Oncolytic Reovirus Research Articles

Application of Bioinformatics Tools for the Prediction of Helper MicroRNAs for Improvement of Oncolytic Virus Efficacy

Purpose. Oncolytic Reoviruses, as a self-limiting virus, can be used in cancer treatment, because they have the ability to replicate in tumor cells selectively and destroy them. Studies show that some immune response proteins may interfere with the virus life cycle. So, the main aim of this bioinformatic study is to check which microRNA is able to target some reovirus inhibitory proteins. Experimental Design. By use of online bioinformatics software, the microRNAs that could target inhibitory genes were selected. Then, other features like content ++ score and cell type were checked and finally the eligible microRNAs were determined. Results. After choosing 15 inhibitory proteins, analysis was performed and finally 37 microRNAs which could target inhibitory proteins in colorectal cell lines were selected. In the end, by investigation of web-based tools, just two microRNAs were finalized. Conclusions and Clinical Relevance. This bioinformatic study shows that microRNA-140 and microRNA-92a have the potential to target some inhibitory proteins which interfere with oncolytic Reovirus replication and it may help in the optimal use of this virus as a cancer treatment. Because selective reproduction of Reovirus in tumor cells, as a nonchemical therapy, can be a good way to overcome this disease with broad advantages.

Oncolytic viruses as emerging therapy against cancers including Oncovirus-induced cancers

There are several human viruses with known potential for causing cancers including, Hepatitis B virus, Hepatitis C virus, Epstein-Barr virus, Kaposi's sarcoma herpesvirus, Human T-cell lymphotropic virus, Human papillomavirus, and Merkel cell polyomavirus. Cancer is the second leading cause of death that affects humans worldwide, especially in developing countries. Surgery, chemotherapy, and radiotherapy can cure about 60% of humans with cancer but recurrent and metastatic diseases remain a major reason for death. In recent years, understanding the molecular characteristics of cancer cells has led to the improvement of therapeutic strategies using novel emerging therapies. Oncolytic viruses with the potential of lysing cancer cells defined the field of oncolytic virology, hence becoming a biotechnology tool rather than just a cause of disease. This study mainly focused on targeting cell proliferation and death pathways in human tumor-inducing viruses by developing innovative therapies for cancer patients based on the natural oncolytic properties of reovirus. To kill tumor cells efficiently and reduce the chance of recurrence both the direct ability of reovirus infection to lyse the tumor cells and the stimulation of a potent host immune response are applied. Hence, bioengineered stem cells can be used as smart carriers to improve the efficacy of oncolytic reovirus and safety profiles.

Potentiating effect of reovirus on immune checkpoint inhibition in microsatellite stable colorectal cancer.

The majority of colorectal cancers (CRCs) are microsatellite stable (MSS) and resistant to immunotherapy. The current study explores the possibility of using oncolytic reovirus to sensitize MSS CRC to immune checkpoint inhibition. While reovirus reduced metabolic activity among KRAS Mut cells, microarray/computational analysis revealed microsatellite status-oriented activation of immune-response pathways. Reovirus plus anti-PD-1 treatment increased cell death among MSS cells ex vivo. Reduced tumorigenicity and proliferative index, and increased apoptosis were evident among CT26 [MSS, KRAS Mut], but not in MC38 [microsatellite unstable/MSI, KRAS Wt] syngeneic mouse models under combinatorial treatment. PD-L1-PD-1 signaling axis were differentially altered among CT26/MC38 models. Combinatorial treatment activated the innate immune system, pattern recognition receptors, and antigen presentation markers. Furthermore, we observed the reduction of immunosuppressive macrophages and expansion of effector T cell subsets, as well as reduction in T cell exhaustion. The current investigation sheds light on the immunological mechanisms of the reovirus-anti-PD-1 combination to reduce the growth of MSS CRC.

Oncolytic avian reovirus σA‐modulated fatty acid metabolism through the PSMB6/Akt/SREBP1/acetyl-CoA carboxylase pathway to increase energy production for virus replication

We have demonstrated previously that the σA protein of avian reovirus (ARV) functions as an activator of cellular energy, which upregulates glycolysis and the TCA cycle for virus replication. To date, there is no report with respect to σA-modulated regulation of cellular fatty acid metabolism. This study reveals that the σA protein of ARV inhibits fatty acids synthesis and enhance fatty acid oxidation by upregulating PSMB6, which suppresses Akt, sterol regulatory element-binding protein 1 (SREBP1), acetyl-coA carboxylase α (ACC1), and acetyl-coA carboxylase β (ACC2). SREBP1 is a transcription factor involved in fatty acid and cholesterol biosynthesis. Overexpression of SREBP1 reversed σA-modulated suppression of ACC1 and ACC2. In this work, a fluorescence resonance energy transfer‐based genetically encoded indicator, Ateams, was used to study σA-modulated inhibition of fatty acids synthesis which enhances cellular ATP levels in Vero cells and human cancer cell lines (A549 and HeLa). By using Ateams, we demonstrated that σA-modulated inhibition of Akt, SREBP1, ACC1, and ACC2 leads to increased levels of ATP in mammalian and human cancer cells. Furthermore, knockdown of PSMB6 or overexpression of SREBP1 reversed σA-modulated increased levels of ATP in cells, indicating that PSMB6 and SREBP1 play important roles in ARV σA-modulated cellular fatty acid metabolism. Furthermore, we found that σA R155/273A mutant protein loses its ability to enter the nucleolus, which impairs its ability to regulate fatty acid metabolism and does not increase ATP formation, suggesting that nucleolus entry of σA is critical for regulating cellular fatty acid metabolism to generate more energy for virus replication. Collectively, this study provides novel insights into σA-modulated inhibition of fatty acid synthesis and enhancement of fatty acid oxidation to produce more energy for virus replication through the PSMB6/Akt/SREBP1/ACC pathway.

Oncolytic virotherapy in head and neck cancer

Oncolytic viruses (OV) infect and kill cancer cells and elicit an antitumoral immune response. With their potential to break through tumor immunoresistance, OV might be a future combination treatment option in patients with advanced head and neck cancer (HNC). Modes of action, biological modifications, handling and side effects of OV for treatment of HNC are reviewed. Results of preclinical and clinical trials are reported. Publications and clinical trials dealing with OV and HNC were searched in PubMed and international platforms for clinical study records. Studies on preclinical and clinical trials regarding oncolytic Herpes Simplex Virus (HSV), Adenovirus, Vacciniavirus and Reovirus were selected. Enhanced infection and killing of tumor cells through capsid and genome modifications of OV were reported in recent preclinical studies. Most of the clinical studies were phase-I/II trials. In phase III studies, tumor regression and prolonged survival were observed after treatment with oncolytic HSV, Adenoviruses and Reoviruses. In most trials, OV were combined with chemoradiotherapy or immunotherapy. In the published studies, OV treatment of HNC patients was safe, often well tolerated and showed promising results with regard to response and survival, especially in combination with chemoradiotherapy or checkpoint inhibitors.

Oncolytic Avian Reovirus p17-Modulated Inhibition of mTORC1 by Enhancement of Endogenous mTORC1 Inhibitors Binding to mTORC1 To Disrupt Its Assembly and Accumulation on Lysosomes.

The mechanism by which avian reovirus (ARV)-modulated suppression of mTORC1 triggers autophagy remains largely unknown. In this work, we determined that p17 functions as a negative regulator of mTORC1. This study suggest novel mechanisms whereby p17-modulated inhibition of mTORC1 occurs via upregulation of p53, inactivation of Akt, and enhancement of binding of the endogenous mTORC1 inhibitors (PRAS40, FKBP38, and FKPP12) to mTORC1 to disrupt its assembly and accumulation on lysosomes. p17-modulated inhibition of Akt leads to activation of the downstream targets PRAS40 and TSC2, which results in mTORC1 inhibition, thereby triggering autophagy and translation shutoff, which is favorable for virus replication. p17 impairs the interaction of mTORC1 with its activator Rheb, which promotes FKBP38 interaction with mTORC1. It is worth noting that p17 activates ULK1 and Beclin1 and increases the formation of the Beclin 1/class III PI3K complex. These effects could be reversed in the presence of insulin or depletion of p53. Furthermore, we found that p17 induces autophagy in cancer cell lines by upregulating the p53/PTEN pathway, which inactivates Akt and mTORC1. This study highlights p17-modulated inhibition of Akt and mTORC1, which triggers autophagy and translation shutoff by positively modulating the tumor suppressors p53 and TSC2 and endogenous mTORC1 inhibitors. IMPORTANCE The mechanisms by which p17-modulated inhibition of mTORC1 induces autophagy and translation shutoff is elucidated. In this work, we determined that p17 serves as a negative regulator of mTORC1. This study provides several lines of conclusive evidence demonstrating that p17-modulated inhibition of mTORC1 occurs via upregulation of the p53/PTEN pathway, downregulation of the Akt/Rheb/mTORC1 pathway, enhancement of binding of the endogenous mTORC1 inhibitors to mTORC1 to disrupt its assembly, and suppression of mTORC1 accumulation on lysosomes. This work provides valuable information for better insights into p17-modulated inhibition of mTORC1, which induces autophagy and translation shutoff to benefit virus replication.

Gastrointestinal cancer-associated fibroblasts expressing Junctional Adhesion Molecule-A are amenable to infection by oncolytic reovirus

Gastrointestinal (GI) cancers are characterized by extensive tumor stroma that both promotes tumor progression and acts as a physical barrier for adjacent tumor cells, limiting the effect of current treatment modalities. Oncolytic virotherapy is currently investigated in clinical trials as a novel therapeutic agent for different malignancies of the GI tract, but it is largely unknown whether these viruses can also target the tumor stroma. Here, we investigated the tropism of two commonly studied OVs, adenovirus and reovirus, towards primary GI fibroblasts from human oesophageal, gastric, duodenal and pancreatic carcinomas (N = 36). GI fibroblasts were susceptible to type 3 Dearing (T3D) strain R124 and bioselected mutant reovirus (jin-3) infection but not oncolytic adenovirus (Ad5-Δ24). Efficient infection and apoptosis of human and mouse GI cancer-derived fibroblasts by these reoviruses was partially dependent on the expression of the reovirus entry receptor, Junctional Adhesion Molecule-A (JAM-A). Moreover, human GI cancer organoid-fibroblast co-cultures showed higher overall infectivity when containing JAM-A expressing fibroblasts as compared to JAM-A negative fibroblasts, indicating a potential role of JAM-A expressing fibroblasts for viral dissemination. We further show that JAM-A is not only necessary for efficient reovirus infection of fibroblasts but also partially mediates reovirus-induced apoptosis, dependent on signaling through the C-terminal PDZ-domain of JAM-A. Altogether, our data show the presence of JAM-A expressing fibroblasts in both human and murine GI cancers that are amenable to infection and induction of apoptosis by reovirus, extending the potential anti-cancer actions of reovirus with stromal targeting.

Abstract 6354: Using imaging mass cytometry to visualize the multiple myeloma tumor microenvironment post immune priming

Abstract Background: Multiple myeloma (MM) is an incurable cancer characterized by clonal plasma cell proliferation in the bone marrow, accounting for approximately 10% of all hematologic malignancies. Recently, patients with relapsed or refractory disease have been treated with a combination of the oncolytic reovirus Pelareorep, bortezomib, and dexamethasone, which was well-tolerated and led to prolonged progression free survival of over 3 years in a subset of patients. To understand the complex tumor immune microenvironment (TiME) and immune responses in patients before and after this treatment, we used imaging mass cytometry (IMC) to perform single cell, highly multiplexed, analysis of these patients’ bone marrow samples. Methods: We comprehensively characterized the changes in the MM TIME in pre and post bone marrow biopsy specimens taken from patients treated on a Phase 1b study with a combination of Pelareorep, bortezomib, and dexamethasone. For analysis with IMC, a marker panel of 35 antibodies was assembled to interrogate the various immune subsets of the bone marrow biopsies; each of these antibodies were conjugated to a unique metal isotope. After validation, the antibody cocktail was used to stain the biopsies. Pixel-based classification was performed in Ilastik to generate cell probability masks and processed in Cellprofiler. PhenoGraph was run in HistoCAT to identify the unique phenotypes. Rstudio was used for t-stochastic neighborhood embedding (tSNE) plot generation, and nearest neighbor analyses. ImaCytE was used for image visualization and spatial analysis. Results: Initial visualization of the raw, unsegmented data showed increased infiltration of natural killer cells and T cells in the post-treatment samples when compared against the pre-treatment samples. These changes correlated with immunohistochemical findings, clinical response to treatment, and changes in T cell clonality. After segmentation, the marker expression heatmaps for each of the clusters identified by PhenoGraph and the further subphenotyping in Rstudio showed complex ecosystems of cell-cell interactions. Nearest neighbor spatial analysis of the post-treatment samples revealed that NK cells (NKG2D+ and NKG2A+ subsets), monocytes (CD14+), macrophages (CD68+), cytotoxic T cells (CD3+, CD8+), and T helper cells (CD3+, CD4+) were significantly closer to the Pelareorep-primed MM than the non-primed MM. Further analysis in ImaCytE highlighted specific instances of these immune neighborhoods. Conclusions: IMC allows us to analyze the potent immune response and cellular interactions in the tumor microenvironment in multiple myeloma treated with Pelareorep and Bortezomib. Characterization of these complex interactions allows for a deeper understanding of the key mechanisms of action of these treatments and planning of future combination studies. Citation Format: Julian Olea, Kaijin Wu, Anthony Colombo, Claudia Villa Celi, Thomas Heineman, Matt Coffey, Steffan T. Nawrocki, Akil Merchant, Kevin R. Kelly. Using imaging mass cytometry to visualize the multiple myeloma tumor microenvironment post immune priming [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 6354.

IMMU-12. Exploring and modulating the tumour immune microenvironment to facilitate the selection of immunotherapies for paediatric-type diffuse high-grade glioma

Abstract Immune cells have the potential to selectively eradicate high-risk brain tumours such as paediatric-type diffuse high-grade glioma (PDHGG). We aim to characterize the tumour immune microenvironment (TIME) of intra-cranial syngeneic mouse models of diffuse hemispheric glioma, H3G34 (DHG-H3G34) and diffuse midline glioma, H3K27 (DMG-H3K27). We also demonstrate how an oncolytic reovirus (Reolysin) can “heat-up” the TIME of our syngeneic models. Orthotopic immunocompetent mouse models of DHG-H3G34 (C57BL/6, NRASG12V + shp53 + shATRX +/- H3.3G34R) and DMG-H3K27 (Nestin-Tv-a/p53fl/fl, RCAS-ACVR1R206H + RCAS-H3.1K27M) were profiled using single-cell RNA-sequencing (scRNA-seq) (10x genomics), a 22-colour custom flow cytometry immune panel and spatial transcriptomics. Differential marker expression was validated with immunohistochemistry and immunofluorescence in tissue sections. Syngeneic mouse tumours treated systemically with Reolysin were also profiled to evaluate the effects of the oncolytic virus on the TIME. Cell type predictions in scRNA-seq using singleR, ssGSEA and expression of individual marker genes suggested that the predominant immune cell types within hemispheric tumours were monocytes (11-21%) and macrophages (10-19%) with much smaller proportions of CD4+ and CD8+ T-cells (4-10%). By contrast, much smaller proportions of monocytes (2%) and macrophages (3%) were observed in the H3.1K27M pontine model. Flow cytometry, immunohistochemistry and immunofluorescence validated scRNA-seq immune profiles and characterised signalling of the PD-1/PD-L1 checkpoint pathway. Spatial transcriptomics allowed immune cell populations to be positioned within tumour sections and showed significant co-localization of CD4+ and CD8+ lymphocytes at tumour margins. Treatment of syngeneic mouse tumours with Reolysin resulted in reduced tumour volumes and altered the TIME, in particular increasing cytotoxic T-cell tumour infiltration. Our results highlight immunological heterogeneity within molecular subgroups of PDHGG and demonstrate ability of a systemically delivered oncolytic virus, Reolysin, to “heat-up” the TIME, contributing to a more immune actionable profile. Future work will help to identify optimal combinations for the next generation of immunotherapies in PDHGG.

P-49 GOBLET: A phase 1/2 multiple indication signal finding and biomarker study in advanced gastrointestinal cancers treated with pelareorep and atezolizumab – safety and preliminary response results

In GI cancers, checkpoint inhibitors are only effective in patients with high microsatellite instability (MSI-H) or deficient mismatch repair (dMMR) tumors. Oncolytic viruses may improve the susceptibility of microsatellite stable (MSS) tumors to immunotherapy by modifying the tumor microenvironment (TME). Pelareorep (pela) is an intravenously delivered, non-genetically modified, oncolytic reovirus that mediates cancer cell killing by activating innate and adaptive immune responses directed against the tumor as well as direct tumor oncolysis. Accordingly, treatment with pelareorep results in increased T cell infiltration and PD-L1 expression in tumors which primes the TME for responsiveness to checkpoint inhibitors. Pela has demonstrated activity in many cancers including colorectal and pancreatic cancer. The GOBLET study is designed to assess the efficacy of pela plus atezolizumab (atezo) with or without chemotherapy in multiple GI cancers. GOBLET is a phase 1/2, open-label, non-randomized study in patients with advanced or metastatic GI cancers and utilizes a Simon two-stage design. In the first stage of the study, four treatment groups are being enrolled: Cohort 1 – First-line metastatic/advanced pancreatic cancer treated with pela plus atezo and chemotherapy (gemcitabine and nab-paclitaxel) (N=12); Cohort 2 – First-line MSI-H/dMMR metastatic CRC treated with pela plus atezo (N=19); Cohort 3 – Third-line metastatic CRC treated with pela plus atezo and chemotherapy (trifluridine/tipiracil) (N=14); and Cohort 4 – Second-line or later advanced squamous cell carcinoma of the anal canal treated with pela plus atezo (N=10). In Cohorts 1 and 3 (chemotherapy-containing cohorts) the first 3-6 patients enrolled comprise a safety run-in. The primary objectives of GOBLET are safety and efficacy measured by the objective response rate (ORR) at week 16. Based on pre-specified response thresholds, any cohort showing a positive ORR signal in Stage 1 may advance to the 2nd Stage and enroll additional patients. The three safety run-in patients in Cohorts 1 and 3 have been enrolled and their safety data reviewed by the independent Data Safety Monitoring Board (DSMB). The DSMB identified no safety signal and recommended that enrollment into these cohort continue without modification. Enrollment into Cohorts 2 and 4 is ongoing as these cohorts do not include safety run-ins. Tumor response results to date indicate that two of the three Cohort 1 patients had a partial response at week 8, the third Cohort 1 patient had a partial response at week 16. No safety signal was observed in either the Cohort 1 (first-line pancreatic cancer) or Cohort 3 (third-line CRC) patients. This is consistent with the favorable safety profile observed in prior studies of pela in multiple cancer indications, and it supports the ability to safely treat patients with advanced GI cancers using pela in combination with checkpoint inhibitors and chemotherapy. Preliminary tumor responses to therapy in first-line pancreatic cancer patients are encouraging.

MP06-04 EXPLORING A NOVEL SYNERGISTIC INTRAVESICAL ONCOLYTIC VIROTHERAPY FOR TREATMENT OF NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-04 EXPLORING A NOVEL SYNERGISTIC INTRAVESICAL ONCOLYTIC VIROTHERAPY FOR TREATMENT OF NON-MUSCLE INVASIVE BLADDER CANCER Woodson Smelser, Jian Wang, Sam Chang, and Austin Kirschner Woodson SmelserWoodson Smelser More articles by this author , Jian WangJian Wang More articles by this author , Sam ChangSam Chang More articles by this author , and Austin KirschnerAustin Kirschner More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: For BCG refractory NMIBC, systemic anti-PD-1 immune checkpoint inhibition is FDA approved as second line therapy. Prior published work by our group demonstrated comparable efficacy and overall survival with intravesical anti-PD-1 treatments in NMIBC in mice. Additionally, oncolytic viral therapy with reovirus has shown pre-clinical efficacy in the bladder & other cancers. We hypothesized that intravesical instilment with both anti-PD-1 inhibitor & oncolytic reovirus would demonstrate a greater synergistic effect than either treatment as monotherapy. METHODS: We investigated an orthotopic mouse model of urothelial bladder cancer using MBT2 cells implanted into the bladders of (n=40) syngeneic, wild-type C3H mice. Groups of 10 mice were treated 14 days after engraftment at the point of palpable tumor for comparison between no treatment, intravesical anti-PD-1, intravesical oncolytic reovirus, & intravesical oncolytic reovirus + anti-PD1. The primary outcome was overall survival; secondary outcomes include long-term immunity & immune milieu changes indicated by mass cytometric evaluation. RESULTS: 40 total mice were engrafted with tumor & then treated. At time of submission, all 10 mice receiving no treatment had died, with median survival of 41 days. Median overall survival for the mice receiving reovirus, anti-PD-1 or anti-PD-1 + reovirus had not been reached. Mice receiving reovirus experienced longer survival than control (HR 14.4; 95% CI 3.9-32.6, p<0.0001), as did mice receiving anti-PD-1 alone (HR 28.4; 95% CI 7.0-115.9, p<0.0001). When compared to control alone, combination therapy with anti-PD-1 & reovirus also demonstrated superior survival (HR 28.4; 95% CI 7.0-115.9, p<0.0001). Comparison between anti-PD-1 and reovirus demonstrated no significant differences in survival (p=0.15). See Figure 1 for Kaplan-Meier analysis comparing survival. CONCLUSIONS: Treatment of mice engrafted with MBT2 orthotopic bladder tumors with intravesical therapy with reovirus, anti-PD-1 antibody, or combination anti-PD-1 antibody & reovirus confer superior survival to intravesical controls. Immune profile analysis examining tumor microenvironment differences in immune response are ongoing with mass cytometry and will further bolster our mechanistic understanding of each therapy. Source of Funding: T32 Grant (WS) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e78 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Woodson Smelser More articles by this author Jian Wang More articles by this author Sam Chang More articles by this author Austin Kirschner More articles by this author Expand All Advertisement PDF DownloadLoading ...

6P The oncolytic virus pelareorep primes the tumor microenvironment for checkpoint blockade therapy in early breast cancer patients: Results from AWARE-1 study

Pelareorep (pela) is an intravenously delivered non-engineered oncolytic reovirus demonstrating anti-tumor activity through innate and adaptive immune responses. Previous data from the AWARE-1 study demonstrated that addition of atezolizumab (atezo) to pela increased the CelTIL score (study’s primary endpoint) by more than 30% in 60% of HR+/HER2- early breast cancer (BC) patients. CelTIL is a composite measure of tumor cellularity and tumor-infiltrating lymphocytes (TILs) and has been associated with a better prognosis in BC.

Oncolytic virus-mediated expansion of dual-specific CAR T cells improves efficacy against solid tumors in mice.

Oncolytic viruses (OVs) encoding a variety of transgenes have been evaluated as therapeutic tools to increase the efficacy of chimeric antigen receptor (CAR)-modified T cells in the solid tumor microenvironment (TME). Here, using systemically delivered OVs and CAR T cells in immunocompetent mouse models, we have defined a mechanism by which OVs can potentiate CAR T cell efficacy against solid tumor models of melanoma and glioma. We show that stimulation of the native T cell receptor (TCR) with viral or virally encoded epitopes gives rise to enhanced proliferation, CAR-directed antitumor function, and distinct memory phenotypes. In vivo expansion of dual-specific (DS) CAR T cells was leveraged by in vitro preloading with oncolytic vesicular stomatitis virus (VSV) or reovirus, allowing for a further in vivo expansion and reactivation of T cells by homologous boosting. This treatment led to prolonged survival of mice with subcutaneous melanoma and intracranial glioma tumors. Human CD19 CAR T cells could also be expanded in vitro with TCR reactivity against viral or virally encoded antigens and was associated with greater CAR-directed cytokine production. Our data highlight the utility of combining OV and CAR T cell therapy and show that stimulation of the native TCR can be exploited to enhance CAR T cell activity and efficacy in mice.

Comparison of the Effect of Adipose Mesenchymal Stem Cells-Derived Secretome with and without Reovirus in CT26 Cells.

Colorectal cancer is the fourth leading cause of cancer-related deaths that has significantly increased over the past three decades. New therapeutic approaches, such as oncolytic viruses, have become very imperative recently to destroy cancer cells. The use of mesenchymal stem cells (MSCs) secretome that is produced in response to variant conditions involves different paracrine molecules secretion that has therapeutic potential in several chronic diseases. Mesenchymal stem cells and their derivatives are employed as regenerative medicine; nevertheless, there is ambiguity in the function of these cells in the control of malignancy. This study aimed to examine the apoptotic effect of secretomes derived from MSCs affected by encompassing oncolytic reoviruses. Mesenchymal stem cells were cultured after separation from abdominal adipose tissue of BALB/c mice. After three passages, the cells were infected by reovirus at the multiplicity of infection of 1 plaque-forming unit per cell. Uninfected and infected secretomes with reovirus were collected separately. The colorectal cancer CT26 cells were confronted with uninfected secretome, infected secretions, reovirus as a positive control, and Dulbecco's Modified Eagle Medium/High Glucose as negative control separately. Finally, apoptosis and necrosis were evaluated by flow cytometry. The infected secretome with reovirus was capable to induce apoptosis more than the uninfected secretome in CT26. However, the supernatant of reovirus infected cells was more capable to induce cell death, in comparison to the infected secretome. Infected MSCs with oncolytic reovirus produced a type of condition media that enhanced apoptosis induction and could have a therapeutic effect on cancer cells. Nonetheless, tumoral cells confronted with the oncolytic reovirus showed more capability in inducing apoptosis in CT26 cells. As a result, the use of oncolytic virus and infected secretome are more effective than uninfected secretome in inducing apoptosis.

NAD+ depletion enhances reovirus-induced oncolysis in multiple myeloma

Cancer cell energy metabolism plays an important role in dictating the efficacy of oncolysis by oncolytic viruses. To understand the role of multiple myeloma metabolism in reovirus oncolysis, we performed semi-targeted mass spectrometry-based metabolomics on 12 multiple myeloma cell lines and revealed a negative correlation between NAD+ levels and susceptibility to oncolysis. Likewise, a negative correlation was observed between the activity of the rate-limiting NAD+ synthesis enzyme NAMPT and oncolysis. Indeed, depletion of NAD+ levels by pharmacological inhibition of NAMPT using FK866 sensitized several myeloma cell lines to reovirus-induced killing. The myelomas that were most sensitive to this combination therapy expressed a functional p53 and had a metabolic and transcriptomic profile favoring mitochondrial metabolism over glycolysis, with the highest synergistic effect in KMS12 cells. Mechanistically, U-13C-labeled glucose flux, extracellular flux analysis, multiplex proteomics, and cell death assays revealed that the reovirus + FK866 combination caused mitochondrial dysfunction and energy depletion, leading to enhanced autophagic cell death in KMS12 cells. Finally, the combination of reovirus and NAD+ depletion achieved greater antitumor effects in KMS12 tumors in vivo and patient-derived CD138+ multiple myeloma cells. These findings identify NAD+ depletion as a potential combinatorial strategy to enhance the efficacy of oncolytic virus-based therapies in multiple myeloma.

P17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories.

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and μNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.

Phase I trial of sargramostim/pelareorep therapy in pediatric patients with recurrent or refractory high-grade brain tumors.

BackgroundBrain tumors are the leading cause of cancer death for pediatric patients. Pelareorep, an immunomodulatory oncolytic reovirus, has intravenous efficacy in preclinical glioma models when preconditioned with GM-CSF (sargramostim). We report a phase I trial with the primary goal of evaluating the safety of sargramostim/pelareorep in pediatric patients with recurrent or refractory high-grade brain tumors and a secondary goal of characterizing immunologic responses.MethodsThe trial was open to pediatric patients with recurrent or refractory high-grade brain tumors (3 + 3 cohort design). Each cycle included 3 days of subcutaneous sargramostim followed by 2 days of intravenous pelareorep. Laboratory studies and imaging were acquired upon recruitment and periodically thereafter.ResultsSix patients participated, including three glioblastoma, two diffuse intrinsic pontine glioma, and one medulloblastoma. Two pelareorep dose levels of 3 × 108 and 5 × 108 tissue culture infectious dose 50 (TCID50) were assessed. One patient experienced a dose limiting toxicity of persistent hyponatremia. Common low-grade (1 or 2) adverse events included transient fatigue, hypocalcemia, fever, flu-like symptoms, thrombocytopenia, and leukopenia. High-grade (3 or 4) adverse events included neutropenia, lymphopenia, leukopenia, hypophosphatemia, depressed level of consciousness, and confusion. All patients progressed on therapy after a median of 32.5 days and died a median of 108 days after recruitment. Imaging at progression did not show evidence of pseudoprogression or inflammation. Correlative assays revealed transient but consistent changes in immune cells across patients.ConclusionsSargramostim/pelareorep was administered to pediatric patients with recurrent or refractory high-grade brain tumors. Hyponatremia was the only dose limiting toxicity (DLT), though maximum tolerated dose (MTD) was not determined.

The Effect of Autophagy Induction in Oncolytic Reovirus Replication in Mesenchymal Stem Cells

Background and Aims: Oncolytic reoviruses can infect and kill malignant cells while sparing their normal counterparts. Reoviral infection can induce or activate autophagy, even though metformin can induce autophagy. Identifying and regulating the cellular pathways important for reovirus replication and oncolysis can improve targeted-biological therapies for cancer. Here, the autophagic process was triggered via metformin, and we investigated the effect of autophagy activation on oncolytic reovirus replication in mesenchymal stem cells as primary cells and L929 cell lines. Materials and Methods: Adipose derived mesenchymal stem cells (AD-MSCs) and L929 cells were treated with metformin and reovirus type-3 strain Dearing (T3D). Twenty-four hours after infection, the viability of AD-MSCs and L929 cells were examined by MTT assay. Also, the effect of metformin-induced autophagy in the reovirus replication in these cells was determined by real-time polymerase-chain-reaction. Results: Our results show that treatment with metformin and reovirus reduced the viability of the cells compared to treatment with metformin or reovirus alone in both cells. Also, coadministration of metformin and reovirus significantly decreased the relative expression level of the Beclin-1 gene compared to treatment with metformin in both cells. However, the expression level of the reovirus L3 gene after treatment with metformin and reovirus in L929 cells increased significantly compared to AD-MSCs. Conclusion: Our data suggest that metformin-induced autophagy enhances reoviral replication in AD-MSCs and L929 cells. These findings represent the role of autophagy induction in facilitating reovirus replication and contribute to a better understanding of reovirus-host interactions.

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome, Creating Novel Antitumor CD8 T Cell Reactivities

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry–based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.

Altered Glycan Expression on Breast Cancer Cells Facilitates Infection by T3 Seroptype Oncolytic Reovirus.

Breast cancer is the most common cancer in women. Although current therapies have increased survival rates for some breast cancer types, other aggressive invasive breast cancers remain difficult to treat. As the onset of breast cancer is often associated with the appearance of extracellular markers, these could be used to better target therapeutic agents. Here, we demonstrated by nanobiophysical approaches that overexpression of α-sialylated glycans in breast cancer provides an opportunity to combat cancer cells with oncolytic reoviruses. Notably, a correlation between cellular glycan expression and the mechanical properties of reovirus attachment and infection is observed in a serotype-dependent manner. Furthermore, we enhance the infectivity of reoviruses in malignant cells by the coinjection of α-sialylated glycans. In conclusion, this study supports both the use of reoviruses as an oncolytic agent in nanomedicine and the role of α-sialylated glycans as adjuvants in oncolysis, offering new perspective in oncolytic cancer therapy.