amplified myeloid-derived suppressor cells. CCL2 and IL10 induction was also observed in serum of patients with recurrent GBM treated with oHSV (rQnestin34.5; NCT03152318). Pharmacologic blockade of NOTCH signaling rescued the oHSV-induced immunosuppressive TME and activated a CD8-dependent antitumor memory response, resulting in a therapeutic benefit. Conclusions: NOTCH-induced immunosuppressive myeloid cell recruitment limited antitumor immunity. Translationally, these findings support the use of NOTCH inhibition in conjunction with oHSV therapy.

Cytomegalovirus (CMV) is widespread in humans and has been implicated in glioblastoma (GBM) and other tumors. However, the role of CMV in GBM remains poorly understood and the mechanisms involved are not well-defined. The goal of this study was to identify candidate pathways relevant to GBM that may be modulated by CMV. Analysis of RNAseq data after CMV infection of patient-derived GBM cells showed significant upregulation of GBM-associated transcripts including the MET oncogene, which is known to play a role in a subset of GBM patients. These findings were validated in vitro in both mouse and human GBM cells. Using immunostaining and RT-PCR in vivo, we confirmed c-MET upregulation in a mouse model of CMV-driven GBM progression and in human GBM. siRNA knockdown showed that MET upregulation was dependent on CMV-induced upregulation of NF-κB signaling. Finally, proneural GBM xenografts overexpressing c-MET grew much faster in vivo than controls, suggesting a mechanism by which CMV infection of tumor cells could induce a more aggressive mesenchymal phenotype. These studies implicate the CMV-induced upregulation of c-MET as a potential mechanism involved in the effects of CMV on GBM growth.

INTRODUCTION Glioblastoma (GBM), an aggressive brain tumor with a poor prognosis, presents an average of 2% of patients surviving beyond 2 years after diagnosis. Therapies to effectively manage glioblastoma are hindered due to the presence of the blood-brain barrier (BBB). Previously, a cell-penetrating peptide, M13, was conjugated to a Pt(IV) cisplatin prodrug, via amide bond formation. The conjugated Pt(IV) releases active cisplatin upon intracellular reduction. Herein, we investigated the BBB-penetrance and biodistribution of M13 conjugated to Pt(IV), as well as its effectiveness against GBM in mouse models. METHODS M13 platinum-conjugate tumor cell killing capacity was assessed by luminescent cell viability assays in vitro. By using Inductively-Coupled Plasma Mass-Spectrometry for platinum detection, BBB penetration and bio-distribution studies were performed in a three-dimensional BBB spheroid in vitro model and in vivo in mouse brain, intracranial tumor, and peripheral organs. Dose-regime studies involved observations of symptomatology and weight variations after bi-weekly injections of platinum compounds at 2mg/kg and 5mg/kg. RESULTS The Pt(IV)-M13 conjugate possesses tumor cell killing effects similar to cisplatin when tested in GBM cell lines in vitro. Platinum increased by using Pt(IV)-M13 when compared to cisplatin in our in vitro BBB-spheroid model (20-fold, p-value=0.0033), in brain tissue (10-fold, p< 0.0001) and GBM tumor-bearing mice models (7.5-fold, p< 0.0001). Bio-distribution of platinum delivered by Pt(IV)-M13 in spleen, heart and blood was significantly different to cisplatin 5hrs. after intravenous injection (p< 0.001). Bi-weekly dose regimes of Pt(IV)-M13 are tolerable in nude mice without toxicity at a similar concentration to reported tolerable cisplatin doses at 5 mg/kg. Finally, Pt(IV)-M13 significantly increased survival in a murine glioblastoma xenograft model compared with controls (median 24 days vs. 29 days, p-value=0.0071). CONCLUSION Overall, our data support the further development of BBB-crossing peptide-drug conjugates for GBM treatment.

Genetic and epigenetic mechanisms determine SCD inhibitor sensitivity and acquired resistance through FOSB in cancer., The lipogenic enzyme stearoyl CoA desaturase (SCD) plays a key role in tumor lipid metabolism and membrane architecture. SCD is often up-regulated and a therapeutic target in cancer. Here, we report the unexpected finding that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and unintentional monoallelic co-deletion with phosphatase and tensin homolog (PTEN) in a subset of patients. Cell lines from this subset expressed undetectable SCD, yet retained residual SCD enzymatic activity. Unexpectedly, these lines evolved to survive independent of SCD through unknown mechanisms. Cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Last, we identify that SCD-dependent lines acquire resistance through a previously unknown FBJ murine osteosarcoma viral oncogene homolog B (FOSB)–mediated mechanism. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor-bearing mice treated with SCD inhibitor.

INTRODUCTION. Glioblastoma is the most aggressive primary malignant brain tumor and has a median survival of approximately 15 months. Treatment is hindered by the blood-brain barrier (BBB) that restricts entry of the vast majority of cancer therapeutics to the brain. We have designed a peptide-drug conjugate, (Pt(IV)-M13), comprised of M13, a perfluoroaryl-stapled cell-penetrating peptide, covalently linked to Pt(IV) via an amide bond. Upon intracellular reduction the Pt(IV) prodrug releases active cisplatin eliciting DNA damage and cell death. To investigate the properties of Pt(IV)-M13 in detail, we determined in vitro cell killing, platinum brain accumulation, biodistribution, and efficacy in mouse models. METHODS. Pt(IV)-M13 tumor cytotoxicity was assessed in vitro using standard approaches. In vivo platinum levels were measured in the brain by ICP-MS, and BBB penetration assessed through a multicellular 3D in vitro BBB model, and biodistribution to peripheral organs was determined. Toxicity from bi-weekly injections of the Pt(IV)-M13 platinum conjugate was monitored from 5mg/kg to 30mg/kg. Animal survival was determined in a glioblastoma xenograft model. DNA damage was quantified by γH2AX staining. RESULTS. Pt(IV)-M13 possesses in vitro tumor cell killing effects similar to cisplatin and Pt(IV) alone. Pt(IV)-M13 showed increased BBB penetration when compared to cisplatin in our in vitro BBB spheroid model (>20-fold), in brain tissue (7-fold) and glioblastoma tumor-bearing mice (8-fold). Five hours after intravenous injection, biodistribution of platinum delivered by Pt(IV)-M13 was higher than cisplatin in the spleen, but lower in the heart. Bi-weekly injections of Pt(IV)-M13 were well-tolerated in nude mice to at least 15mg/kg, three times the reported cisplatin MTD. Finally, Pt(IV)-M13 treatment increased survival in a murine glioblastoma xenograft model (median survival= 33 days for Pt(IV)-M13 vs. 22.5 days for cisplatin and 24 days Pt(IV) alone. Elevated γH2AX foci were observed in Pt(IV)-M13 treated mice providing direct support for the improved delivery of cisplatin to brain tumors using this approach. CONCLUSION. Pt(IV)-M13 leads to enhanced BBB penetration and cisplatin accumulation in glioblastoma models, with consequent animal survival benefit. Overall, our data support further investigation and development of Pt(IV)-M13 for glioblastoma treatment. Future studies will include additional glioblastoma models, and further optimization of this approach. Citation Format: Jorge-Luis Jimenez-Macias, Yen-Chun Lee, Tomer Finkelberg, Gilles Berger, Michal Nowicki, Choi-Fong Cho, Bogdan Fedeles, Andrei Loas, Brad Penetelute, Sean E. Lawler. Pt(IV)-M13, a blood-brain-barrier penetrant macrocyclic peptide-platinum(IV) conjugate, leads to enhanced drug uptake and efficacy in murine glioblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5329.

Background Gene-mediated cytotoxic immunotherapy (GMCI) is a local tumor immunotherapy that uses aglatimagene besadenovec (a non-replicating serotype 5 adenovirus, expressing HSV1 thymidine kinase) with the prodrug ganciclovir to induce DNA double strand breaks (DSB), leading to immunogenic tumor cell death and intratumoral immune cell invasion. Here we investigate potential repressors and enhancers of GMCI’s effectiveness. GMCI is currently in clinical trials in combination with immune checkpoint blockade in glioblastoma. Thus we set out to identify potential areas to improve this approach for future application. Dexamethasone is used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. However, the influence of dexamethasone on the efficacy of GMCI has not been explored. In contrast, DNA damage response inhibitors like the ATR inhibitor (ATRi) AZD6738 might not only amend the cytotoxic but also the immunogenic profile of GMCI, rendering it an attractive combination partner. Methods We investigated the effects of ATR-inhibition and dexamethasone on GMCI in vitro using cytotoxicity, flow cytometry and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone and ATRi in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. Tumor immune infiltrates were analyzed with flow cytometry. Results Cytotoxicity assays showed that dexamethasone has a slight impact on GMCI in vitro. In T-cell-functional assays, we observed a significantly impaired tumor cell killing. Immune cell response assays revealed a reduced immune cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells. In vivo, while treatment with GMCI alone resulted in longer median symptom-free survival (39.5d) versus no treatment (23d), the combination of GMCI and dexamethasone resulted in the significant reduction of this effect (29d vs 39.5d ; p = 0.0184). The combination of ATRi with GMCI proved to be synergistic in cytotoxicity assays. Flow cytometry revealed a significant increase in DSB-associated H2AX foci as well as an improved immune profile by downregulation of GMCI-induced PD-L1 expression. In vivo, the combination with ATRi led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant compared to the untreated control (p=0.0022). Conclusions Our data suggest that dexamethasone may decrease the efficacy of immunotherapy for glioma through impaired T cell function: this emphasizes the need in identifying alternatives to dexamethasone to prevent attenuated responses in immunotherapies. The combination of GMCI with ATRi however points to additional therapeutic benefit through enhanced cytotoxic efficacy, improved immunogenicity in vitro and increased long-term survival in vivo, making it a promising future approach for the treatment of glioblastoma. Acknowledgements This was supported by NCI P01CA069246 (Chiocca)

Simple Summary Oncolytic Herpes simplex virus-1 (HSV-1) offers the dual potential of both lytic tumor-specific cell killing and inducing anti-tumor immune responses. The HSV-1 genome can be altered to enhance both components and this may be applicable for the treatment of a broad range of cancers. Several engineered oncolytic viruses based on the HSV-1 backbone are currently under investigation in various clinical trials, both as single agents and in combination with various immunomodulatory drugs. Abstract Herpes simplex virus 1 (HSV-1) provides a genetic chassis for several oncolytic viruses (OVs) currently in clinical trials. Oncolytic HSV1 (oHSV) have been engineered to reduce neurovirulence and enhance anti-tumor lytic activity and immunogenicity to make them attractive candidates in a range of oncology indications. Successful clinical data resulted in the FDA-approval of the oHSV talimogene laherparepvec (T-Vec) in 2015, and several other variants are currently undergoing clinical assessment and may expand the landscape of future oncologic therapy options. This review offers a detailed overview of the latest results from clinical trials as well as an outlook on newly developed HSV-1 oncolytic variants with improved tumor selectivity, replication, and immunostimulatory capacity and related clinical studies.

Glioblastoma (GBM) is amongst the deadliest human cancers and is characterized by high levels of vascularisation. Angiogenesis is highly dynamic during brain development and almost quiescent in the adult brain, but is reactivated in vascular-dependent CNS pathologies such as brain tumors. Nucleolin (NCL) is a known regulator of cell proliferation and angiogenesis, but its roles on physiological and pathological brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit (NVU) in human fetal brains and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial- and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression in tumor- and blood vessel cells correlated with glioma malignancy in vivo. In culture, siRNA-mediated NCL knockdown reduced human umbilical vein endothelial cell (HUVEC) sprouting angiogenesis, proliferation and filopodia extension, and reduced glucose metabolism. Mechanistically, RNA sequencing of Nucleolin knockdown in HUVECs revealed a putative p53-TIGAR-HK2 regulation of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that positively regulates sprouting angiogenesis and endothelial metabolism. Our findings have important implications in therapeutic targeting of glioma.

Lung cancer is one of the leading causes of cancer-related deaths worldwide and is characterized by hijacking immune system for active growth and aggressive metastasis. Neutrophils, which in their original form should establish immune activities to the tumor as a first line of defense, are undermined by tumor cells to promote tumor invasion in several ways. In this study, we investigate the mutual interactions between the tumor cells and the neutrophils that facilitate tumor invasion by developing a mathematical model that involves taxis-reaction-diffusion equations for the critical components in the interaction. These include the densities of tumor and neutrophils, and the concentrations of signaling molecules and structure such as neutrophil extracellular traps (NETs). We apply the mathematical model to a Boyden invasion assay used in the experiments to demonstrate that the tumor-associated neutrophils can enhance tumor cell invasion by secreting the neutrophil elastase. We show that the model can both reproduce the major experimental observation on NET-mediated cancer invasion and make several important predictions to guide future experiments with the goal of the development of new anti-tumor strategies. Moreover, using this model, we investigate the fundamental mechanism of NET-mediated invasion of cancer cells and the impact of internal and external heterogeneity on the migration patterning of tumour cells and their response to different treatment schedules., Author summary When cancer patients are diagnosed with tumours at a primary site, the cancer cells are often found in the blood or already metastasized to the secondary sites in other organs. These metastatic cancer cells are more resistant to major anti-cancer therapies, and lead to the low survival probability. Until recently, the role of neutrophils, specifically tumor-associated neutrophils as a member of complex tumor microenvironment, has been ignored for a long time due to technical difficulties in tumor biology but these neutrophils are emerging as an important player in regulation of tumor invasion and metastasis. The mutual interaction between a tumor and neutrophils from bone marrow or in blood induces the critical transition of the naive form, called the N1 type, to the more aggressive phenotype, called the N2 TANs, which then promotes tumor invasion. In this article, we investigate how stimulated neutrophils with different N1 and N2 landscapes shape the metastatic potential of the lung cancers. Our simulation framework is designed for boyden invasion chamber in experiments and based on a mathematical model that describes how tumor cells interact with neutrophils and N2 TANs can promote tumor cell invasion. We demonstrate that the efficacy of anti-tumor (anti-invasion) drugs depend on this critical communication and N1→N2 landscapes of stimulated neutrophils.

The mode of action for oncolytic viruses (OVs) in cancer treatment is thought to depend on a direct initial cytotoxic effect against infected tumor cells and subsequent activation of immune cell responses directed against the neoplasm. To study both of these effects in a mouse model of glioblastoma (GBM), we employed murine GBM cells engineered to constitutively express the type I Herpes Simplex Virus (HSV1) HSV-1 receptor, nectin-1, to allow for more efficient infection and replication by oncolytic HSV (oHSV). These cells were further engineered with a surrogate tumor antigen to facilitate assays of T cell activity. We utilized MRI-based volumetrics to measure GBM responses after injection with the oHSV and bioluminescent imaging (BLI) to determine oHSV replicative kinetics in the injected tumor mass. We found increased infiltration of both surrogate tumor antigen- and oHSV antigen-specific CD8+ T cells within 7 days after oHSV injection. There was no increase in tumor infiltrating CD8+ T cells expressing “exhaustion” markers, yet oHSV infection led to a reduction in PD-1+ CD8+ T cells in injected GBMs and an increase in IFNγ+ CD8+ T cells. There was a significant direct correlation between oHSV-mediated reduction in GBM volume and increased infiltration of both viral and tumor antigen-specific CD8+ T cells, as well as oHSV intratumoral gene activity. These findings imply that CD8+ T cell cytotoxicity against both tumor and viral antigens as well as intratumoral oHSV gene expression are important in oHSV-mediated GBM therapy.

Oncolytic viruses are of growing interest to cancer researchers and clinicians, due to their selectivity for tumor cells over healthy cells and their immunostimulatory properties. The immune response to an oncolytic virus plays a critical role in treatment efficacy. However, uncertainty remains regarding the circumstances under which the immune system either assists in eliminating tumor cells or inhibits treatment via rapid viral clearance, leading to the cessation of the immune response. In this work, we develop an ordinary differential equation model of treatment for a lethal brain tumor, glioblastoma, using an oncolytic Herpes Simplex Virus. We use a mechanistic approach to model the interactions between distinct populations of immune cells, incorporating both innate and adaptive immune responses to oncolytic viral therapy (OVT), and including a mechanism of adaptive immune suppression via the PD-1/PD-L1 checkpoint pathway. We focus on the tradeoff between viral clearance by innate immune cells and the innate immune cell-mediated recruitment of antiviral and antitumor adaptive immune cells. Our model suggests that when a tumor is treated with OVT alone, the innate immune cells' ability to clear the virus quickly after administration has a much larger impact on the treatment outcome than the adaptive immune cells' antitumor activity. Even in a highly antigenic tumor with a strong innate immune response, the faster recruitment of antitumor adaptive immune cells is not sufficient to offset the rapid viral clearance. This motivates our subsequent incorporation of an immunotherapy that inhibits the PD-1/PD-L1 checkpoint pathway by blocking PD-1, which we combine with OVT within the model. The combination therapy is most effective for a highly antigenic tumor or for intermediate levels of innate immune localization. Extreme levels of innate immune cell activity either clear the virus too quickly or fail to activate a sufficiently strong adaptive response, yielding ineffective combination therapy of GBM. Hence, we show that the innate and adaptive immune interactions significantly influence treatment response and that combining OVT with an immune checkpoint inhibitor expands the range of immune conditions that allow for tumor size reduction or clearance.

Extracellular vesicles (EVs) are now well established as important mediators of intercellular communication. EVs constitute a diverse group of secreted vesicles which function by the delivery of protein and nucleic acid cargoes from donor to recipient cells. In cancer, tumor cell-derived EVs are shown to promote disease progression by facilitating local reprogramming of the tumor microenvironment. EVs also have more distant systemic effects via transport in biofluids, and therefore have great potential as biomarkers for disease detection and monitoring. Recently, the discovery that EVs derived from glioblastoma cells can mediate immunosuppression by activation of immune checkpoint signaling and T cell dysfunction was reported. Mechanistically we showed that this occurs via direct binding of PD-L1 secreted in EVs, to its receptor PD1 expressed on the surface of activated T cells. This previously unidentified mechanism of tumor immunosuppression has been confirmed in subsequent independent studies, which have demonstrated the biologic importance of this mechanism across multiple tumor types. These studies have established a new and significant paradigm in which PD-L1 containing tumor cell-derived EVs cause immune suppression by the direct engagement of PD1 on T cells, decreasing their activation and providing a further barrier to protect tumors from T cell killing.

BACKGROUND CAN-2409 is a replication-deficient adenovirus that delivers HSV thymidine kinase to cancer cells, resulting in local conversion of orally administered valacyclovir into a toxic metabolite. Previously, a phase 1b/2 clinical trial of CAN-2409 combined with standard-of-care (SOC) demonstrated safety and improved survival in HGG patients. Addition of CAN-2409 to nivolumab has the potential to activate locally recruited lymphocytes and teach them to recognize tumor neoantigens, changing the ‘cold’ immunosuppressive tumor microenvironment, and synergizing with the activity mediated by immune checkpoint inhibitors. This notion is supported by preclinical experiments showing that the combination of CAN-2409 with anti-PD1 therapy was more effective than either treatment alone. METHODS This ongoing phase 1 clinical trial evaluates safety and initial efficacy of CAN-2409 combined with nivolumab and SOC in newly diagnosed HGG. CAN-2409 is injected during neurosurgery into the tumor bed, followed by 14-days of valacyclovir. Radiation starts within 8 (+/-4) days of surgery. Temozolomide is administered to MGMT-methylation positive patients only. Nivolumab is given every 2 weeks, up to 52-weeks. Deep immune profiling studies are ongoing and initial results will be available shortly. RESULTS From February 2019 to March 2021, 41 patients were enrolled and 35 were evaluable for safety from the combination of CAN-2409, nivolumab and SOC: 24 male and 11 female; 34 glioblastoma, 1 diffuse astrocytoma; 33 IDH-wildtype, 2 IDH-mutant; 15 MGMT-methylated, 20 unmethylated. Median age was 62-years (range 28-79), median KPS 90 (range 80-100). With 13 months median follow-up, no unexpected serious adverse events were observed, and 23 patients are still alive. The most frequent possibly related adverse events (>10%) were nausea, fatigue, fever, headache, and increased ALT. CONCLUSIONS The combination of CAN-2409 + nivolumab + SOC was well tolerated. Clinical follow-up and extensive biomarker analyses will provide a better understanding of the therapeutic potential of this approach.

RATIONALE Herpes virus thymidine kinase (HSV-TK) suicide gene therapy is a well-established approach for in situ tumor cell killing after administration of ganciclovir (GCV), due to the induction of lethal DNA damage in HSV-TK expressing cells. Here we investigated the effects of HSV-TK gene delivery with a non-replicating serotype 5 adenovirus (AdTK) in murine glioblastoma models in combination with the ATR-inhibitor AZD6738 to disrupt the DNA damage response (DDR). METHODS We investigated the effects of disrupted DDR signaling on AdTK therapy in vitro using cytotoxicity, cytokine and flow cytometry assays in glioblastoma cell lines and in vivo with an orthotopic syngeneic murine glioblastoma model. Therapy response was monitored with MRI. Changes in the tumor microenvironment were analyzed with CyTOF. RESULTS The combination of AZD6738 with AdTK was synergistic in cytotoxicity assays, which was complemented by a significant increase of γH2AX foci. Complex modulations of the tumor microenvironment were observed with significantly reduced expression of PD-L1, MICA/B and the pro-tumorigenic cytokines IL1b and IL-4. In vivo, the combination with AZD6738 led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant in contrast to untreated controls (p=0.0022). However, the combination treatment did not block the growth of tumors upon rechallenge in long-term survivors. CONCLUSION DDR signaling is crucial in the therapeutic efficacy of AdTK/GCV. It significantly enhances cytotoxicity in vitro and in vivo while having complex ramifications at the immunological level, requiring further studies to determine ideal conditions for a maximized therapeutic benefit.

BackgroundRecurrent high-grade glioma (HGG) represents a significant clinical unmet need with expected survival between 6 to 9 months. Oncolytic viruses are a new therapeutic approach for solid tumors that deploy oncolytic activity combined with local and systemic immune activation. CAN-3110 (rQNestin34.5v2) is an oncolytic herpes simplex virus (HSV), modified to encode the HSV1 ICP34.5 protein under control of the nestin promoter. Selective expression of nestin in brain tumors confers tumor-restricted replication of CAN-3110. We conducted an open-label dose-escalation phase 1 clinical trial in patients with recurrent HGG to evaluate safety, tolerability, and immunological changes after CAN-3110 treatment.MethodsThirty patients with biopsy-confirmed recurrent HGG were enrolled from September 2017 to February 2020. CAN-3110 was injected intratumorally starting at 1x106 plaque forming units (pfu) and dose-escalated by half log to 1x1010 pfu. Patients also received standard of care. Peripheral blood mononuclear cells (PBMCs), plasma and tumor samples were collected for analysis at different time-points post treatment. We evaluated HSV antigen expression in tumor tissue. RNA sequencing and T cell receptor (TCR) rearrangement analysis was performed in matched tissue and PBMCs. Cytokine profiling was completed in 29 patients at baseline, day 2, and day 28 post treatment.ResultsEighteen patients were recruited at their first recurrence and 12 at the second recurrence. Three patients presented with multifocal disease. Tumor volume ranged from 357.4 to and 54,036.1mm3 (median 7,733.9mm3, SDV 15,610.2). CAN-3110 was well-tolerated with no dose-limiting toxicity. Median overall survival was 11.7 months. We demonstrated persistence of HSV antigen and CD8+ T cell infiltrates at the site of injected tumor. Preliminary analysis revealed expansion of shared TCR clonotypes and upregulation of pro-inflammatory genes in post-treatment tumors and peripheral blood samples. Longitudinal modeling of cytokine profiling demonstrated increased levels of IL-6, VEGF alpha, CCL2 and IL1-RA and a decrease in GCP-2 levels at day 2 post-treatment (p ConclusionsIntratumoral administration of CAN-3110 appears well-tolerated in recurrent HGG. Histologic, molecular, and cytokine analyses demonstrate persistence of viral antigen as well as local and systemic immune activation after treatment.Ethics ApprovalThe study was approved by the Office for Human Research Studies at Dana-Farber Cancer Institute, Protocol Number 16–557.

Background CNS tumors, including medulloblastoma and pediatric glioblastoma (pGBM) account for the majority of solid pediatric malignancies. There remains an unmet need to identify novel treatment approaches in poor prognosis and relapsed pediatric brain tumors, where therapeutic options are limited. Small-molecule B-cell lymphoma 2 (BCL-2) family inhibitors may enhance tumor cell killing when combined with conventional and targeted chemotherapeutic agents. We investigated the effect of disrupting BCL-2 and B cell lymphoma-extra large (BCL-XL) protein function using ABT-263, ABT-199 and WEHI-539 in medulloblastoma and pGBM cells following treatment with MLN8237, an Aurora kinase inhibitor under investigation as a novel agent for the treatment of malignant brain tumors. Methods Tumor cell growth and viability were determined by MTT/WST-1 assays and flow cytometry. Effects on cell phenotype, cell cycle progression, and ploidy were determined by live cell imaging and DNA content analysis. Apoptosis was determined by annexin V/propidium iodide staining and time-lapse microscopy and confirmed by measuring caspase-3/7 activity and western blotting and by short interfering RNA (siRNA) knockdown of BCL-2 associated X protein/BCL-2 antagonist killer (BAX/BAK). Results ABT-263, in combination with MLN8237, reduced mitotic slippage and polyploidy and promoted the elimination of mitotically defective cells via a BAX/BAK-dependent, caspase-mediated apoptotic pathway. The BCL-XL antagonist, WEHI-539, significantly augmented tumor cell killing when used in combination with MLN8237, as well as sensitized resistant brain tumor cells to a novel BAX activator, SMBA1. In addition, siRNA-mediated knockdown of BCL-XL sensitized pGBM and medulloblastoma cells to MLN8237 and mimicked the effect of combination drug treatment. Conclusion Selective small-molecule inhibitors of BCL-XL may enhance the efficacy of MLN8237 and other targeted chemotherapeutic agents.

SCOPUS: ed.j, info:eu-repo/semantics/published

High-grade gliomas are deadly cancers, and current standard-of-care has demonstrated limited success. The ability to specifically target glioma cells allows for the development of safer and more efficacious brain cancer therapy strategies. Brevican, a CNS-specific extracellular matrix protein is upregulated in glioma cells and its expression correlates with tumor progression. Particularly, a brevican isoform lacking glycosylation, B/bΔg is a unique glioma marker and not expressed in non-cancerous tissues. Therefore, B/bΔg represents a valuable target for anti-cancer strategies. Here, we describe the utilization of state-of-the-art platforms to screen a one-bead-one-compound combinatorial peptide library to discover a novel “B/bΔg-Targeting Peptides”, called BTP-7 that can bind B/bΔg with high affinity and specificity. BTP-7 displayed 260 nanomolar affinity for recombinant B/bΔg protein. Binding to a specific site on B/bΔg was confirmed in a competitive binding assay using BTP-7 functionalized with a UV-crosslinker and BTP-7 had little association with the fully glycosylated isoform of brevican (control). Scrambling of the BTP-7 sequence led to complete abrogation of B/bΔg binding. Furthermore, BTP-7 is preferentially taken up by B/bΔg-expressing glioma cells compared with non-expressing cells. We also discovered that BTP-7 can cross the blood-brain barrier in both the in vitro BBB organoid model and in mice. BTP-7 displayed 10x greater binding to intracranial GBM-6 tumors than control peptides, and 4x higher tumor uptake than in normal brain tissues. Conjugation of BTP-7 to camptothecin (an anti-tumor drug) via a cleavable linker led to increased DNA damage in intracranial GBM-6 tumors and prolonged survival in tumor-bearing mice. Our results show the potential of BTP-7 for the development of next-generation targeted therapeutics that could greatly benefit the outcome of patients with advanced brain cancer.

The use of oncolytic viruses (OV) as therapeutic agents for glioblastoma multiforme (GBM) is an emerging clinical focus. OV have the potential to reinvigorate the immune contexture in the tumor microenvironment (TME). However, current pre-clinical models are insufficient to study the immune effects of OV. Here, we describe an imaging approach using a human ex-vivo platform to study the effects of OV on tumor-associated immune cells in GBM. Using the rQNestin34.5v.1 herpes simplex virus (rQNestin) we treated human GBM ex-vivo, incorporating autologous patient-derived peripheral-blood mononucleated cells and whole tissue fragments. We treated GBM (n=10) with rQNestin, capable of expressing GFP, and profiled GFP expression as a proxy for replication. We analyzed serial sections of H&E, HSV-1, and multiplex immunohistochemistry (mIHC) to investigate the impact of rQNestin on the spatial context of the immune compartment. Cell Signaling Technology antibodies, CD3e (D7A6E™,ID:85061), CD68 (D4B9C,ID:76437), CD11c (D3V1E,49420), MHC-Class II ((HLA-DRB)LGII-612.14,NA) and Pan-keratin (C11,4545) were optimized for identifying intratumoral T-cells (CD3e+), macrophages (CD68) and dendritic cells (CD11c+MHCII+) in relation to the surrounding tissue architecture (pan cytokeratin). Using the serial-section imaging approach, we overlaid infiltration and replication of rQNestin. Additionally, strategic sectioning identified dynamic spatial alterations in the immune compartment, via visualization of the tumor-immune interface with indications of infiltration and replication. Quantitative pathology showed dynamic re-arrangement of immune cells - in spatial orientation, quantity and localization, relative to the tumor, under rQnestin pressure, ex-vivo. Here, we report that our ex-vivo human tumor model can be used to evaluate the effects of OV in the TME – not only viral replication but also the direct effect on the immune compartment. In particular, we can assess changes in immune subsets post-OV treatment in a 3-D manner, therefore providing greater insight into their spatial arrangement and distribution. These findings could develop our understanding of anti-tumor OV mechanisms.

INTRODUCTION Cytomegalovirus (CMV), a member of the betaherpesvirinae subfamily widespread in human populations has been reported to be detectable in glioblastoma (GBM). We have recently shown that CMV latent infection promotes GBM growth in a mouse model. The mechanisms involved are not well-defined and treatment responses have not been evaluated. METHODS To investigate the effects of CMV on GBM cells we performed an RNAseq study. Differentially expressed genes were identified and validated in vitro and in vivo. RESULTS RNAseq analysis showed multiple alterations induced by CMV infection of GBM cells including significant upregulation of c-MET (8.2 log2 fold change) and MGMT (3.9 log2 fold change) transcripts after CMV infection. c-MET is a tyrosine kinase receptor known to promote GBM growth, and MGMT expression leads to resistance to the alkylating agent temozolomide (TMZ). These findings were validated in vitro in both mouse and human GBM cells, and shown to be dependent on CMV-induced upregulation of NF-kB by siRNA knockdown of the essential RelA NF-kB subunit. The c-MET co-receptor CD44 was also upregulated suggesting a role for the c-MET signalling axis in CMV induced tumor progression. CMV-infected tumor cells were resistant to TMZ treatment as expected due to MGMT upregulation. Treatment of our murine GBM model with TMZ led to a significant increase in survival in control mock-infected mice (median survival 38 days control, 48 days TMZ), whereas the CMV infected mice did not respond (median survival 35 days control, 35 days TMZ; p value between TMZ treated groups = 10–4). CONCLUSIONS Our results define novel and relevant mechanisms by which CMV may influence both GBM growth and resistance to treatment. Further therapeutic and mechanistic studies are underway to investigate these pathways in the context of CMV in GBM. These data support further study of CMV targeting in GBM.

Introduction Targeting the infiltrative nature of GBMs with anti-migratory drugs as combination treatment for prevention of tumour recurrence is an attractive disease management option. One such class of drugs are the GSK-3 inhibitors 6-bromoindirubin-3′-oximes (BIO). BIO has been proposed for use in combination therapy, however, the administration of indirubins remains problematic due to low solubility causing low bioavailability. We describe the characterisation of a BIO derivative, VTIND42, identified from a screen of 450 compounds with improved solubility. Method The compounds were initially screened on the following criteria, selective anti-migratory effect with enhanced potency over the lead compound BIO. Effect on cell migration in two cell subpopulations (core obtained, edge obtained) from the patient derived GBM40 was assessed by 3D migration/invasion assays and for cytoskeletal rearrangements by immunofluorescence. Results VTIND42 (1 µM) showed the best performance overall in terms of specificity and efficacy in comparison to BIO with a pronounced effect on GBM40 edge cell populations. Anti-migratory effects over 72 hours in GBM40 edge (24h, 10.74% p=0.04; 48h, 6.52% p=0.06, 72h, 4.26% p=0.06) in comparison to the control were observed; BIO (5 µM) significantly reduced migration over 72 hours (24h, 12.79% p Discussion VTIND42 displayed increased efficacy in comparison to BIO, further investigations will determine its potential as a bioavailable anti-migratory drug for glioblastoma treatment potentially improving disease outcome in patients.

BackgroundMicroRNAs play an important role in the regulation of mRNA translation, and have therapeutic potential in cancer and other diseases.MethodsTo profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen using a synthetic oligonucleotide library representing all known human microRNAs in adult and pediatric GBM cells. Bio-informatics analysis were used to refine this list and the top seven microRNAs were validated in a larger panel of cells by flow-cytometry, and RTqPCR. The downstream mechanism of the strongest and most consistent candidate was investigated by siRNAs, 3’UTR luciferase assays and Western Blotting.ResultsOur screen identified ∼100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 expressing cells and characterized the mechanism of action as cytokinesis failure followed by apoptosis, which was observed in an extended GBM cell panel including two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In glioblastoma cells, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target of miR-1300. ECT2 siRNA phenocopied the effects of miR-1300, and its overexpression led to a significant rescue of miR-1300 induced binucleation.ConclusionMiR-1300 was identified as a novel regulator of endomitosis with translatable potential for therapeutic application. The datatasets will be a resource for the neuro-oncology community.Key points (2 or 3 key points 85 characters plus spaces each)70% of cytotoxic microRNAs were shared between adult and pediatric glioblastoma cellsMiR-1300 expression is restricted to endomitosis within megakaryocyte differentiationMiR-1300’s ectopic expression is a potent and promising therapeutic tool in cancerImportance of StudyPrevious functional studies of microRNAs involved in the regulation of glioblastoma cell proliferation and/or survival have focused on adult glioblastoma alone and are restricted to only a few microRNAs at a time. Our study provides the first encompassing landscape of potent cytotoxic microRNAs in pediatric and adult glioblastoma.Not only, does our data provide an invaluable resource for the research community but it also revealed that 70% of microRNAs with significant cytotoxicity were shared by adult and pediatric cells. Finally, we identified and characterized the previously undescribed role of microRNA-1300 in the tight regulation of megakaryocyte differentiation into platelets and how, when expressed outside of this context, miR-1300 consistently causes cytokinesis failure followed by apoptosis, and thus represents a powerful cytotoxic tool with potential for translation towards therapeutic applications.

The advent of immunotherapy in recent years has shown the potential to revolutionize the treatment of cancer. Unleashing antitumor T cell responses via immune checkpoint blockade has led to remarkable responses in previously untreatable tumors. The master regulator of interferon-mediated antiviral responses – stimulator of interferon genes (STING) – has now emerged as a critical mediator of innate immune sensing of cancer, and is a promising target for local immunostimulation, promoting intratumoral inflammation, and facilitating antitumor T cell responses. Pharmacological activation of the STING pathway can lead to T cell-mediated tumor regression in preclinical tumor models, and novel STING activating small molecules are now being tested in clinical trials. Here we will introduce the STING pathway and review the current state of drug development., SCOPUS: re.j, info:eu-repo/semantics/published

We previously showed lithium chloride (LiCl) and other inhibitors of glycogen synthase kinase-3 (GSK-3) including 6-bromo-indirubin-3-oxime (BIO), can block glioblastoma (GBM) cell migration. To investigate the mechanisms involved we used two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry to identify proteins altered after treatment of U251 GBM cells with 20 mM LiCl. Downregulation of the intermediate filament protein vimentin was the most significant change identified. Analysis of patient tumor samples revealed that vimentin is expressed abundantly in GBM, and is prognostic especially in lower grade tumors. Additionally, siRNA-mediated vimentin knockdown impaired GBM migration. Western blotting showed that treatment with LiCl or small molecule GSK-3 inhibitors led to the rapid downregulation of detergent soluble vimentin levels across a panel of GBM-derived cells. Fluorescence reactivation after photobleaching (FRAP) microscopy studies showed a significant reduction in the ability of the vimentin cytoskeleton to recover from photo-bleaching in the presence of LiCl or BIO. Biochemical studies revealed that GSK-3 and vimentin directly interact, and analysis of vimentin revealed a GSK-3 consensus phosphorylation site. We conclude that anti-migratory compounds with the ability to inhibit GSK-3 have effects on vimentin cytoskeletal dynamics, which may play a role in their anti-invasive activity.

MicroRNA deregulation is a consistent feature of glioblastoma, yet the biological effect of each single gene is generally modest, and therapeutically negligible. Here we describe a module of microRNAs, constituted by miR-124, miR-128 and miR-137, which are co-expressed during neuronal differentiation and simultaneously lost in gliomagenesis. Each one of these miRs targets several transcriptional regulators, including the oncogenic chromatin repressors EZH2, BMI1 and LSD1, which are functionally interdependent and involved in glioblastoma recurrence after therapeutic chemoradiation. Synchronizing the expression of these three microRNAs in a gene therapy approach displays significant anticancer synergism, abrogates this epigenetic-mediated, multi-protein tumor survival mechanism and results in a 5-fold increase in survival when combined with chemotherapy in murine glioblastoma models. These transgenic microRNA clusters display intercellular propagation in vivo, via extracellular vesicles, extending their biological effect throughout the whole tumor. Our results support the rationale and feasibility of combinatorial microRNA strategies for anticancer therapies., The delivery of single therapeutic microRNAs in brain cancers is challenging. Here, the authors engineer three neuronal microRNAs (miR-124, 128 and 137) into a cluster that, targeting oncogenic chromatin repressors, increases survival of GBM-bearing mice when combined with chemotherapy.

2009 Background: Recurrent glioma patients have few therapeutic options and an expected survival of only 7 to 10 months. New treatments to improve the prognosis of this patient population are a dire medical need. Oncolytic viruses (OVs) are emerging as important new agents for cancer treatment. The first FDA approved OV was talimogene laherparepvec (Imlygic, T-Vec) for treatment of melanoma. T-Vec, as most other clinical HSV-1 based OVs, is deleted in the ICP34.5 gene, which is responsible for HSV-1 neurovirulence. However, deletion of ICP34.5 also impedes efficient viral replication. CAN-3110 (rQNestin34.5v2) maintains a copy of the HSV1 ICP34.5 gene under transcriptional control of the tumor-specific promoter for nestin to drive robust tumor-selective replication. CAN-3110 replicates in malignant glioma cells far above levels seen with ICP34.5 deleted viruses. This potency also created the hypothetical risk for increased neurovirulence, thus the regulatory advice to conduct a cautious nine-dose-level Phase-1 dose escalation study in patients with recurrent high-grade glioma (HGG). Methods: From September 2017 to February 2020, thirty patients with biopsy-confirmed recurrent high-grade glioma were treated in an open label clinical trial. Patients with multifocal, multicentric, tumors larger than 5 cm, and tumors that had recurred multiple times were eligible. All patients received best standard of care treatments as indicated by their physician. CAN-3110 was injected intratumorally starting at 1x106 plaque forming units (pfu) and dose-escalating (3+3 design) by half log increments up to 1x1010 pfu. Tissue (when possible) and blood samples were obtained before and during treatment for experimental medicine analyses. Results: CAN-3110 was well tolerated with no dose limiting toxicity observed. The initial tissue diagnosis of the recurrent tumor for the 30 subjects was 26 glioblastoma, 3 anaplastic oligodendroglioma, and 1 anaplastic astrocytoma. The median overall survival (mOS) of the entire study group is 13.25 months. Post-treatment tissue is available for 18/30 subjects and revealed persistence of HSV antigen and CD8+ T cell infiltrates. Additional response, immunologic (including T cell receptor repertoire), transcriptomic and single cell RNA sequencing analyses are ongoing. Conclusions: Administration of CAN-3110 into recurrent glioma was well tolerated without evidence of ICP34.5-induced encephalitis/meningitis. Histological and molecular analyses showed evidence of biological activity and that CAN-3110 injection was associated with immune activation and viral antigen persistence. Although definitive clinical efficacy cannot be determined in this small phase 1 study, OS of CAN-3110 treated subjects compares favorably to historical reports and warrants further clinical studies. Clinical trial information: NCT03152318.

Glioblastoma (GBM) is the most common and deadliest form of brain tumor and remains amongst the most difficult cancers to treat. Brevican (Bcan), a central nervous system (CNS)-specific extracellular matrix protein, is upregulated in high-grade glioma cells, including GBM. A Bcan isoform lacking most glycosylation, dg-Bcan, is found only in GBM tissues. Here, dg-Bcan is explored as a molecular target for GBM. In this study, a d-peptide library is screened to identify a small 8-amino acid dg-Bcan-Targeting Peptide (BTP) candidate, called BTP-7 that binds dg-Bcan with high affinity and specificity. BTP-7 is preferentially internalized by dg-Bcan-expressing patient-derived GBM cells. To demonstrate GBM targeting, BTP-7 is radiolabeled with 18F, a radioisotope of fluorine, and increased radiotracer accumulation is found in intracranial GBM established in mice using positron emission tomography (PET) imaging. dg-Bcan is an attractive molecular target for GBM, and BTP-7 represents a promising lead candidate for further development into novel imaging agents and targeted therapeutics.

Glioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome.In vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy.In vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells.This report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment.

Tumor interferon signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the interferon- (IFN)-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2 and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. INCR1’s primary transcript binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2 enabling their expression. Together, these findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

Long noncoding RNAs (lncRNAs) are key regulators of gene expression both in physiologic and pathologic conditions. Although the role of lncRNAs in tumorigenesis is now emerging, their function in cancer-mediated immunosuppression remains unexplored. We have identified the interferon-γ (IFNγ)-stimulated noncoding RNA 1 (INCA1) as a novel lncRNA expressed from the PD-L1 locus and showed that INCA1 is a key regulator of PD-L1 expression. Silencing INCA1 decreased PD-L1 expression both at basal level and in response to IFNγ stimulation in multiple tumor types. INCA1 knockdown sensitized tumor cells to cytotoxic T cell-mediated killing. Moreover, tumors with silenced INCA1 showed increased CAR T trafficking and activity in vivo. These findings provide novel insights into the biology of PD-L1 regulation and tumor immune evasion. INCA1 is thus a new target for cancer immunotherapy. Citation Format: Marco Mineo, Shawn M. Lyons, Ruben Ferrer-Luna, Hirotaka Ito, Niklas von Spreckelsen, Quazim A. Alayo, Jasneet K. Khalsa, Khalid Shah, Keith L. Ligon, Rameen Beroukhim, Pavel Ivanov, Paul J. Anderson, Hiroshi Nakashima, Sean E. Lawler, E. Antonio Chiocca. The long noncoding RNA INCA1 is a novel regulator of PD-L1 expression in tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B46.

PURPOSE: Glioblastoma (GBM) is resistant to standard of care. Immune checkpoints inhibitors (such as anti-PD-1 mAbs) efficiently restore antitumor T-cell activity. We engineered a new oncolytic herpes simplex virus (oHSV) expressing a single-chain antibody against PD-1 (scFvPD-1) to evaluate its efficacy in mouse models of GBM. EXPERIMENTAL DESIGN: NG34scFvPD-1 expresses the human GADD34 gene transcriptionally controlled by the Nestin promoter to allow replication in GBM cells and a scFvPD-1 cDNA transcriptionally controlled by the CMV promoter. ELISA assays were performed to detect binding of scFvPD-1 to mouse and human PD-1. In vitro cytotoxicity and replication assays were performed to measure NG34scFvPD-1 oncolysis, and scFvPD-1 expression and secretion were determined. In vivo survival studies using orthotopic mouse GBM models were performed to evaluate the therapeutic potency of NG34scFvPD-1. RESULTS: NG34scFvPD-1–infected GBM cells express and secrete scFvPD-1 that binds mouse PD-1. The introduction of the scFvPD-1 sequence in the viral backbone does not alter the oncolytic properties of NG34scFvPD-1. In situ NG34scFvPD-1 treatment improved the survival with a tail of durable survivorship in 2 syngeneic immunocompetent mouse models of GBM. Mice that survived the first GBM challenge rejected the second challenge of GBM when implanted in the contralateral hemisphere. However, this was not true when athymic mice were employed as the recipients of the second challenge, consistent with the need for an intact immune system to obtain a memory response. CONCLUSIONS: NG34scFvPD-1 treatment induces a durable antitumor response in 2 preclinical mouse models of GBM with evidence for antitumor memory.

High-grade gliomas are deadly cancers, and current standard-of-care has demonstrated limited success. The ability to specifically target glioma cells can allow for the development of safer and more efficacious brain cancer therapy strategies. Brevican, a CNS-specific extracellular matrix protein is upregulated in glioma cells and its expression correlates with tumor progression. Particularly, a brevican isoform lacking glycosylation, B/bΔg is a unique glioma marker and not expressed in non-cancerous tissues. Therefore, B/bΔg represents a valuable target for anti-cancer strategies. Here, we describe the utilization of state-of-the-art platforms to screen a one-bead-one-compound combinatorial peptide library to discover a novel “B/bΔg-Targeting Peptides”, called BTP-7 that can bind B/bΔg with high affinity and specificity. BTP-7 displayed 260 nanomolar affinity for recombinant B/bΔg protein, and had little association with the fully glycosylated isoform of brevican (control). Scrambling of the BTP-7 sequence led to complete abrogation of B/bΔg binding. Furthermore, BTP-7 is preferentially taken up by B/bΔg-expressing glioma cells compared with non-expressing cells. We also discovered that BTP-7 can cross the blood-brain barrier using both the in vitro BBB organoid model and in mice. BTP-7 displayed 10x greater binding to intracranial GBM-6 tumors than control peptides, and 4x higher tumor uptake than in normal brain tissues. Conjugation of BTP-7 to camptothecin (an anti-tumor drug) via a cleavable linker led to increased DNA damage in intracranial GBM-6 tumors and prolonged survival in tumor-bearing mice. Our results show the potential of BTP-7 for the development of next-generation targeted therapeutics that could greatly benefit the outcome of patients with advanced brain cancer.

Neutrophils display rapid and potent innate immune responses in various diseases. Tumor-associated neutrophils (TANs) however either induce or overcome immunosuppressive functions of the tumor microenvironment through complex tumor-stroma crosstalk. We developed a mathematical model to address the question of how phenotypic alterations between tumor suppressive N1 TANS, and tumor promoting N2 TANs affect nonlinear tumor growth in a complex tumor microenvironment. The model provides a visual display of the complex behavior of populations of TANs and tumors in response to various TGF-β and IFN-β stimuli. In addition, the effect of anti-tumor drug administration is incorporated in the model in an effort to achieve optimal anti-tumor efficacy. The simulation results from the mathematical model were in good agreement with experimental data. We found that the N2-to-N1 ratio (N21R) index is positively correlated with aggressive tumor growth, suggesting that this may be a good prognostic factor. We also found that the antitumor efficacy increases when the relative ratio (Dap) of delayed apoptotic cell death of N1 and N2 TANs is either very small or relatively large, providing a basis for therapeutically targeting prometastatic N2 TANs.

Purpose: Glioblastoma (GBM) is resistant to standard of care. Immune checkpoints inhibitors (such as anti-PD-1 mAbs) efficiently restore antitumor T-cell activity. We engineered a new oncolytic herpes simplex virus (oHSV) expressing a single-chain antibody against PD-1 (scFvPD-1) to evaluate its efficacy in mouse models of GBM. Experimental Design: NG34scFvPD-1 expresses the human GADD34 gene transcriptionally controlled by the Nestin promoter to allow replication in GBM cells and a scFvPD-1 cDNA transcriptionally controlled by the CMV promoter. ELISA assays were performed to detect binding of scFvPD-1 to mouse and human PD-1. In vitro cytotoxicity and replication assays were performed to measure NG34scFvPD-1 oncolysis, and scFvPD-1 expression and secretion were determined. In vivo survival studies using orthotopic mouse GBM models were performed to evaluate the therapeutic potency of NG34scFvPD-1. Results: NG34scFvPD-1–infected GBM cells express and secrete scFvPD-1 that binds mouse PD-1. The introduction of the scFvPD-1 sequence in the viral backbone does not alter the oncolytic properties of NG34scFvPD-1. In situ NG34scFvPD-1 treatment improved the survival with a tail of durable survivorship in 2 syngeneic immunocompetent mouse models of GBM. Mice that survived the first GBM challenge rejected the second challenge of GBM when implanted in the contralateral hemisphere. However, this was not true when athymic mice were employed as the recipients of the second challenge, consistent with the need for an intact immune system to obtain a memory response. Conclusions: NG34scFvPD-1 treatment induces a durable antitumor response in 2 preclinical mouse models of GBM with evidence for antitumor memory.

Glioblastoma can suppress immunity by using surface PD-L1 on extracellular vesicles to block T cell receptor–mediated T cell activation., Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1–dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers and is characterized by fast growth and aggressive invasion. GBM also communicates with microglia and macrophages which are recruited by tumor cells to facilitate growth and invasion. In this study we investigate the biochemical and cell-mechanical interactions between the glioma cells and the microenvironment including resident glial cells and M1/M2 microglia that enhance tumor invasion. We develop various types of mathematical models that involve reaction-diffusion equations or multi-scale hybrid models for the important components in this mutual interaction. In particular, we investigate the dynamics of intracellular signaling including miR-451 and AMPK, biochemical interaction of a glioma with M1/M2 microglia via CSF-1-EGF-TGF-β signaling, and glioma cell infiltration through the narrow intercellular space via the regulation of myosin II. We show that these models can replicate the key features of the experimental findings and make novel predictions to guide future experiments aimed at the development of new anti-invasive strategies.

One of the more polarized ongoing debates in the brain tumor field over recent years has centered on the association of cytomegalovirus (CMV) with glioblastoma. Several laboratories have reported the presence of CMV antigens in glioblastoma patient specimens, whereas others have failed to detect them. CMV genomic DNA and mRNAs have been detected by PCR, but not in next-generation sequencing studies. CMV promotes high grade glioma progression in a mouse genetic model, and many CMV proteins promote cancer hallmarks in vitro, but actively replicating virus has not been isolated from tumor samples. A consensus is gradually emerging in which the presence of CMV antigens in glioblastoma is increasingly accepted. However, it remains challenging to understand this mechanistically due to the low levels of CMV nucleic acids and the absence of viral replication observed in tumors thus far. Nonetheless, these observations have inspired the development of novel therapeutic approaches based on anti-viral drugs and immunotherapy. The potential benefit of valganciclovir in glioblastoma has generated great interest, but efficacy remains to be established in a randomized trial. Also, early stage immunotherapy trials targeting CMV have shown promise. In the near future we will know more answers to these questions, and although areas of controversy may remain, and the mechanisms and roles of CMV in tumor growth are yet to be clearly defined, this widespread virus may have created important new therapeutic concepts and opportunities for the treatment of glioblastoma.

Background: Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed. Methods: Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays. Results: All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging. Conclusions: Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

High-grade gliomas are deadly cancers, and current standard-of-care has demonstrated limited success. The ability to specifically target glioma cells can allow for the development of safer and more efficacious brain cancer therapy strategies. Brevican (BCAN), a CNS-specific extracellular matrix protein is upregulated in glioma cells and its expression correlates with tumor progression. Particularly, a membrane-bound BCAN isoform lacking normal glycosylation, called B/bΔg is a unique glioma marker and is not expressed in non-cancerous tissues. Therefore, B/bΔg represents a valuable target for anti-cancer strategies. Here, we describe the utilization of state-of-the-art technologies that we have recently developed to discover novel “B/bΔg-Targeting Peptides” (BTP). Briefly, small magnetic beads displaying B/bΔg were used to screen a one-bead-one-compound combinatorial library, enabling high-throughput labeling of library beads presenting peptide candidates with high affinity for B/bΔg. The “hit” beads were rapidly sorted using a microfluidic magnetic-activated sorter of our own design. The hits were then, exposed to cells expressing B/bΔg, and beads with the highest cell association were isolated and sequenced. Characterization of the purified peptides through kinetic binding analysis and cell uptake studies revealed BTP-7 as the lead candidate for B/bΔg binding. BTP-7 displayed 260 nanomolar affinity for recombinant B/bΔg protein, and had little association with the fully glycosylated isoform of BCAN. Scrambling of the BTP-7 sequence led to complete abrogation of B/bΔg binding. For in vivo evaluation, GBM-6 tumors derived from human patients were established intracranially in nude mice, and tumor formation was verified by MRI. Upon systemic administration, BTP-7 displayed approximately 10x greater binding to GBM-6 tumors than the control, as well as 4x higher tumor uptake compared to normal brain tissue. We are currently working on conjugating BTP-7 to a variety of toxic payload to selectively target gliomas, with the goal of improving brain cancer therapeutic efficacy that can ultimately benefit patient.

Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor with a median patient survival of less than two years, even with the optimal standard of care, namely, surgical resection followed by radiotherapy with adjuvant temozolomide chemotherapy. Long-term survival is extremely rare and there is a tremendous need for novel GBM therapies. Following our prior reports on the anticancer activity of osmium(VI) nitrido compounds and their effectiveness against cancer initiating cells, we investigated the efficacy of Os(VI) on GBM initiating cells in vitro and in vivo. Conventional MTT and 3D cytotoxicity assays revealed that patient-derived GBM models were sensitive to cisplatin, TMZ, and two Os(IV) derivatives. Rapid cell death occurred at low micromolar concentrations of the Os(IV) compounds. Cell cycle analysis, Os uptake studies, and cellular distribution experiments provided further insight into the anticancer properties of these compounds, indicating differential uptake for both compounds and a modest G2/M arrest after treatment. Moreover, in vivo experiments showed a significant increase in survival after a single intracranial chemotherapeutic injection, results that warrant further studies using this approach.

Background Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Developing therapeutics that target multiple receptor signaling pathways in tumors is critical as therapies targeting single specific biomarker/pathway have shown limited efficacy in patients with cancer. In this study, we extensively characterized a bi-functional molecule comprising of epidermal growth factor receptor (EGFR) targeted nanobody (ENb) and death receptor (DR) targeted ligand TRAIL (ENb-TRAIL). We show that ENb-TRAIL has therapeutic efficacy in tumor cells from different cancer types which do not respond to either EGFR antagonist or DR agonist monotherapies. Utilizing pharmacological inhibition, genetic loss of function and FRET studies, we show that ENb-TRAIL blocks EGFR signalling via the binding of ENb to EGFR which in turn induces DR5 clustering at the plasma membrane and thereby primes tumor cells to caspase-mediated apoptosis. In vivo, using a clinically relevant orthotopic resection model of primary glioblastoma and engineered stem cells (SC) expressing ENb-TRAIL, we show that the treatment with synthetic extracellular matrix (sECM) encapsulated SC-ENb-TRAIL alleviates tumor burden and significantly increases survival. This study is the first to report novel mechanistic insights into simultaneous targeting of receptor-mediated proliferation and cell death signaling pathways in different tumor types and presents a promising approach for translation into the clinical setting.

Objective: Cytomegalovirus (CMV) is a ubiquitous member of the herpes virus family with a prevalence of between 50-100% in the human population and lifelong persistence. While usually clinically benign, severe cases are observed in neonates and immunocompromised patients. CMV has been associated with various tumors, including glioblastoma (GBM). The aim of this study is to clarify the interrelationship between CMV and GBM, as to elucidate its contribution to GBM progression. Methods: Pericytes, proneural (PN) and mesenchymal (MES) human GBM cell lines were assessed via flow cytometry for permissiveness to CMV (Towne Strain) infection. The transcriptome of infected cells was assessed using RT-qPCR and RNA-Seq analysis. Transwell migration and endothelial tube formation were evaluated in co-culture experiments. C57BL/6 mice were infected with murine CMV (MCMV-Δm157) at P2 and syngeneic GL261 tumor cells were implanted at week 14 after latency was established. Tumor composition was analyzed using flow cytometry. Murine and human brain sections were analyzed by immunofluorescence staining. Results: CMV was found to closely co-localize with tumor vessels in patient derived GBM specimens. In vitro studies confirmed a high permissiveness of pericytes for CMV infection. GBM cells, permissive for CMV infection had a proneural, rather than a mesenchymal signature. The transcription of pro-angiogenic cytokines such as IL-6, TGF-beta and angiogenesis inducing receptor PAR-1 were up-regulated upon CMV infection. Transwell assays revealed increased migration towards CMV infected cells after 48h (GBM cells towards infected pericytes: 1.5-fold (p=0.07); pericytes towards infected GBM cells: 1.48-fold (p=0.009)). Further, co-culture of human brain microvascular endothelial cells (HBMEC) and infected PN cells led to the establishment of larger (160%, p=