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BackgroundOncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials. Results from these studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we assess the extent to which targeting specific mechanisms underlying the immunosuppressive tumor microenvironment optimizes viroimmunotherapy.MethodsWe used RNA-seq analyses to analyze the transcriptome, and validated the results using Q-PCR, flow cytometry, and immunofluorescence. Viral activity was analyzed by replication assays and viral titration. Kyn and Trp metabolite levels were quantified using liquid chromatography–mass spectrometry. Aryl hydrocarbon receptor (AhR) activation was analyzed by examination of promoter activity. Therapeutic efficacy was assessed by tumor histopathology and survival in syngeneic murine models of gliomas, including Indoleamine 2,3-dioxygenase (IDO)-/- mice. Flow cytometry was used for immunophenotyping and quantification of cell populations. Immune activation was examined in co-cultures of immune and cancer cells. T-cell depletion was used to identify the role played by specific cell populations. Rechallenge experiments were performed to identify the development of anti-tumor memory.ResultsBulk RNA-seq analyses showed the activation of the immunosuppressive IDO-kynurenine-AhR circuitry in response to Delta-24-RGDOX infection of tumors. To overcome the effect of this pivotal pathway, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. The combination therapy increased the frequency of CD8+ T cells and decreased the rate of myeloid-derived suppressor cell and immunosupressive Treg tumor populations in animal models of solid tumors. Functional studies demonstrated that IDO-blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of the effectors and suppressors of the tumor immune landscape significantly prolonged the survival in animal models of orthotopic gliomas.ConclusionsOur data identified for the first time the in vivo role of IDO-dependent immunosuppressive pathways in the resistance of solid tumors to oncolytic adenoviruses. Specifically, the IDO-Kyn-AhR activity was responsible for the resurface of local immunosuppression and resistance to therapy, which was ablated through IDO inhibition. Our data indicate that combined molecular and immune therapy may improve outcomes in human gliomas and other cancers treated with virotherapy.

Background Oncolytic adenoviruses are promising new treatments against solid tumors, particularly for glioblastoma (GBM), and preclinical models are required to evaluate the mechanisms of efficacy. However, due to the species selectivity of adenovirus, there is currently no single animal model that supports viral replication, tumor oncolysis, and a virus-mediated immune response. To address this gap, we took advantage of the Syrian hamster to develop the first intracranial glioma model that is both adenovirus replication-permissive and immunocompetent. Methods We generated hamster glioma stem-like cells (hamGSCs) by transforming hamster neural stem cells with hTERT, simian virus 40 large T antigen, and h-RasV12. Using a guide-screw system, we generated an intracranial tumor model in the hamster. The efficacy of the oncolytic adenovirus Delta-24-RGD was assessed by survival studies, and tumor-infiltrating lymphocytes (TILs) were evaluated by flow cytometry. Results In vitro, hamGSCs supported viral replication and were susceptible to Delta-24-RGD mediated cell death. In vivo, hamGSCs consistently developed into highly proliferative tumors resembling high-grade glioma. Flow cytometric analysis of hamster gliomas revealed significantly increased T-cell infiltration in Delta-24-RGD infected tumors, indicative of immune activation. Treating tumor-bearing hamsters with Delta-24-RGD led to significantly increased survival compared to hamsters treated with phosphate buffered saline (PBS). Conclusions This adenovirus-permissive, immunocompetent hamster glioma model overcomes the limitations of previous model systems and provides a novel platform to study the interactions between tumor cells, the host immune system, and oncolytic adenoviral therapy; understanding of which will be critical to implementing oncolytic adenovirus in the clinic.

Gastric cancer is the third leading cause of cancer-related mortality worldwide, and current standard-of-care treatment allows for a 5-year survival rate of 20%. Newer treatments against solid tumors include an oncolytic adenovirus armed with T-cell activator OX40L, named Delta-24-RGDOX, which expresses the positive immune checkpoint OX-40L. While studies have revealed the interaction between the gut microbiome and immunotherapy agents, it’s still unknown how the gut microbiome might influence viroimmunotherapy efficacy. We hypothesized that the gut microbiota plays an important role in modulating the virus-driven anti-tumor response. To test this hypothesis, immunocompetent C57BL/6 mice were subcutaneously implanted M12Luc gastric cancer cells and were administered PBS (control) or Delta-24-RGDOX by intratumoral injections. Mice fecal pellets were collected at three different time points: (1) before tumor implantation, (2) after tumor establishment, and (3) 14 days after the first dose of the treatment. Genomic DNA was isolated with the QIAGEN DNeasy Powersoil Kit from fecal pellets of cancer-bearing mice (n=60), and the V4 region of the 16S ribosomal RNA gene (prokaryotes) was sequenced using the Illumina platform. Data was deposited in QIITA for quality assessment, followed by microbial community analysis using the taxonomic reference database SILVA and the downstream platforms QIIME2 and R with a rarefaction depth of 9,187 reads per sample. We found significant differences in the gut microbiome community structure of viroimmunotherapy-treated mice compared to controls (ANOSIM p-value=0.025). No differences in gut diversity were observed, although control animals tended to show higher richness (Kruskal-Wallis p-value>0.05). Of interest, the bacterial composition of responders to the viroimmunotherapy had a decrease in Actinobacteria and an increase of Patescibacteria. Additionally, responders had higher levels of butyrate-producer bacteria such as Ruminococcaceae and Clostridium sensu stricto. These findings suggest that butyrate producers may be important in regulating host gut homeostasis associated with the clinical efficacy of Delta-24-RGDOX, suggesting possible benefits of microbiota modulation in patient survival. This project was sponsored by a pilot award within The University of Puerto Rico/University of Texas MD Anderson Cancer Center (MDACC) Partnership for Excellence in Cancer Research Grant (2U54CA096297-16). Citation Format: Natalie M. Melendez-Vazquez, Sagar Sohoni, Arie Van Wieren, Juan Fueyo, Candelaria Gomez-Manzano, Filipa Godoy-Vitorino. Changes in the gut microbiota associated with oncolytic virotherapy efficacy in a gastric cancer murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5894.

Purpose of the work: Pediatric High Grade Gliomas (pHGGs) and Diffuse Midline Gliomas (DMGs) are two of the most aggressive tumors developed during childhood. Its poor overall survival emphasizes the need of alternative treatments. ONC201, an imipiridone small molecule, and the oncolytic virus Delta-24-RGD have shown safety and effectiveness in preclinical and clinical settings for these diseases. In this work, we set to characterize the antitumor effect of these two agents combined for the treatment of pHGGs and DMGs. Experimental procedures: A panel of human pHGG (CHLA-03-AA, SF188, SJ-GBM2), DMG (SU-DIPG IV, SF8628, TP-54, HSJD-DIPG-007, UMPED83), and murine DMG cell lines (XFM, NP-53) were used. We assessed viral replication by hexon titration, protein expression by immunoblotting/immunohistochemistry, and oxygen consumption by Seahorse analyses. Viability was measured by MTS. Mitochondrial DNA (mtDNA) were measured by qPCR. CHLA-03-AA/XFM were orthotopically engrafted in athymic nude/Balb/c mice. For in vivo experiments, animals were treated with PBS/Delta-24-RGD (107 pfu/mice) intratumorally, followed by ONC201 oral gavage administration (125 mg/kg/twice/weekly). The tumor microenvironment immune infiltration was evaluated by flow cytometry. Results: We checked the potential negative interaction between both agents in vitro. Adenovirus replication was not affected by ONC201 cotreatment, indicating the suitability of this combination. In vitro citotoxycity analyses showed the cotreatment is either synergistic or additive in 80% of the cells evaluated. Then, we set to characterize the mechanistic effects of the combination. Analysis of mitochondria status showed the decrease in oxygen consumption previously reported as ONC201 effect is maintained with Delta-24-RGD presence. Confirmation of higher mitochondria damage was assessed by a reduction of mtDNA in the combination. Independently, endoplasmic reticulum stress (ERS) previously described in both agents was found potentiated in cotreatment via ATF4. Altogether, these data suggest an improved response in the combination by mitochondria and ERS perturbation. In vivo, cotreatment led to a significant increase in the median OS (Control: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; Delta-24-RGD/ONC201: 95 days (P=0.0008)) in CHLA-03-AA bearing mice, leading to 20% long-term survivors, free of disease. Of importance, a reshaping of the tumor microenvironment towards a proinflammatory phenotype were described (increases of lymphoid (T and NK cells) and myeloid (macrophages, granulocytes, monocytes, denditric cells and microglia) linage. Conclusions: Our data support the combination ONC201/Delta-24-RGD leads to a superior therapeutic effect due to the generation of a proinflammatory microenvironment in pHGG and DMGs preclinical models. Citation Format: Daniel de la Nava, Virginia Laspidea, Iker Ausejo-Mauleón, Marc García-Moure, Sabine Mueller, Javad Nazarian, Joshua E. Allen, Oren Becher, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-Garcia, Carl Koschmann, Marta M. Alonso. Depicting ONC201/Delta-24-RGD combination for the treatment of pHGGs and DMGs reveals a therapeutic benefit and a proinflammatory tumor microenvironment remodelation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4659.

Oncolytic virotherapy shows great potential for treating brain tumors and other solid tumors that metastasize to the brain. A phase I clinical trial testing the oncolytic adenovirus Delta-24-RGD (DNX-2401) in patients with recurrent malignant gliomas demonstrated significant clinical benefits for a subset of patients, and another phase I trial testing Delta-24-RGD in patients with pediatric brain tumors also indicated similar clinical benefits. However, virus injection in tumors initiates both anti-virus and anti-tumor immune responses by activating respective clones of CD8+ T-cells, which compete for limited resources for clonal expansion. Expansion of T-cells against highly immunogenic viral antigens might limit the expansion of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for the dominant viral antigens will allow the expansion of previously subdominant tumor-specific T-cells. In this work, we observed that nanoparticles encapsulating adenoviral antigens successfully induced immune tolerance for viral antigens. These nanoparticles were taken up by liver resident macrophages, which are involved in maintaining peripheral immune tolerance. RNA-sequencing revealed that T-cells recognizing the encapsulated antigens had unique transcriptome signatures with upregulation of genes specifically involved in peripheral immune tolerance, including immune coinhibitory molecules. Virus-specific immune tolerance using nanoparticles redirected the focus of the immune response towards antigens for melanoma as measured by interferon-gamma secretion (P < 0.0001). Reduction of virus-specific T-cells and simultaneous expansion of tumor-specific T-cell clones were confirmed with tetramer staining (P < 0.05). Importantly, virotherapy in combination with nanoparticle-induced immune tolerance towards viral antigens increased the percentage of long-term survivors compared to virus treatment alone (100% versus 50%). Our data provide the basis to develop future clinical trials that aim to maximize the potential of cancer virotherapy in solid tumors.

Purpose: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma. Patients and Methods: Dose-escalation phase I study with 3+3 cohorts, dosing 107 to 1 × 1011 viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding. Results: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2-associated cytokine levels and numbers of CD3+ T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+ and CD8+ T cells. No evidence of viral shedding in excreta was observed. Conclusions: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses.

Oncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials in a small fraction of patients. To improve the response rate, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. We engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus to express the T-cell activator OX40L, which triggered, in murine glioblastoma models, a dramatic reshaping of the tumor microenvironment dominated by changes in immune processes in an RNA-seq analysis of ingenuity pathways. Paradoxically, network analyses also revealed that Delta-24-RGDOX induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of gliomas to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma-bearing mice. The combined therapy decreased the activation of the IDO network without affecting viral activity. Flow cytometry assays revealed a decrease in the presence of the immunosuppressive cell populations, MDSCs and Tregs, which presence was higher in Delta-24-RGDOX-treated tumors. These data were further confirmed in gene set enrichment analyses. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens and that this activation was reversed by Kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing the immunosuppression caused by the virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy.

The median survival for GBM patients is 15 months with a five-year survival rate of less than 10%. Current conventional and experimental treatments, including immune checkpoint blockade, have not improved outcomes in the vast majority of glioma patients. Oncolytic viruses (OVs) are designed to specifically infect and lyse cancer cells to elicit an anti-tumor immune response. In a clinical trial developed by the MDACC team, 20% of GBM patients treated with the oncolytic adenovirus Delta-24-RGD showed complete response and survival longer than three years (NCT00805376). Although these studies have shown that robust immune responses can be invoked using oncolytic virotherapy, the molecular mechanisms underlying OV-mediated anti-tumor immunity remain elusive. Exposure to viruses by innate immune sensors induces protective antiviral immunity. Using bulk RNA sequencing of cells infected with adenovirus we identified the main upstream regulators of the innate immune response including IFN gamma, Interferon responsive genes 3 and 7, and STAT3. However, RNAseq unexpectedly uncovered NONO (Non-POU Domain Containing Octamer Binding) as the most relevant upstream regulator. In agreement with the RNAseq data, Protein analyses demonstrated the upregulation of NONO after adenovirus infection. In addition, immunofluorescence and confocal microscopy examination of the infected cells showed the overexpression and translocation to the nucleus of NONO upon virus infection. We are the first laboratory that has identified NONO as a sensor of adenovirus infection, which may have great significance for the future development of oncolytic viruses. Importantly, we discovered the network of proteins that are upregulated after NONO activation and, finally, we have established that changes in the expression level and subcellular localization of NONO are coincident in time during the infection of cultured cells. Future work is focused on examining the immunomodulatory functions of NONO in adenoviral infected tumors and subsequent generation of NONO-resistant oncolytic adenovirus.

Tumor-targeting biological therapies, including adoptive T cell therapy (ACT) and oncolytic viruses, are being tested clinically to address the challenges in treating advanced solid tumors with heterogeneity and immune suppressive microenvironment (TME). Since complementary properties of these two approaches have been observed, we hypothesize that localized intratumoral treatment with combination of ACT and Delta-24-RGDOX (third generation oncolytic adenovirus developed by our group) induces not only potent therapeutic effect in treated tumor but also abscopal benefit to eliminate distant disseminated tumors. Using a B16-C57BL/6 melanoma model with disseminated tumors in C57BL/6 mice, we found gp100-specific CD8+ T cells injected into the first subcutaneous (s.c.) tumor showed tropism for distant s.c. and intracranial tumors, tumor draining lymph nodes, compared to spleen, blood and normal brain hemisphere. In a B16-OVA-C57BL/6 s.c./s.c. tumor model, Delta-24-RGDOX injections following gp100-specific T cells in the first tumor increased total cytotoxic T cell presence within both tumors, including gp100- and OVA- specific T cells. Further analysis revealed that Delta-24-RGDOX reduced the exhaustion of OVA-specific T cells while that of gp100-specific T cells kept at a low level. Moreover, Delta-24-RGDOX augmented the activity of the splenocytes against the tumor cells and other tumor-associated antigens (OVA, TRP2) than gp100. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected and untreated distant tumor growth and caused improved survival rate than either of the agent alone (p < 0.05). Importantly, we observed relapse of the regressed tumors in the T cell treatment group, but not in the combination group. The survivors developed immune memory against rechallenging with B16-OVA cells. Collectively, our data demonstrate that localized treatment with intratumoral ACT followed by Delta-24-RGDOX induces global immune activation and antigen spread to expand antitumor T cell repertoire, leading to efficacious systemic anti-tumor immunity and sustainable tumor regression.

BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.MethodsThe in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.ResultsDelta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.ConclusionsIn summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032)., The oncolytic virus Delta-24-RGD is in clinical trial for adult glioma. Here, the authors show that this virus elicits an immune response in mouse models of pediatric high-grade glioma and diffuse pontine intrinsic glioma, resulting in improved survival.

Viroimmunotherapy aims to infect cancer cells to elicit anti-tumor immune responses. In clinical trials, glioma treatment with oncolytic viruses induced durable clinical responses in a small fraction of patients. To improve the percentage of responders, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. Thus, we engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus that carries the cDNA of the T-cell activator, OX40L. In this work, we observed that Delta-24-RGDOX triggered a dramatic reshaping of the tumor microenvironment dominated by strong changes in immune processes as indicated by RNA-sequencing via ingenuity pathway analyses in a murine glioblastoma model. Paradoxically, network analyses revealed that Delta-24-RGDOX also induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of the cancer cells to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma bearing mice. Importantly, addition of the IDO inhibitor to Delta-24-RGDOX decreased the activation of the IDO network. IDO inhibition did not affect virus infection or replication in human or murine glioma cells. Flow cytometry assays revealed that the combination therapy increased the frequency of activated CD8+ T cells and decreased the presence of the immunosuppressive cell populations, MDSCs and Tregs. Gene set enrichment analyses confirmed the decrease of MDSCs and Tregs in the combination treated glioma-bearing mice compared to the virus alone. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens, and that this activation was reversed by kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing immunosuppression caused by virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Sagar Sohoni, Sanjay K. Singh, Yisel Rivera-Molina, Hong Jiang, Xuejun Fan, Joy Gumin, Frederick F. Lang, Christopher Alvarez-Breckenridge, Marta M. Alonso, Filipa Godoy-Vitorino, Lijie Zhai, Erik Ladomersky, Kristen L. Lauing, Derek A. Wainwright, Juan Fueyo, Candelaria Gomez-Manzano. RNA-seq analyses reveal remodeling of tumor microenvironment and reversal of glioma resistance to oncolytic viruses by targeting immunometabolism [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 4184.

Recent clinical observations that some coronavirus infections induced complete remissions in lymphoma patients emphasized again the potential of cancer virotherapy. Infection of cancer cells with oncolytic viruses reshapes the tumor microenvironment by activating anti-viral and anti-tumor immunity. A phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD (DNX-2401) to treat recurrent malignant gliomas demonstrated activation of CD8+ T-cells and significant clinical benefits for a subset of patients. However, both anti-virus and anti-tumor immune responses are contingent on the activation of respective clones of CD8+ T-cells, which compete for clonal expansion. Thus, overexpansion of T-cells against viral antigens reduces the frequency of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for viral antigens will decrease anti-viral immunity and in turn derepress anti-tumor immunity, resulting in enhanced efficacy of cancer virotherapy. In this work, we used nanoparticles encapsulating adenoviral antigens E1A, E1B and hexon that distributed to liver resident macrophages (P Citation Format: Dong Ho Shin, Hong Jiang, Debora Kim, Teresa T. Nguyen, Sagar Sohoni, Xuejun Fan, Yanhua Yi, Arie C. Van Wieren, Gheath Al-Atrash, Chantale Bernatchez, Ramon Alemany, Marta Alonso, Frederick Lang, Candelaria Gomez-Manzano, Bulent Ozpolat, Juan Fueyo. Redirection of anti-virus immunity towards anti-tumor immunity using nanoparticle-induced immune tolerance for oncolytic adenovirus therapy [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 3559.

CAR T therapy greatly improves the survival of patients with hematological malignancies. But its effect in solid tumors is suboptimal. Intratumoral injections of oncolytic viruses, including Delta-24-RGDOX (DNX-2440) from our group, directly lyse cancer cells and activate tumor microenvironment, leading to adaptive antitumor immunity. To take advantage of instant antitumor activity of T cells and virus-mediated in situ autovaccination against heterogeneous cancer cells, we hypothesize that intratumorally injections of tumor-targeting T cells followed by Delta-24-RGDOX overcome antigen escape of CAR T therapy, leading to more sustainable systemic anti-cancer immunity. Thus, we used subcutaneous (s.c.)/s.c. B16 melanoma mouse models to assess the effect of localized treatment in disseminated tumors. OVA (or gp100)-specific CD8+ T cells or CD19 CAR T cells were injected into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. T cells and tumor cells from the mice were profiled for surface markers with flow cytometry and immune staining. Activity of splenocytes against tumor cells and specific tumor-associated-antigens (TAAs) was measured with ELISA. Tumor growth was monitored through measuring tumor size. The animal survival curves were plotted according to the Kaplan-Meier method. We found TAA-specific T cells injected into the first s.c. tumor showed tropism for disseminated s.c. and intracranial tumors, tumor draining lymph nodes, but not for spleen, peripheral blood and normal brain. Unlike untreated and Delta-24-RGDOX-treated tumor, T cell-treated tumor showed decreased expression of the target TAA which was depleted in the recurrent tumor. Moreover, Delta-24-RGDOX increased total T cell presence within the tumors, and the activity of the splenocytes against the tumor cells and other antigens than the one targeted by injected T cells. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected and untreated disseminated tumor growth and caused improved survival rate than either of the agent alone (p < 0.05). Importantly, we observed relapse of the regressed tumors in the group treated by T cells alone, but not in the combination group. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory. In summary, our study indicates the virus induces antigen spread, resulting in expansion of antitumor T cell repertoire to prevent cancer relapse in adoptive T cell therapy (ACT). Our data demonstrate that Delta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to sustainable tumor regression. Citation Format: Hong Jiang, Dong Ho Shin, Yanhua Yi, Xuejun Fan, Joy Gumin, Marta M. Alonso, Frederick F. Lang, Candelaria Gomez-Manzano, Juan Fueyo. Combining oncolytic adenovirus Delta-24-RGDOX with adoptive T cell therapy in localized treatment induces sustainable regression of disseminated solid tumors through antigen spreading [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 3569.

Purpose of Study: Diffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children, with a peak of incidence between 5-10 years old. The median survival of DMG patients is only 9 months, being the leading cause of pediatric death caused by a brain tumor. On this project we set to characterize the efficacy of Delta-24-RGDOX, an oncolytic adenovirus based on Delta-24-RGD platform, which has demonstrated safety and a therapeutic benefit in different pediatric tumors, armed with the ligand of OX40. The binding of OX40 to OX40L leads to the co-stimulation of CD4 and CD8 cells, generating effector and memory T cells. Therefore, the aim of this project is to improve the antitumor effect of the virus, providing a greater co-stimulation in the tumor. Experimental Procedures: Murine and human DMG cell lines were used. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. OX40L expression was measured by flow cytometry and qPCR. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system. The adenovirus was administered once into the tumor using the same procedure. Tumor immune populations were analyzed by flow cytometry. Results: We first confirmed the oncolytic effect of Delta-24-RGDOX in DMG murine and human cell lines in vitro. The virus was able to infect the cells, produce viral proteins, and to cause cell death in a dose-dependent manner. In addition, we observed effective viral replication in human cell lines but not in mouse cells, as previously described. We then measured OX40L expression in infected cells both at protein and mRNA level, observing that at 50 PFU/cell almost 100% of cells expressed the ligand on their membrane. More importantly, the OX40L was functional as it activated CD8 lymphocytes in vitro. Once we confirmed that the viral administration in vivo was safe, we assess the efficacy of the virus in murine DMG models. We observed a significant survival benefit in mice bearing NP53 tumor treated with Delta-24-RGDOX, which lead to 30% of long-term survivors (P=0.003, median OS PBS 25.5 days vs 35.5 days for treated mice). More importantly, we administered the virus in an already stablished tumor model using XFM cells and also obtained a significant improve of survival (P=0.018, median OS PBS 9 days vs 12.5 treated mice). Finally, we analyzed the immune mechanisms underlying the survival benefit, both in the tumor and in spleen. We observed a significant increase of activated immune cells in the tumor microenvironment 7 days after the viral administration. In addition, splenocytes from virus-treated mice were also significantly more activated. Conclusions: These data show that Delta-24-RGDOX adenovirus expresses a functional OX40L that can modulate the immune response, leading to a significantly improved survival outcome in DMG models. Citation Format: Virginia Laspidea, Sara Labiano, Sumit Gupta, Hong Jiang, Iker Ausejo-Mauleon, Daniel de la Nava, Marc García-Moure, Javier Marco-Sanz, Reyes Hernández-Osuna, Oren J Becher, Juan Fueyo, Ana Patiño-García, Candelaria Gomez-Manzano, Marta M Alonso. Delta-24-RGDOX oncolytic adenovirus improves the survival by modulating the immune system in DMG 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 2001.

Oncolytic viruses are a promising experimental treatment for solid tumors. Recently, several phase 1 clinical trials have reported encouraging therapeutic effects of oncolytic viruses in adult and pediatric patients with malignant gliomas. To further improve the therapeutic outcome of viroimmunotherapy, we have developed Delta-24-RGDOX (DNX-2440), a replication competent adenovirus encompassing the T-cell activator OX40L in the genetic backbone of Delta-24-RGD. We have previously reported the effect of Delta-24-RGDOX in murine brain tumors supporting the translation of this new agent to treat patients with recurrent malignant gliomas (NCT03714334). In the work presented here, we have tested the therapeutic effect of Delta-24-RGDOX in murine syngeneic models of breast (4T1), gastric (M12) and lung cancer (LLC and CMT 167). We found that Delta-24-RGDOX infected all cancer cell lines efficiently. In addition, infection of cells was followed by the expression of the ectopic ligand in vitro and in vivo. Because the elicitation of an anti-tumor immunity is part of the mechanisms underlying the therapeutic effect of oncolytic viruses, we examined whether infection of tumors led to the reshaping of the tumor microenvironment. We observed that Delta-24-RGDOX infection was followed by increased frequencies of tumor infiltrating lymphocytes, particularly CD8+ T cells and NK cells. In addition, the CD8+/CD4+ ratio was increased in Delta-24-RGDOX-treated tumor versus PBS-treated tumors. Interestingly, abscopal modifications were observed in breast cancer brain metastases with increased frequency of CD8+ T cells at the distal, untreated site. Delta-24-RGDOX treatment induced an anti-cancer effect in orthotopically implanted breast cancer and subcutaneously implanted lung and gastric tumors, as well as in metastatic niches. In summary, our data showed that treatment of solid tumors with Delta-24-RGDOX induces robust remodeling of the tumor microenvironment and produces anti-tumor effects leading to decrease in tumor volume, along with a delay in the development and in the reduction of the number of metastases. These data suggest that Delta-24-RGDOX should be tested in the clinical setting in patients with metastatic breast, gastric and lung cancers. Citation Format: Arie C. Van Wieren, Sagar Sahoni, Teresa Nguyen, Ashley Ossimetha, Yisel Rivera-Molina, Hong Jiang, Dong Ho Shin, Debora Kim, Xuejun Fan, Yanhua Yi, Natalie M. Melendez-Vazquez, Filipa Godoy-Vitorino, Joy Gumin, Frederick F. Lang, Marta M. Alonso, Juan Fueyo, Candelaria Gomez-Manzano. Viroimmunotherapy for solid tumors results in local and abscopal anti-cancer effects and the remodeling of tumor microenvironment [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 3565.

Purpose of the work: Pediatric High Grade Gliomas (pHGGs), including Diffuse Midline Gliomas (DMGs), are very aggressive solid tumors developed during childhood with a poor overall survival underscoring the need for effective treatments. The adenovirus Delta-24RGD and the imipridone ONC201 have demonstrated safety and effectiveness in preclinical models and clinical trials. Thus, we evaluated the therapeutic efficacy of the Delta-24-RGD/ONC201 combination and analyzed possible changes in the tumor microenvironment in preclinical models of pHGGs and DMGs. Experimental procedures: A battery of pHGGs (SF188 and CHLA-03-AA), human DMG (SU-DIPG IV), and murine DMG cell lines (XFM and NP-53) were used. We assessed the viral replication by hexon titration, the protein expression amount (E1A, fiber, and mTORC1 key proteins)by immunoblotting, and oxygen consumption by Seahorse Analyses. Viability was measured by MTS. CHLA-03-AA and XFM were engrafted orthotopically in athymic nude mice and Balb/c mice, respectively. To assess in vivo efficacy, the animals were treated with Delta-24-RGD (107 PFU/animal) or PBS, followed by ONC201administration (125 mg/kg/twice/weekly). The tumor immune microenvironment was evaluated by flow cytometry. Results: First, the potential adverse interactions between both agents were checked in vitro, showing that ONC201 cotreatment did not interfere with the Delta-24-RGD replication capacity. As expected, ONC201 treatment led to a reduction in oxygen consumption, which was also observed in the combination treatment. Evaluation of cytotoxicity in a panel of cell lines showed that the combination was significantly better than either agent alone. In vivo, CHLA-03-AA bearing mice treated with the combination showed a significant increase in the median overall survival (PBS: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; Delta-24-RGD/ONC201: 95 days (P=0.0008)), leading to 20%long-term survivors, free of disease. Evaluation of the combination in an immunocompetent orthotopic DMG model also revealed the superior efficacy of the Delta-24-RGD/ONC201 (PBS: 11 days; ONC201: 12 days; Delta-24-RGD: 12 days; ONC201+Delta-24-RGD: 15 days (P=0.02)). Of importance, the combination treatment resulted in the reshaping of the tumor microenvironment towards a proinflammatory phenotype (T cells, NK cells, macrophages, granulocytes, monocytes, dendritic cells, and microglia). Conclusions: Our data indicate that the combination Delta-24-RGD/ONC201 generates a pro-inflammatory microenvironment that ultimately leads to a superior therapeutic effect in preclinical pHGG and DMGs models. Citation Format: Daniel de la Nava, Javier Marco-Sanz, Virginia Laspidea, Sara Labiano, Iker Ausejo-Mauleon, Reyes Hernandez-Osuna, Sabine Mueller, Javad Nazarian, Joshua E. Allen, Oren Becher, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-Garcia, Marta M. Alonso. The adenovirus Delta-24-RGD in combination with ONC201 provides a therapeutic benefit and a proinflammatory recruitment in preclinical models of pHGGs and DMGs [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 2000.

With a 2-year survival less than 20%, Diffuse Intrinsic Pontine Glioma (DIPG) is the principal cause of pediatric death. Despite recent advances in the current treatments, the outcome for children with DIPGs remains dismal. Since the approval of T-VEC for melanoma by the FDA, oncolytic adenoviruses have emerged as a promising therapeutic strategy for brain tumors. Thus, our group launched the first world clinical trial phase I with the oncolytic adenovirus Delta-24-RGD (DNX-2401 in the clinic) for newly diagnosed DIPG (NCT03178032), which has shown safety and feasibility. Despite DNX-2401 increases the recruitment of T cells into the tumor, they usually become inactive due to the non-responsive tumor microenvironment evidencing the urgent need to improve this strategy focusing on the generation of effective long-term immune responses. Therefore, we decided to combine the Delta-24-RGD with the targeting of the costimulatory molecule CD40 in immunocompetent mice bearing orthotopic DIPG. The activation of the CD40 receptor, which is expressed by antigen presenting cells (APC) such as microglia, macrophages, and dendritic cells, is known to increase antigen presentation and enable T-cell priming and activation. Here, we observed that in addition to Delta-24-RGD anti-tumor effects, the stimulation of CD40 (using an agonistic antibody) on the tumor APCs results in a remodeling of the tumor immune compartment towards a proinflammatory scenario and a more efficient T-cell infiltration. Of importance, the combination therapy extends survival of treated mice as compared to single treatments or non-treated mice. In addition, we observe a complete regression of tumors in more than 40% of treated mice and the development of long-term anti-tumor immunity. We believe that these results provide a translational breakthrough in the treatment of these lethal tumors and open the door for a future innovative clinical trial.

Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells. Experimental Design: Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in vitro in a battery of CNS-PNET and AT/RT cell lines. In vivo, efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34+-NSG-SGM3). Results: Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC50 value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. In vivo, a single intratumoral Delta-24-RGD injection (107 or 108 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD–treated tumors revealed increased CD8+ T-cell infiltration. Conclusions: Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting.

With a 2-year survival less than 20%, Diffuse Intrinsic Pontine Glioma (DIPG) is the principal cause of pediatric death. Despite recent advances in the current treatments, the outcome for children with DIPGs remains dismal. Since the approval of T-VEC for melanoma by the FDA, oncolytic adenoviruses have emerged as a promising therapeutic strategy for brain tumors. Thus, our group launched the first world clinical trial phase I with the oncolytic adenovirus Delta-24-RGD (DNX-2401 in the clinic) for newly diagnosed DIPG (NCT03178032), which has shown safety and feasibility. Despite DNX-2401 increases the recruitment of T cells into the tumor, they usually become inactive due to the suppressive tumor microenvironment evidencing the urgent need to improve this strategy focusing on the generation of effective long-term immune responses. Therefore, we decided to combine the Delta-24-RGD with the targeting of the costimulatory molecule CD40 in two unique orthotopic immunocompetent mouse models of DIPG. The activation of the CD40 receptor, which is expressed by antigen presenting cells (APC) such as microglia, macrophages and dendritic cells, is known to increase antigen presentation and enable T-cell priming and activation. Here, we have observed that in addition to Delta-24-RGD anti-tumor effects, the stimulation of CD40 on the tumor APCs results in a remodeling of the tumor immune compartment with a more efficient T-cell tumor infiltration. Of importance, there is an increase of the survival of mice treated with the combination as compared to single treatments or non-treated mice. In addition, the combination therapy induced a complete regression of tumors in 25% of treated mice indicating the development of long-term anti-tumor immunity. We believe that these results provide a translational breakthrough in the treatment of these lethal tumors and open the door for a future innovative clinical trial.

Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases proliferation and induces apoptosis of surrounding effector T-cells. Kynurenine, a metabolite of tryptophan, induces T-cell differentiation into immunosuppressive Tregs. Excess kynurenine elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, Delta-24-RGDOX, triggers IFNγ production contributing to a positive IDO-Kynurenine-AhR feedback loop. We hypothesized that combining Delta-24-RGDOX with IDO inhibitors will synergize to effectively treat glioblastoma. We characterized IDO and AhR in Delta-24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo. We also observed induction of AhR in CD4+ and CD8+ T-cells by Delta-24-RGDOX in vivo. Delta-24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. We used a murine glioblastoma model to test the efficacy of combining Delta-24-RGDOX with IDO inhibitor, 1MT/indoximod; the combination produced 30% more long-term survivors compared Delta-24-RGDOX alone (P=0.03), which we showed, through lymphocytic depletion studies, was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and myeloid derived suppressor cells compared to Delta-24-RGDOX single treatment (P≤0.05). This microenvironment remodeling correlated with complete tumor elimination. Altogether, Delta-24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression.

Atypical teratoid/rhabdoid tumors (AT/RTs) and primitive neuroectodermal tumors of the central nervous system (CNS-PNET) are rare pediatric brain tumors affecting mainly infants and young children. Current therapies for these diseases are inefficient and often cause severe side effects; thus, new therapeutic strategies are urgently needed. Here, we evaluate the anti-tumor effect of the Delta-24-RGD oncolytic adenovirus in preclinical models of CNS-PNET and AT/RT. In vitro, the virus infects and efficiently replicates in different cell culture models of AT/RT and CNS-PNET, inducing a dose-dependent cytotoxic effect (IC50 values below 1 PFU/cell) and the release of immunogenic cell death stimuli. Survival studies in mice bearing orthotopic AT/RTs (supra and infratentorial) or CNS-PNETs demonstrate the therapeutic benefit of a single Delta-24-RGD intratumoral administration (107 or 108 PFU). In virus-treated mice, median overall survival increased between 50–300% (depending on the model), obtaining up to 60% of long-term survivors. Intra-ventricular injection of Delta-24-RGD hinders the development of secondary AT/RT spinal lesions. Moreover, response to the treatment was observed even delaying viral administration up to 30 days after tumor engraftment; thus, demonstrating therapeutic benefit even in animals challenged with large well-established tumor masses. The effect of the oncolytic virus was also assessed in an immunocompetent environment using hCD34+ humanized NSG-SGM3 mice. In this model, Delta-24-RGD significantly extended overall survival (from 23 to 24 days in AT/RT model and from 28 to 55 days in CNS-PNET) with no signs of toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD treated tumors by flow cytometry, RNAseq and multiplexed histology revealed that Delta-24-RGD contributes to generate a pro-inflammatory status, allowing the infiltration of immune cells and the trigger of an antitumor immune response. In conclusion, these results demonstrate that Delta-24-RGD could be a feasible therapeutic choice for AT/RT and CNS-PNET, paving the way for upcoming clinical trials.

Despite the advances in our understanding of pediatric diffuse midline gliomas (DMG) they remain the leading cause of pediatric death caused by cancer. Our group has demonstrated that the administration of Delta-24-RGD in DIPG models is safe and therapeutically efficacious. An on-going clinical trial with this virus has proven to be safe for these patients. To further improve the anti-tumoral response of the virus, we armed Delta-24-RGD with 4-1BBL (Delta-24-ACT). 41BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. Here, we showed that in vitro Delta-24-ACT can infect and express 4-1BBL in murine and human DIPG cell lines. Importantly, 4-1BBL expression in DIPG cell lines was able to activate lymphocytes and increase their IFN-gamma production. In addition, Delta-24-ACT triggered immunogenic cell death in DIPG cell lines, as shown by the release of DAMPs such as ATP, HMGB1, Hsp90a and calreticulin translocation. Delta-24-ACT administration in orthotopic DIPG models was well tolerated and safe. We confirmed the expression of the ligand within the tumor. Moreover, using flow cytometry and multispectral immunohistochemistry we observed profound changes in the tumor microenvironment with an increase in the T-cell populations and a remodeling of the myeloid component before and after treatment. Functional studies showed no differences in lymphocytes isolated from mice treated splenocytes but uncovered TILS that showed a significantly increased in the production of IFN-gamma. Delta-24-ACT treatment of mice bearing orthotopic DIPG murine tumors resulted in a significant increase in the median survival and led to free of disease long-term survivors. In summary, Delta-24-ACT is a virus that builds in our clinical experience with Delta-24-RGD in DIPG patients going a step further to boost the antitumor effect of viral therapy while maintaining a safe profile.

BackgroundDiffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children between 5–10 years old. DMGs are the leading cause of pediatric death caused by a brain tumor, with a median survival of only 9 months.1 2 We have previously shown that the administration of the oncolytic adenovirus Delta-24-RGD is safe and lead to an increase in long-term survivors in murine models.3 4 In order to further increase the antitumor effect of Delta-24-RGD by boosting the immune response, we have constructed a new adenovirus, Delta-24-ACT, which incorporates the 4-1BBL (CD137L) into its backbone. 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells and NK cells and the generation and maintenance of memory CD8+ T cells, among other functions.5 6MethodsMurine and human DMG cell lines were used. 4-1BBL expression was assessed in infected cells by flow cytometry and immunofluorescence. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system.7 A single administration of the adenovirus was injected intratumorally using the same procedure. The tumor immune populations were analyzed by flow cytometry.ResultsWe first confirmed by flow cytometry that DMG cells infected with Delta-24-ACT expressed 4-1BBL in their membrane in a dose-dependent manner. Afterwards, we analyzed the oncolytic effect of Delta-24-ACT in vitro. Delta-24-ACT was able to express viral early and late proteins in murine and human DMG cell lines and to replicate efficiently in human cells. In addition, the virus caused cell death in a dose-dependent manner. In vivo, Delta-24-ACT administration demonstrated to be safe and to produce a significant survival benefit in murine DMG models, obtaining 30–50% of long-term survivors depending on the model. More importantly, Delta-24-ACT generated immune memory, as long-term survivors were disease-free after cell rechallenge. On the other hand, we analyzed immune infiltration 7 or 10 days after the viral administration into the tumor and observed a significant increase of tumor infiltration in treated mice, which showed an activated state.ConclusionsDelta-24-ACT administration into DMG murine tumor models significantly increases the recruitment and activation of immune cells, which leads to long term survivors and immunological memory.ReferencesCooney T, Lane A, Bartels U, Bouffet E, Goldman S, Leary S, Foreman NK, Packer RJ, Broniscer A, Minturn JE, Shih C, Chintagumpala M, Hassall T, Gottardo NG, Dholaria H, Hoffman L, Chaney B, Baugh J, Doughman R, Leach JL, Jones BV, Fouladi M, Warren KE, Monje M. Contemporary survival endpoints: an International diffuse Intrinsic pontine glioma registry study. Neuro Oncol 2017;19(9):1279–1280.Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, Ponnuswami A, Chen S, Johung TB, Sun W, Kogiso M, Du Y, Qi L, Huang Y, Hütt-Cabezas M, Warren KE, Le Dret L, Meltzer PS, Mao H, Quezado M, van Vuurden DG, Abraham J, Fouladi M, Svalina MN, Wang N, Hawkins C, Nazarian J, Alonso MM, Raabe EH, Hulleman E, Spellman PT, Li XN, Keller C, Pal R, Grill J, Monje M. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med 2015;21(6):555–9.Martínez-Vélez N, Garcia-Moure M, Marigil M, González-Huarriz M, Puigdelloses M, Gallego Pérez-Larraya J, Zalacaín M, Marrodán L, Varela-Guruceaga M, Laspidea V, Aristu JJ, Ramos LI, Tejada-Solís S, Díez-Valle R, Jones C, Mackay A, Martínez-Climent JA, García-Barchino MJ, Raabe E, Monje M, Becher OJ, Junier MP, El-Habr EA, Chneiweiss H, Aldave G, Jiang H, Fueyo J, Patiño-García A, Gomez-Manzano C, Alonso MM. The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models. Nat Commun 2019;10(1):2235.Garcia-Moure M, Gonzalez-Huarriz M, Labiano S, Guruceaga E, Bandres E, Zalacain M, Marrodan L, de Andrea C, Villalba M, Martinez-Velez N, Laspidea V, Puigdelloses M, Gallego Perez-Larraya J, Iñigo-Marco I, Stripecke R, Chan JA, Raabe EH, Kool M, Gomez-Manzano C, Fueyo J, Patiño-García A, Alonso MM. Delta-24-RGD, an oncolytic adenovirus, increases survival and promotes proinflamatory immune landscape remodeling in models of AT/RT and CNS-PNET. Clin Cancer Res 2021;27(6):1807–1820.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Yonezawa A, Dutt S, Chester C, Kim J, Kohrt HE. Boosting cancer immunotherapy with anti-CD137 antibody therapy. Clin Cancer Res 2015;21(14):3113–20.Marigil M, Martinez-Velez N, Domínguez PD, Idoate MA, Xipell E, Patiño-García A, Gonzalez-Huarriz M, García-Moure M, Junier MP, Chneiweiss H, El-Habr E, Diez-Valle R, Tejada-Solís S, Alonso MM. Development of a DIPG orthotopic model in mice using an implantable guide-screw system. PLoS One 2017;12(1):e0170501.

BackgroundUnlike its remarkable success in treating hematological malignancies, adoptive T cell therapy (ACT), such as CAR T therapy, targeting limited tumor-associated antigens (TAAs) is far less effective in patients with heterogeneous solid tumors. Oncolytic viruses, including oncolytic adenovirus Delta-24-RGDOX from our group [1, 2], are emerging as promising immunotherapy drugs. To take advantage of instant antitumor activity of T cells and oncolytic adenovirus mediated in situ autovaccination against heterogeneous cancer cells, we hypothesize that intratumorally injected Delta-24-RGDOX complements with ACT to overcome antigen escape, leading to more sustainable anti-cancer effect.MethodsWe used B16-OVA-C57BL/6 subcutaneous (s.c.)/s.c. melanoma model [2] to assess the systemic therapeutic effect in disseminated tumors. OVA (or gp100)-specific CD8+ T cells were injected into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. T cells from the mice were profiled for surface markers with flow cytometry. Activity of splenocytes against tumor cells and specific TAAs was measured with ELISA. Tumor growth was monitored through measuring tumor size three times a week. The animal survival curves were plotted according to the Kaplan–Meier method.ResultsTAA-specific T cells injected into the first s.c. tumor showed tropism for disseminated s.c. and intracranial tumors, tumor draining lymph nodes, compared to normal tissue, spleen and peripheral blood. Delta-24-RGDOX increased total T cell presence within the tumors, and the activity of the T cells against the tumor cells and targeting other antigens than the one targeted by the transferred T cells. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected first tumor and untreated disseminated second tumor growth and resulted in improved survival rate than either of the agent alone (p < 0.05). Interestingly, we observed relapse of the regressed tumors in the group treated by T cells alone. But the relapse was not observed in most of the mice in the combination group. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory.ConclusionsDelta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to sustainable tumor regression and improved survival rate. Our study indicates the virus induces antigen spread, resulting in expansion of T cell repertoire to prevent cancer relapse in ACT.ReferencesJiang, H., et al., Localized Treatment with Oncolytic Adenovirus Delta-24-RGDOX Induces Systemic Immunity against Disseminated Subcutaneous and Intracranial Melanomas. Clin Cancer Res, 2019. 25(22): p. 6801–6814. https://cancerres.aacrjournals.org/content/77/14/3894.long.Jiang, H., et al., Oncolytic Adenovirus and Tumor-Targeting Immune Modulatory Therapy Improve Autologous Cancer Vaccination. Cancer Res, 2017. 77(14): p. 3894–3907.https://clincancerres.aacrjournals.org/content/25/22/6801.full-text.pdf

Oncolytic virotherapy and adoptive T cell therapy (ACT) are promising strategies for cancer treatment, but both are underdeveloped into routine clinical interventions for solid tumors. We reported previously that localized treatment with oncolytic adenovirus Delta-24-RGDOX (DNX-2440), which expresses immune co-stimulator OX40 ligand, stimulated tumor-specific T cells proliferation and migration to untreated disseminated tumors. We hypothesize that intratumorally injected Delta-24-RGDOX complements with ACT to overcome antigen escape, leading to more sustainable anti-cancer effect. To test the hypothesis, we used B16-OVA-C57BL/6 subcutaneous (s.c.)/s.c. melanoma model to assess the effect of the combination therapy. Briefly, 10 days after the first tumor implantation, we injected OVA-specific CD8+ T cells from OT-I mice into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. We found the combination was more potent to inhibit the injected 1st tumor and untreated disseminated 2nd tumor growth and resulted in improved survival rate than either of the agent alone (p less than 0.05). Interestingly, we observed relapse of the regressed tumors in the group treated by T cells alone, which recapitulated the effect of CAR T therapy in cancer patients. But the relapse was not observed in most of the mice from the combination group, indicating the combination induces more sustainable tumor regression than T cells alone. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory against the same tumor cells. Furthermore, profiling the tumor-infiltrating lymphocytes with flow cytometry revealed that the combination promotes the T cell presence within the tumor and increase the population of the T cells targeting other antigens in addition to the original ACT targeted antigen. Collectively, the results demonstrate that Delta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to more sustainable tumor regression and improved survival rate. Our study suggests the virus induces antigen spread, resulting in T cell repertoire to target more tumor-associated antigens to overcome cancer relapse due to cancer heterogeneity and limited targets in ACT and CAR T therapy. Citation Format: Hong Jiang, Teresa T. Nguyen, Dong Ho Shin, Sagar S. Sohoni, Sumit Gupta, Yanhua Yi, Xuejun Fan, Candelaria Gomez-Manzano, Juan Fueyo. Oncolytic adenovirus Delta-24-RGDOX collaborates with adoptive cell therapy to sustain anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1579.

Metastatic breast cancer is the second leading cause of cancer related death among women. Although, conventional therapies such as surgery, adjuvant and neoadjuvant therapies have shown promising outcomes in preclinical and clinical settings, long-term survival rates remain unacceptably low for patients with recurrent disease or disseminated metastases. Breast primary and metastasic niches are characterized by an immunosuppressive microenvironment. Hence, novel therapeutic regimens that modulate this challenging phenotype are desperately needed. Our group has developed and tested in preclinical models and in clinical trials for patients with recurrent glioblastoma the anti-tumor effect of oncolytic adenovirus Delta-24-RGD, also named DNX-2401, resulting with a 20% of long-term survivors, and the awaking of a antitumoral immunity after the administration of the pathogen. This paradigm shift suggests that viroimmunotherapy can be used for the treatment of metastasic cancer. Thus, here we hypothesize that viroimmunotherapy is capable of exerting abscopal effects to eradicate primary breast tumors and their metastatic niches. To test the hypothesis, 4T1 tumor bearing mice were subjected to the local administration of Delta-24-RGD and its armed derivatives, expressing expressing immune co-stimulatory ligands OX40L (Delta-24-RGDOX or DNX-2440) and GITRL (Delta-24-GREAT). We found that, the tumors treated with oncolytic adenoviruses exhibited increase of both CD8+ T cell infiltration and CD8+/CD4+ ratio. Additionally, T-cell activity measured by IFN-γ secretion of splenocytes when in co-culture with breast cancer cells was found to be increased in mice that underwent viroimmunotherapy (P Citation Format: Sagar Shashikant Sohoni, Teresa Nguyen, Dong Ho Shin, Xuejun Fan, Jiang Hong, Sumit Gupta, Ashley Ossimetha, Yanhua Yi, Chandra Bartholomeusz, Marta Alonso, Juan Fueyo, Candelaria Gomez-Manzano. Targeting the immunosuppressive microenvironment of metastatic breast cancer with viroimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1786.

Oncolytic viruses are a new form of cancer immunotherapy that harnesses the combined abilities of the virus to directly lyse cancer cells and to induce immunogenic environments to activate anti-tumor immunity. Our oncolytic adenovirus Delta-24-RGD has demonstrated significant clinical benefits for a subset of patients with recurrent malignant gliomas in a recent phase 1 clinical trial. However, the backbone of Delta-24-RGD is derived from adenovirus serotype 5 to which 40 percent of the human population has prior exposure and harbor neutralizing antibodies that can block virus infection and replication. We hypothesized that replacing the major targets of antibodies in the oncolytic virus will protect it from neutralizing antibodies, resulting in enhanced efficacy of the cancer virotherapy. Because hexon hypervariable regions are the dominant targets of antibodies, we replaced these regions with those from a lesser prevalent serotype 43 to create a hybrid virus called Delta-24-RGD-H43m. Transmission electron microscopy imaging of the viruses revealed the assembly of viable virions after infection with either Delta-24-RGD or Delta-24-RGD-H43m in human and mouse cancer cell lines, signifying successful virus replication, that was corroborated by quantification of viral replication using hexon-staining dilution method. Accordingly, the inherent anti-tumor effect of the virus was not perturbed after the genetic modification, as Delta-24-RGD and Delta-24-RGD-H43m showed comparable efficacies both in vitro (P= 0.568) and in vivo using mice without prior immunity to adenovirus (P= 0.228). Importantly, the oncolytic ability of Delta-24-RGD-H43m was not compromised by neutralizing antibodies generated against adenovirus serotype 5 that efficiently neutralized Delta-24-RGD (P< 0.0001) as assessed by measuring cell viability after virus infection. In immunized mice with prior adenovirus exposure, Delta-24-RGD-H43m treatment yielded higher occurrences of complete tumor remission compared to Delta-24-RGD (89% versus 67%). Despite our growing understanding of viruses, the immune system, and cancer, the specific interaction between oncolytic viruses and the host immune system is still very limited. Strategies to protect the therapeutic viruses from virus-specific immune responses should prolong their persistence in tumors and result in better therapeutic outcomes. Further research in this field will aid the development of enhanced cancer virotherapies. Citation Format: Dong Ho Shin, Teresa T. Nguyen, Hong Jiang, Xuejun Fan, Yanhua Yi, Sagar S. Sohoni, Sumit Gupta, Ashley Ossimetha, Candelaria Gomez-Manzano, Juan Fueyo. Evasion of neutralizing antibodies and enhanced anti-tumor effects of hexon-modified Delta-24-RGD oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1912.

Gastric cancer is the third leading cause of cancer-related mortality worldwide, with a 5-year survival rate of 30% in the US despite standard treatment. Black, Asian, and non-white Hispanic populations have a 40-50% higher risk of gastric cancer than white people. The poor prognosis and limited therapies associated with gastric cancer has led us to investigate the use of these oncolytic adenoviruses as an alternative treatment. Viroimmunotherapy is showing promising results in preclinical and clinical studies. Our group developed an oncolytic adenovirus, Delta-24-RGD, also named DNX-2401, which has been tested in clinical trials for recurrent glioblastoma, with complete responses and durable survival in 20% of patients, which was associated with an anti-tumor immune response. To further enhance the immune effect of Delta-24-RGD, we developed the next generation virus armed with T cell co-stimulatory agonist, OX40L, termed Delta-24-RGDOX, clinically known as DNX-2440. To explore the sensitivity of gastric cancer to oncolytic adenoviruses, we performed viral infection and replication experiments in a panel of human gastric cancer cells (AGS, MKN-45, SNU-1) and a murine gastric cancer cell line, M12. Cell lines were infected in a dose-dependent experiment using an Ad-GFP-RGD vector, showing that >80% of the culture was infected using a multiplicity of infection of 25 for AGS, 50 for MKN-45 and SNU-1, and 100 for M12. Viral replication in gastric cancer was assessed by hexon staining of 293HEK cells incubated with cell lysates obtained from infected gastric cancer cells, illustrating that infection with either virus resulted in the production of new virions. Consistent with these data, we detected in Delta-24-RGD- and Delta-24-RGDOX-infected gastric cancer cells expression of E1A and hexon, confirming viral infection and replication, respectively. Of interest, 74% to 81% Delta-24-RGDOX-infected gastric cancer cells expressed in their surface OX40L 48 hours post-infection as assessed by flow cytometry. Efficacy of the proposed virotherapy in gastric cancer was visualized by using cell titer blue viability assay, which showed anti-cancer efficacy of the proposed therapy. Results on the efficacy and immune effects of armed-oncolytic adenovirus expressing OX40L in a M12-C57BL/6 gastric cancer immunocompetent mouse model are in progress. Current results suggest that armed oncolytic adenoviruses with positive immunomodulators can effectively infect, replicate, and lyse gastric cancer cells. Ultimately, we aim to translate these oncolytic adenoviruses to the clinic to improve the survival of patients with gastric cancer. Citation Format: Ashley Ossimetha, Teresa Nguyen, Natalie Melendez, Xuejun Fan, Marta Alonso, Juan Fueyo, Filipa Godoy-Vitorino, Candelaria Gomez-Manzano. Armed oncolytic adenovirus expressing the positive immune checkpoint OX40L for gastric cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1748.

Diffuse Intrinsic Pontine Glioma (DIPG) is the leading cause of pediatric death by brain tumors with a median survival of only 9 months, in spite of the current standard of therapy which includes radiation. The purpose of our study is to explore the use of oncolytic adenoviruses as a new therapeutic modality. Our group is leading a phase I clinical trial in naive DIPGs treated with the oncolytic adenovirus Delta-24-RGD, also named DNX-2401 (NCT03178032), which results are still pending. Of interest, clinical trials using Delta-24-RGD in adult gliomas resulted in encouraging results with long-term survivors and the presence of a strong anti-tumor immunity. In this study, we aim to test the effect of armed Delta-24-RGD with immune costimulatory ligands such as GITRL (Delta-24-GREAT) and OX-40L (Delta-24-RGDOX or DNX-2440) for the treatment of DIPGs. We used a panel of murine DIPG cell lines (NP53 and XFMA) to test the effect of these armed-oncolytic adenoviruses. Infectivity assay using an Ad-RGD-GFP vector and flow cytometry showed the expression of the ectopic GFP in more than 80% of the NP53 cells (50 MOIs) and XFMA cells (30 MOIs). Consistent results were obtained using flow cytometry and western blot to detect the expression of the ectopic ligands GITRL and OX-40L in these cultures. These cells were semipermissive for viral replication as assessed by the detection of the viral structural protein hexon in western blot analysis. Preliminary in vivo data using immunocompetent mice models where DIPG cells have been implanted into the pons showed a dose-dependent effect and the generation of immune memory. Our results suggest that oncolytic adenoviruses expressing positive immunocheckpoints might prove be a new effective modality for treating DIPG and hence, increase survival in children suffering this dismal disease. Citation Format: Sumit Gupta, Virginia Laspidea, Juan Fueyo, Oren Becher, Teresa Nguyen, Dong Ho Shin, Hong Jiang, Sagar Sohoni, Xuejun Fan, Yanhua Yi, Joy Gumin, Frederick Lang, Candelaria Gomez-Manzano, Marta M. Alonso. Armed oncolytic virus for treatment of pediatric diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3053.

Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases viral replication and diminishes cell proliferation and induces apoptosis of surrounding effector T-cells. Furthermore, kynurenine (kyn), a metabolite of tryptophan, induces T-cell differentiation into Tregs. Excess kynurenine also elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, D24-RGDOX, clinically known as DNX-2440, triggers IFNγ production contributing to a positive IDO-Kyn-AhR feedback loop. Thus, we hypothesized that combining D24-RGDOX with IDO inhibitors will synergize to treat glioblastoma. We studied the IDO-Kyn-AhR cascade in response to D24-RGDOX infection in human and murine glioblastoma cells. Orthotopic syngeneic glioblastoma mouse models were used to determine efficacy and tumor immune effects of D24-RGDOX and IDO inhibitors as single agents or as a combination treatment. We characterized IDO and AhR in D24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo compared to mock infected controls. D24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. Furthermore, we showed that viral protein expression and viral activity was enhanced in D24-RGDOX infected cultures under IDO inhibition. To test the therapeutic efficacy of combining D24-RGDOX with IDO inhibitors, we used a murine immunocompetent glioblastoma model; the combination produced 30% more long-term survivors compared D24-RGDOX alone (P=0.03, log-rank test). Next, lymphocytic depletion studies illustrated that this anticancer effect was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to D24-RGDOX-treated mice (P=0.009, student t-test). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and MDSCs compared to D24-RGDOX alone (P≤0.05 in all populations, student t-test). This microenvironment remodeling correlated with complete tumor elimination. Altogether, D24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Derek A. Wainwright, Sagar Sohoni, Sumit Gupta, Ashley Ossimetha, Frederick Lang, Marta Alonso, Candelaria Gomez-Manzano, Juan Fueyo. Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1785.

Pediatric High Grade Gliomas (pHGGs), including Diffuse Midline Gliomas (DMGs), are aggressive pediatric tumors with a poor overall survival. In the last years, ONC201 has emerged as a promising agent in the field of pediatric brain tumors. Another interesting approach is virotherapy; Delta-24-RGD, which is an oncolytic virus, has demonstrated safety and effectiveness in different preclinical models and in clinical trials. Therefore, in this work we set to evaluate whether the combination of ONC201 with Delta-24-RGD could result in an increased therapeutic benefit in pHGGs and DMGs. Given that ONC201 targets mitochondrial metabolism in a preclinical setting, we assessed potential negative interactions of the combination therapy. While ONC201 treatment resulted in decreased viral protein load (E1A and fiber), there was no significant negative impact on the viral replication (measured by hexon staining). ONC201 did not disrupt the activation of mTORC1 pathway by the adenovirus. Furthermore, Delta-24-RGD did not affect the decrease in basal oxygen consumption rate induced by ONC201. Our results suggested that ONC201 and Delta-24-RGD are not antagonistic. Evaluation of the in vitro cytotoxicity in different human pHGG (CHLA-03-AA and SF188) and DMG (TP-54 and SU-DIPG-IV) cell lines showed that the combination treatment was significantly better that either agent alone. In vivo, a single local injection of Delta-24-RGD followed by a weekly ONC201 of mice bearing CHLA-03-AA cell line orthotopically significantly increased the median overall survival (PBS: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; ONC201+Delta-24-RGD: 95 days (P=0.0008)) of these mice leading to 20% long-term survivors, free of disease. Currently, we are evaluating the effect of the combination in immunosuppressed and immunocompetent models of DMGs. In summary, our data indicate ONC201 in combination with Delta-24-RGD could be a potential therapeutic choice for patients affected by pHGGs and DMGs.

Diffuse Intrinsic Pontine Gliomas (DIPG) are aggressive pediatric brain tumors that arise in the pons of children, being the leading cause of pediatric death caused by cancer. We have previously demonstrated that Delta-24-RGD administration is safe and efficacious in DIPG preclinical models, indicating that it could be a good candidate as therapeutic approach for DIPG. However, our data underscore that there is still room to improve the anti-DIPG effect obtained with Delta-24-RGD. For that purpose, we have constructed three new virus by engineering Delta-24-RGD with different T cell activators: 4-1BBL (Delta-24-ACT), OX40-L (Delta-24-RGDOX) and GITRL (Delta-24-GREAT), to further increase the immune response generated by the viral effect. In vitro, the three virus were able to infect murine and human DIPG cell lines, produce oncolytic effect in a dose-dependent manner and express the corresponding functional ligand (4-1BBL, OX40L or GITRL) in the membrane of infected cells (almost 100% of cells expressing them at 10 MOIs). As expected, viral replication was optimal in human cell lines but semipermissive in murine cells. In vivo, the intratumoral administration of armed oncolytic viruses was safe and significantly increased survival of mice bearing orthotopic DIPG murine tumors, leading to long-term survivors. In addition, we analyzed the effect of the virus in the tumor microenvironment by flow cytometry and immunohistochemistry, which indicated that there was a significant increase of immune infiltration in brains of treated mice. Moreover, the immune infiltrated showed a functional active phenotype. Although deeper characterization is needed, these data show that the incorporation of a positive immune modulator into Delta-24-RGD could improve the oncolytic effect of the virus by boosting the immune response, while maintaining a safe profile in immunocompetent models offering a feasible option treatment for DIPG.

Background A Phase 1, single center study is ongoing to evaluate the conditionally replicative oncolytic adenovirus, DNX-2401 (tasadenoturev), followed by radiotherapy (RT) in pediatric patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Methods Patients 1–18 years with newly diagnosed DIPG with no prior treatment, Lansky/Karnofsky performance score ≥ 70, and adequate organ function were enrolled. A tumor biopsy was performed followed by a single intratumoral injection of 1e10-5e10 virus particles (vp) DNX-2401. Conventional radiotherapy was initiated within 1 month of DNX-2401 administration. Results Enrolled subjects (n=12) had a median age of 9 (range 3–18) and performance scores of 90–100 (n=4; 33%) or 70–80 (n=8; 67%). As part of a dose escalation design, subjects were treated with 1e10 vp (n=4) or 5e10 vp DNX-2401 (n=8), which was then followed by standard RT in 11 of 12 subjects (92%). No dose-limiting toxicities were observed and the treatment regimen was well-tolerated. Adverse events (AEs) have been primarily mild to moderate and consistent with underlying disease. The most commonly reported AEs (≥ 5 subjects), regardless of study drug relationship, include headache, asthenia, vomiting, anemia, leukocytosis, and fever. Two SAEs have been reported including grade 3 lymphopenia and grade 3 abdominal pain. Tumor reductions have been observed and efficacy evaluations are ongoing. As of 09Dec2020, 12-month survival (OS-12) was 71% and 4 of 12 patients had survived > 20 months. Four subjects continue to be followed for survival. Correlative analysis of tumor biopsy and peripheral samples is ongoing. Conclusions DNX-2401 followed by RT can be safely administered to pediatric subjects with newly diagnosed DIPG; clinical activity and preliminary survival are encouraging.

Background MicroRNAs (miRs) are promising new therapeutics for glioblastoma. However, which miRs are most effective against glioblastomas and how these miRs should be delivered are major unanswered problems. Methods To identify potent antiglioma miRs, we selected 8 miRs based on a literature search and screened them against a panel of glioma stem cell (GSC) lines, representing all of the glioblastoma subtypes defined by The Cancer Genome Atlas. To address delivery, we tested the hypothesis that ex vivo cultured bone marrow-derived mesenchymal stem cells (MSCs) can package miRs into exosomes and that these engineered exosomes can systemically deliver antiglioma miRs to glioblastomas. Results Of the screened miRs, we identified miR-124a as the most effective antiglioma agent against GSCs. We then transduced MSCs with lentivirus vectors containing miR-124a and isolated vesicles from the medium. Electron microscopy, western blotting, and Nanosight proved that the isolated vesicles were exosomes. Quantitative PCR documented that these exosomes contained high levels of miR-124a, which was not present in control exosomes. In vitro treatment of GSCs with exosomes containing miR-124a (Exo-miR124) resulted in a significant reduction in viability and clonogenicity of GSCs compared with controls. In vivo treatment of mice harboring intracranial GSC267 with systemically delivered Exo-miR124 resulted in 50% of animals living long term. No evidence of tumor was present on histological analysis of the survivors. Mechanistic studies showed that miR-124a acts by silencing Forkhead box (FOX)A2, resulting in aberrant intracellular lipid accumulation. Conclusion MSCs can be used as natural biofactories to produce Exo-miR124, which is an effective antiglioma agent worthy of further clinical evaluation.

The idea of targeting vessels for cancer therapy, termed antiangiogenesis, was coined by Folkman in his pioneer report in 1971 (1,2). Folkman’s seminal contribution to cancer biology was the early description of what we now understand as the tumor microenvironment (2). In this manuscript, he postulated that tumor growth depends on vessel recruitment and that the growth of vascular and tumor cells are interdependent. Per instance, he stated that the maintenance of the mitotic index of the two cell populations depends on each other, and that the secretion of diffusible factors from tumor cells influences the formation of tumor capillaries (1). Furthermore, he described that the blockade of pro-angiogenic signals resulted in the regression of the new blood vessels. In addition, he hypothesized that central tumor necrosis is the consequence of poor perfusion due to increased internal pressure and decreased of the blood flow at the tumor site (1,2). This hypothesis was tested by Rakesh Jain who demonstrated that Folkman’s assumption was correct, and further suggested that increased interstitial fluid pressure might impede the delivery of large anticancer agents to tumors (2,3).

Atypical teratoid/rhabdoid tumors (AT/RTs) are rare pediatric brain tumors affecting mainly infants and young children. However, AT/RTs encompass almost 10% of death caused by pediatric brain tumors, and the 2-year overall survival for these children remains below 20%. For this reason, AT/RT ranks among the deadliest pediatric brain tumors. Therefore, it is clear we need to find out new therapeutic options for these children. Delta-24-RGD oncolytic adenovirus has already demonstrated its efficacy in Phase I/II clinical trials in adult patients affected by high grade gliomas with no evidence of severe side effects. Of interest for pediatric brain tumors, the safety of Delta-24-RGD is has been demonstrated in an ongoing Phase I clinical trial for the treatment of DIPGs (NCT03178032). For these reasons, we propose to evaluate the anti-tumor effect of Delta-24-RGD in preclinical models of AT/RT. In vitro, the virus was able to infect and replicate in three different cell culture models of AT/RT, inducing a dose-dependent cytotoxic effect that results in IC50 values below 1 PFU/cell. In vivo, intratumor administration of the virus in mice bearing orthotopic localized AT/RT (supratentorial and infratentorial) extended significantly the survival the animals, leading to up to 20% of long-term survivors. We have also generated models of disseminated disease through intraventricular injection of the tumor cells, thus mimicking the lesions found in patients. AT/RT cell lines were transduced with a luc-expressing lentivirus in order to facilitate the follow up of these tumors. In disseminated AT/RT models, light emission reveals reduction of tumor growth in Delta-24-RGD treated animals in comparison to those mock treated, thus obtaining an increased overall survival. In conclusion, these results demonstrate that Delta-24-RGD could be a feasible therapeutic choice for patients affected by AT/RT.

Based on promising results of recent clinical trials using oncolytic viruses, virotherapy is evolving as an alternative to treat patients with malignant glioma. Our group developed the oncolytic adenovirus Delta-24-RGD (DNX-2401) that is being tested, alone or in combination with anti-PD1, in clinical trials for recurrent glioblastoma (NCT00805376; NCT01956734; NCT02798406). The results suggest that, besides the expected oncolytic effect, the injection of the pathogen initiated, in a subset of patients, an anti-tumoral immunity that led to 20% of long-term survivors (3.5–5 years). To further enhance this effect, we have armed Delta-24-RGD to express the co-stimulatory ligand GITRL, and generated Delta-24-GREAT. The intracranial injection of Delta-24-GREAT prolonged the survival of GL261 glioma-bearing immunocompetent mice when compared to Delta-24-RGD treatment (P=0.002, log-rank test). Delta-24-GREAT treatment resulted in enhanced frequency of tumor-infiltrating lymphocytes: T lymphocytes (CD45+/CD3+) and cytotoxic T lymphocytes (CD45+CD3+CD8+). Functional studies performed by culturing splenocytes from Delta-24-GREAT-treated mice with glioma cells and analyzing secretion of Th1 cytokines, such as IL2 and IFN-γ, showed that lymphocytes recognized not only viral antigens but also tumoral antigens, suggesting the triggering of anti-tumoral immunity. Of interest, Delta-24-GREAT treatment resulted in an antigen-restricted anti-tumor memory effect and in the generation of central immune memory. Thus, rechallenging the survivor mice from the first experiment with a second implantation of glioma cells did not lead to tumor growth, and we detected an increased frequency of central memory CD8+ T cells (CD45+CD62L+). However, survivor mice developed lethal tumors when implanted intracranially with B16/F10 melanoma cells, strongly indicating that the developed immune response was specific for GL261 glioma antigens. This is a novel approach using an oncolytic adenovirus expressing GITRL to target cancer and to stimulate the immunity within the tumor. Our data strongly indicate that this type of strategy may be further developed to treat patients with malignant glioblastoma.

Immune checkpoint blockade has revolutionized cancer therapy; however the therapeutic benefit is limited to only a subset of patients with immunogenic (“hot”) tumors and is compromised by immune-related adverse events. We have reported the efficacy of oncolytic adenovirus Delta-24-RGDOX (DNX-2440) in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas, including intracranial melanomas. We tested the hypothesis in the subcutaneous (s.c.)/s.c. and s.c./intracranial (i.c.) melanoma models derived from luciferase-expressing B16-Red-FLuc cells in C57BL/6 mice. First, through monitoring tumor growth with bioluminescence imaging, we found that, in both s.c./s.c. and s.c./i.c. models, three injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. or i.c. tumor, thereby prolonging survival. Next, through cell profiling with flow cytometry, we observed that the virus increased the presence of T cells and effector T cell frequency in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, tumor-draining lymph nodes (TDLNs), spleens, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the frequency of exhausted T cells and regulatory T cells in the virus-injected and untreated i.c. tumors. Consequently, the virus promoted recruitment and/or in situ expansion of antigen-specific T cells in tumors expressing the target antigen. Therefore, we concluded that local intratumoral injection of Delta-24-RGDOX resulted in systemic immune activity against the disseminated tumors. Furthermore, we speculate that given the immunogenicity, cancer-selectivity and intratumoral administration of the virus, Delta-24-RGDOX is expected to have an improved safety profile when compared to immune checkpoint blockade treatment strategies. This is the first report demonstrating that local administration of oncolytic adenovirus results in eradication of intracranial tumors, suggesting Delta-24-RGDOX could be used to manage brain metastases of melanoma.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

BACKGROUND Despite our increased understanding of the genetic make-up and new therapies for pediatric high grade glioma (pHGG) and Diffuse Intrinsic Pontine Glioma (DIPG) the outcome remains grim. Delta-24-RGD (DNX-2401 in the clinic) has been tested for adult glioblastoma presenting a safe profile and promising efficacy. Recently our group has showed that the virus is safe and effective in preclinical models of pHGG and DIPG. Moreover, we showed that the virus is able to trigger an antitumor immune response. These outstanding preclinical results allowed us to propel a phase I clinical trial for newly diagnosed DIPGs (NCT03178032) where the patients received an intratumoral viral injection followed by standard radiotherapy. MATERIALS AND METHODS A phase I clinical trial with DNX-2401 for patients with newly diagnosed DIPG to assess the MTD is taking place in our hospital (N=12). Tumor biopsy is performed through the cerebellar peduncle, followed by virus injection. The virus is injected using a cannula, MEMS cannula (Alcyone Lifesciences) that prevents the reflux. Virus will be injected starting with 1010 pv. The trial is uncontrolled, unicentric with a 3 + 3 design. The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in subjects with DIPG and to collect tumor samples of this type of tumor. Secondary endpoints are overall survival at 12 months (OS12), percentage of responses and induced immune response against tumor. The follow up includes close monitoring of neurological status, blood tests and brain MRI. If this trial shows evidence of safety and efficacy will propel a multicenter clinical trial. RESULTS All the clinical data from the trial available until September 2019 will be presented during the congress, to date 8 patients have been treated within the trial. Three patients were treated with the D1=1x1010vp and because the lack of toxicity we escalated to the D2= 5x1010vp. The procedure was well tolerated and safe. Patients were home 3–4 days after the injection. All the patients displayed a reduced tumor volume after combined treatment. We performed molecular studies in 6 out of the 8 patients. We are currently assessing the immune responses to the virus. CONCLUSIONS Information acquired within this clinical study would aid to understand the response of DIPGs to viral therapies and therefore to better tailor this strategy to improve the survival and the quality of life of pediatric brain tumors.

Purpose: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas. Experimental Design: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry. Results: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen. Conclusions: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors.

Pediatric high grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPGs), are aggressive tumors with a dismal outcome. Radiotherapy (RT) is part of the standard of care of these tumors; however, radiotherapy only leads to a transient clinical improvement. Delta-24-RGD is a genetically engineered tumor-selective adenovirus that has shown safety and clinical efficacy in adults with recurrent gliomas. In this work, we evaluated the feasibility, safety and therapeutic efficacy of Delta-24-RGD in combination with radiotherapy in pHGGs and DIPGs models. Our results showed that the combination of Delta-24-RGD with radiotherapy was feasible and resulted in a synergistic anti-glioma effect in vitro and in vivo in pHGG and DIPG models. Interestingly, Delta-24-RGD treatment led to the downregulation of relevant DNA damage repair proteins, further sensitizing tumors cells to the effect of radiotherapy. Additionally, Delta-24-RGD/radiotherapy treatment significantly increased the trafficking of immune cells (CD3, CD4+ and CD8+) to the tumor niche compared with single treatments. In summary, administration of the Delta-24-RGD/radiotherapy combination to pHGG and DIPG models is safe and significantly increases the overall survival of mice bearing these tumors. Our data offer a rationale for the combination Delta-24-RGD/radiotherapy as a therapeutic option for children with these tumors. Significance Delta-24-RGD/radiotherapy administration is safe and significantly increases the survival of treated mice. These positive data underscore the urge to translate this approach to the clinical treatment of children with pHGG and DIPGs. Electronic supplementary material The online version of this article (10.1186/s40478-019-0714-6) contains supplementary material, which is available to authorized users.

Cancer virotherapy is a paradigm-shifting treatment modality based on virus-mediated oncolysis and subsequent antitumor immune responses. Clinical trials of currently available virotherapies showed that robust antitumor immunity characterizes the remarkable and long-term responses observed in a subset of patients. These data suggest that future therapies should incorporate strategies to maximize the immunotherapeutic potential of oncolytic viruses. In this review, we highlight the recent evidence that the antiviral immunity of the patients may limit the immunotherapeutic potential of oncolytic viruses and summarize the most relevant approaches to strategically redirect the immune response away from the viruses and toward tumors to heighten the clinical impact of viro-immunotherapy platforms.

Current therapies for atypical teratoid/rhabdoid tumors (AT/RTs) are suboptimal, resulting in a 2-year OS below 20% and the development of severe side effects. Therefore, we need to explore alternative therapeutic approaches for this disease. Since the virus Delta24-RGD has already demonstrated its efficacy and safety as a therapeutic agent for brain tumors, including pediatric patients, here we propose to evaluate the anti-tumor effect of Delta24-RGD in AT/RT. In vitro, Delta24-RGD infects and replicates in AT/RT cultures followed by oncolysis, obtaining IC50 values below 1 PFU/cell. In vivo, a single local injection of Delta-24-RGD in three infratentorial AT/RT models (BT-12, CHLA-06 and CHLA-266) extended significantly the median OS (50 to 78 days BT-12; 21 to 31 days CHLA-06; 64 to 110 days CHLA-266). Delta-24-RGD also increased the survival of mice bearing supratentorial CHLA-266 tumors (from 93 to 132 days). Next, we evaluated the efficacy of Delta24-RGD in a model mimicking metastatic disease through intraventricular injection of BT-12-luciferase cells. Administration of Delta24-RGD inhibited tumor growth and development of metastases, leading to an increased OS and nearly 70% of long-term survivors. The interaction between Delta24-RGD and the immune system was evaluated in humanized mice models bearing CHLA-06. In this model, Delta24-RGD treatment extended OS (from 23 to 34 days) and we characterized the anti-tumor immune landscape in control and Delta24-RGD treated mice by transcriptional and functional analyses. These results underscore the potential of Delta24-RGD as a promising therapeutic choice for patients affected by AT/RT.

The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in newly diagnosed DIPG patients (NCT03178032) followed by radiotherapy. Secondary endpoints are overall survival at 12 months, percentage of responses and induced immune response against tumor. Tumor biopsy was performed through the cerebellar peduncle, followed by intratumoral injection of DNX-2401 (N=12). Three patients were treated with 1x1010vp and given the lack of toxicity we escalated to 5x1010vp. The procedure was well tolerated and reduced tumor volume was demonstrated in all patients after combined treatment (virus + radiotherapy). We performed molecular studies (RNAseq and the Oncomine Childhood Research Panel from Thermo Fisher). The immune cell composition of the biopsies pre-virus injection was assessed using multiplexed quantitative immunofluorescence. T cells were hardly detectable in these tumors while macrophages were abundant. Using a multiplexed TCR-sequencing mRNA-based assay to analyze 18 available paired pre- and post-treatment samples from the trial, we detected increased clonal T cell diversity following treatment with the virus. We also measured pre and post treatment neutralizing antibodies and their relationship with survival. Finally, we performed functional studies using 2 cell lines isolated from patients included in this trial to assess the response to the virus (infectivity, viability, T-cell recognition). In summary, the virus has shown safety and efficacy in some patients. The information obtained in this clinical study would aid understanding the response of DIPG patients to viral therapies and, therefore, to better tailor this strategy to improve the survival of these patients.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumour, being the leading cause of paediatric death caused by cancer. Despite all the advances made regarding effective therapies, the survival is dismal. Our lab has engineered the oncolytic virus Delta-24-ACT armed with the costimulatory ligand 41BBL in order to increase the antitumoral effect of the adenovirus. 41BB is a costimulatory receptor which promotes the expansion of activated T cells and the generation and maintenance of CD8 T memory cells. Therefore, we propose the use of Delta-24-ACT as a therapeutic approach for DIPG tumours. We observed that Delta-24-ACT is able to infect and replicate in NP53 and PDGFB-driven, two DIPG murine cell lines. Furthermore, 41BBL is expressed in the membranes of the infected cells and results with immunogenic cell death as shown by the different DAMPs. Injection of Delta-24-ACT in DIPG model was safe, showed no sign of toxicity and led to a significantly increase in the median overall survival, generating anti-glioma memory in long-term survivors. Mechanistic experiments, showed an increase of T cell infiltration (mainly CD8), decrease of proliferating cells and a reduction of the number of vessels in FFPE brain samples in the treated mice. We are currently performing nanostring analyses to assess the changes in the transcriptional immune phenotype of treated versus control mice. In summary, our data suggest that Delta-24-ACT is safe and induces a potent antitumor immune response in DIPG models mainly based in the activation of CD8 lymphocytes recruited by the viral activity.

Glioblastoma is the most frequent malignant brain tumor. Even with aggressive treatment, prognosis for patients is poor. One characteristic of glioblastoma cells is its intrinsic resistance to apoptosis. Therefore, drugs that induce alternative cell deaths could be interesting to evaluate as alternative therapeutic candidates for glioblastoma. Salinomycin (SLM) was identified through a chemical screening as a promising anticancer drug, but its mechanism of cell death remains unclear. In the present work we set out to elucidate how SLM causes cell death in glioblastoma cell lines (both established cell lines and brain tumor stem cell lines), aiming to find a potential antitumor candidate. In addition, we sought to determine the mechanism of action of SLM so that this mechanism can be can be exploited in the fight against cancer. Our data showed that SLM induces a potent endoplasmic reticulum (ER) stress followed by the trigger of the unfolded protein response (UPR) and an aberrant autophagic flux that culminated in necrosis due to mitochondria and lysosomal alterations. Of importance, the aberrant autophagic flux was orchestrated by the production of Reactive Oxygen Species (ROS). Alleviation of ROS production restored the autophagic flux. Altogether our data suggest that in our system the oxidative stress blocks the autophagic flux through lipid oxidation. Importantly, oxidative stress could be instructing the type of cell death in SLM-treated cells, suggesting that cell death modality is a dynamic concept which depends on the cellular stresses and the cellular mechanism activated.

Background Endoplasmic reticulum (ER) stress results from protein misfolding imbalance and has been postulated as a therapeutic strategy. ER stress activates the unfolded protein response which leads to a complex cellular response, including the upregulation of aberrant protein degradation in the ER, with the goal of resolving that stress. O(6)-methylguanine DNA methyltransferase (MGMT), N-methylpurine DNA glycosylase (MPG), and Rad51 are DNA damage repair proteins that mediate resistance to temozolomide in glioblastoma. In this work we sought to evaluate whether ER stress-inducing drugs were able to downmodulate DNA damage repair proteins and become candidates to combine with temozolomide. Methods MTT assays were performed to evaluate the cytotoxicity of the treatments. The expression of proteins was evaluated using western blot and immunofluorescence. In vivo studies were performed using 2 orthotopic glioblastoma models in nude mice to evaluate the efficacy of the treatments. All statistical tests were 2-sided. Results Treatment of glioblastoma cells with ER stress-inducing drugs leads to downregulation of MGMT, MPG, and Rad51. Inhibition of ER stress through pharmacological treatment resulted in rescue of MGMT, MPG, and Rad51 protein levels. Moreover, treatment of glioblastoma cells with salinomycin, an ER stress-inducing drug, and temozolomide resulted in enhanced DNA damage and a synergistic antitumor effect in vitro. Of importance, treatment with salinomycin/temozolomide resulted in a significant antiglioma effect in 2 aggressive orthotopic intracranial brain tumor models. Conclusions These findings provide a strong rationale for combining temozolomide with ER stress-inducing drugs as an alternative therapeutic strategy for glioblastoma.