Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity.

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where antiviral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, inducing STAT1 and STAT4 signalling in both CD56dim and CD56bright NK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dim and CD56bright subsets. However, reovirus treatment inhibited IL-15 induced NK cell proliferation in an IFN-I dependent manner and was associated with reduced AKT signalling. In vivo, human CD56dim and CD56bright NK cells responded with similar kinetics to reovirus treatment, but CD56bright NK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. Coupled with the direct, OV-mediated killing of tumour cells, the activation of both CD56dim and CD56bright NK cells by antiviral pathways induces a spectrum of activity that includes the NK cell-mediated killing of tumour cells and modulation of adaptive responses via the trafficking of IFN-γ expressing CD56bright NK cells to lymph nodes.

The immunological consequences of radiation-induced DNA damage.

Historically, our understanding of the cytotoxicity of radiation has centred on tumour cell-autonomous mechanisms of cell death. Here, tumour cell death occurs when a threshold number of radiation-induced non-reparable double-stranded DNA breaks is exceeded. However, in recent years, the importance of immune mechanisms of cell death has been increasingly recognised, as well as the impact of radiotherapy on non-malignant cellular components of the tumour microenvironment. Conserved antiviral pathways that detect foreign nucleic acid in the cytosol and drive downstream interferon (IFN) responses via the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of IFN genes (cGAS/STING) pathway are key components of the immune response to radiation-induced DNA damage. In preclinical models, acute induction of a type 1 IFN response is important for both direct and abscopal tumour responses to radiation. Inhibitors of the DNA damage response show promise in augmenting this inflammatory IFN response. However, a substantial proportion of tumours show chronic IFN signalling prior to radiotherapy, which paradoxically drives immunosuppression. This chronic IFN signalling leads to treatment resistance, and heterotypic interactions between stromal fibroblasts and tumour cells contribute to an aggressive tumour phenotype. The effect of radiotherapy on myeloid cell populations, particularly tumour-associated macrophages, has an additional impact on the immune tumour microenvironment. It is not yet clear how the above preclinical findings translate into a human context. Human tumours show greater intratumoural genomic heterogeneity and more variable levels of chromosomal instability than experimental murine models. High-quality translational studies of immunological changes occurring during radiotherapy that incorporate intrinsic tumour biology will enable a better understanding of the immunological consequences of radiation-induced DNA damage in patients. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Potentiating Oncolytic Virus-Induced Immune-Mediated Tumor Cell Killing Using Histone Deacetylase Inhibition.

A clinical oncolytic herpes simplex virus (HSV) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), talimogene laherparepvec, causes regression of injected and non-injected melanoma lesions in patients and is now licensed for clinical use in advanced melanoma. To date, limited data are available regarding the mechanisms of human anti-tumor immune priming, an improved understanding of which could inform the development of future combination strategies with improved efficacy. This study addressed direct oncolysis and innate and adaptive human immune-mediated effects of a closely related HSV encoding GM-CSF (HSVGM-CSF) alone and in combination with histone deacetylase inhibition. We found that HSVGM-CSF supported activation of anti-melanoma immunity via monocyte-mediated type I interferon production, which activates NK cells, and viral maturation of immature dendritic cells (iDCs) into potent antigen-presenting cells for cytotoxic T lymphocyte (CTL) priming. Addition of the histone deacetylase inhibitor valproic acid (VPA) to HSVGM-CSF treatment of tumor cells increased viral replication, viral GM-CSF production, and oncolysis and augmented the development of anti-tumor immunity. Mechanistically, VPA increased expression of activating ligands for NK cell recognition and induced expression of tumor-associated antigens, supporting innate NK cell killing and CTL priming. These data support the clinical combination of talimogene laherparepvec with histone deacetylase inhibition to enhance oncolysis and anti-tumor immunity.

Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) carries a dismal prognosis, with advanced disease being resistant to both radiotherapy and conventional cytotoxic drugs, whilst anti-angiogenic drugs are marginally efficacious. Oncolytic viruses (OVs) offer the promise of selective cancer therapy through direct and immune-mediated mechanisms. The premise of OVs lies in their preferential genomic replication, protein expression and productive infection of malignant cells. Numerous OVs are being tested in preclinical models of HCC, with good evidence of direct and immune-mediated anti-tumour efficacy. Efforts to enhance the performance of these agents have concentrated on engineering OV cellular specificity, immune evasion, enhancing anti-tumour potency and improving delivery. The lead agent in HCC clinical trials, JX-594, a recombinant Wyeth strain vaccinia virus, has demonstrated evidence for significant benefit and earned orphan drug status. Thus, JX-594 appears to be transcending the barrier between novel laboratory science and credible clinical therapy. Relatively few other OVs have entered clinical testing, a hurdle that must be overcome if significant progress is to be made in this field. This review summarizes the preclinical and clinical experience of OV therapy in the difficult-to-treat area of HCC.

Cytotoxic and immune-mediated killing of human colorectal cancer by reovirus-loaded blood and liver mononuclear cells.

Reovirus is a promising oncolytic virus, acting by both direct and immune-mediated mechanisms, although its potential may be limited by inactivation after systemic delivery. Our study addressed whether systemically delivered reovirus might be shielded from neutralising antibodies by cell carriage and whether virus-loaded blood or hepatic innate immune effector cells become activated to kill colorectal cancer cells metastatic to the liver in human systems. We found that reovirus was directly cytotoxic against tumour cells but not against fresh hepatocytes. Although direct tumour cell killing by neat virus was significantly reduced in the presence of neutralising serum, reovirus was protected when loaded onto peripheral blood mononuclear cells, which may carry virus after intravenous administration in patients. As well as handing off virus for direct oncolytic killing, natural killer (NK) cells within reovirus-treated blood mononuclear cells were stimulated to kill tumour targets, but not normal hepatocytes, in a Type I interferon-dependent manner. Similarly, NK cells within liver mononuclear cells became selectively cytotoxic towards tumour cells when activated by reovirus. Hence, intravenous reovirus may evade neutralisation by serum via binding to circulating mononuclear cells, and this blood cell carriage has the potential to investigate both direct and innate immune-mediated therapy against human colorectal or other cancers metastatic to the liver.

Reovirus exerts potent oncolytic effects in head and neck cancer cell lines that are independent of signalling in the EGFR pathway.

BACKGROUND: Reovirus exploits aberrant signalling downstream of Ras to mediate tumor-specific oncolysis. Since ~90% squamous cell carcinomas of the head and neck (SCCHN) over-express EGFR and SCCHN cell lines are sensitive to oncolytic reovirus, we conducted a detailed analysis of the effects of reovirus in 15 head and neck cancer cell lines. Both pre- and post-entry events were studied in an attempt to define biomarkers predictive of sensitivity/resistance to reovirus. In particular, we analysed the role of EGFR/Ras signalling in determining virus-mediated cytotoxicity in SCCHN. METHODS: To test whether EGFR pathway activity was predictive of increased sensitivity to reovirus, correlative analyses between reoviral IC50 by MTT assay and EGFR levels by western blot and FACS were conducted. Inhibition or stimulation of EGFR signalling were analysed for their effect on reoviral oncolysis by MTT assay, and viral growth by TCID50 assay. We next analysed the effects of inhibiting signalling downstream of Ras, by specific inhibitors of p38MAPK, PI3-K or MEK, on reoviral killing examined by MTT assay. The role of PKR in reoviral killing was also determined by blockade of PKR using 2-aminopurine and assaying for cell survival by MTT assay. The apoptotic response of SCCHN to reovirus was examined by western blot analysis of caspase 3 cleavage. RESULTS: Correlative analyses between reoviral sensitivity and EGFR levels revealed no association. Intermediate sub-viral and core particles showed the same infectivity/cytotoxicity as intact reovirus. Therefore, sensitivity was not determined by cell entry. In 4 cell lines, oncolysis and viral growth were both unaffected by inhibition or stimulation of EGFR signalling. Inhibition of signalling downstream of Ras did not abrogate reoviral oncolysis and, in addition, modulation of PKR using 2-aminopurine did not alter reovirus sensitivity in resistant cell lines. Caspase 3 cleavage was not detected in infected cells and oncolysis was observed in pan-caspase inhibited cells. CONCLUSIONS: In summary, reovirus is potently oncolytic in a broad panel of SCCHN cell lines. Attempts to define sensitivity/resistance by analysis of the EGFR/Ras/MAPK pathway have failed to provide a clear predictive biomarker of response. Further analysis of material from in vitro and clinical studies is ongoing in an attempt to shed further light on this issue.

Enhancing anti-tumour innate immunity by targeting the DNA damage response and pattern recognition receptors in combination with radiotherapy.

Radiotherapy is one of the most effective and frequently used treatments for a wide range of cancers. In addition to its direct anti-cancer cytotoxic effects, ionising radiation can augment the anti-tumour immune response by triggering pro-inflammatory signals, DNA damage-induced immunogenic cell death and innate immune activation. Anti-tumour innate immunity can result from recruitment and stimulation of dendritic cells (DCs) which leads to tumour-specific adaptive T-cell priming and immunostimulatory cell infiltration. Conversely, radiotherapy can also induce immunosuppressive and anti-inflammatory mediators that can confer radioresistance. Targeting the DNA damage response (DDR) concomitantly with radiotherapy is an attractive strategy for overcoming radioresistance, both by enhancing the radiosensitivity of tumour relative to normal tissues, and tipping the scales in favour of an immunostimulatory tumour microenvironment. This two-pronged approach exploits genomic instability to circumvent immune evasion, targeting both hallmarks of cancer. In this review, we describe targetable DDR proteins (PARP (poly[ADP-ribose] polymerase); ATM/ATR (ataxia-telangiectasia mutated and Rad3-related), DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) and Wee1 (Wee1-like protein kinase) and their potential intersections with druggable immunomodulatory signalling pathways, including nucleic acid-sensing mechanisms (Toll-like receptors (TLR); cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and retinoic acid-inducible gene-I (RIG-I)-like receptors), and how these might be exploited to enhance radiation therapy. We summarise current preclinical advances, recent and ongoing clinical trials and the challenges of therapeutic combinations with existing treatments such as immune checkpoint inhibitors.

Intravenous Oncolytic Vaccinia Virus Therapy Results in a Differential Immune Response between Cancer Patients.

Pexa-Vec is an engineered Wyeth-strain vaccinia oncolytic virus (OV), which has been tested extensively in clinical trials, demonstrating enhanced cytotoxic T cell infiltration into tumours following treatment. Favourable immune consequences to Pexa-Vec include the induction of an interferon (IFN) response, followed by inflammatory cytokine/chemokine secretion. This promotes tumour immune infiltration, innate and adaptive immune cell activation and T cell priming, culminating in targeted tumour cell killing, i.e., an immunologically 'cold' tumour microenvironment is transformed into a 'hot' tumour. However, as with all immunotherapies, not all patients respond in a uniformly favourable manner. Our study herein, shows a differential immune response by patients to intravenous Pexa-Vec therapy, whereby some patients responded to the virus in a typical and expected manner, demonstrating a significant IFN induction and subsequent peripheral immune activation. However, other patients experienced a markedly subdued immune response and appeared to exhibit an exhausted phenotype at baseline, characterised by higher baseline immune checkpoint expression and regulatory T cell (Treg) levels. This differential baseline immunological profile accurately predicted the subsequent response to Pexa-Vec and may, therefore, enable the development of predictive biomarkers for Pexa-Vec and OV therapies more widely. If confirmed in larger clinical trials, these immunological biomarkers may enable a personalised approach, whereby patients with an exhausted baseline immune profile are treated with immune checkpoint blockade, with the aim of reversing immune exhaustion, prior to or alongside OV therapy.

Harnessing radiotherapy-induced NK-cell activity by combining DNA damage-response inhibition and immune checkpoint blockade.

BACKGROUND: Despite therapeutic gains from immune checkpoint inhibitors (ICI) in many tumor types, new strategies are needed to extend treatment benefits, especially in patients failing to mount effective antitumor T-cell responses. Radiation and drug therapies can profoundly affect the tumor immune microenvironment. Here, we aimed to identify immunotherapies to increase the antitumor response conferred by combined ataxia telangiectasia and Rad3-related kinase inhibition and radiotherapy. METHODS: Using the human papillomavirus (HPV)-negative murine oral squamous cell carcinoma model, MOC2, we assessed the nature of the antitumor response following ataxia telangiectasia and Rad3-related inhibitor (ATRi)/radiotherapy (RT) by performing RNA sequencing and detailed flow cytometry analyses in tumors. The benefit of immunotherapies based on T cell immunoreceptor with Ig and ITIM domains (TIGIT) and Programmed cell death protein 1 (PD-1) immune checkpoint blockade following ATRi/RT treatment was assessed in the MOC2 model and confirmed in another HPV-negative murine oral squamous cell carcinoma model called SCC7. Finally, immune profiling was performed by flow cytometry on blood samples in patients with head and neck squamous cell carcinoma enrolled in the PATRIOT clinical trial of combined ATRi/RT. RESULTS: ATRi enhances radiotherapy-induced inflammation in the tumor microenvironment, with natural killer (NK) cells playing a central role in maximizing treatment efficacy. We demonstrated that antitumor activity of NK cells can be further boosted with ICI targeting TIGIT and PD-1. Analyses of clinical samples from patients receiving ATRi (ceralasertib) confirm the translational potential of our preclinical studies. CONCLUSION: This work delineates a previously unrecognized role for NK cells in the antitumor immune response to radiotherapy that can be augmented by small-molecule DNA damage-response inhibitors and immune checkpoint blockade.

Neoadjuvant Intravenous Oncolytic Vaccinia Virus Therapy Promotes Anticancer Immunity in Patients.

Improving the chances of curing patients with cancer who have had surgery to remove metastatic sites of disease is a priority area for cancer research. Pexa-Vec (Pexastimogene Devacirepvec; JX-594, TG6006) is a principally immunotherapeutic oncolytic virus that has reached late-phase clinical trials. We report the results of a single-center, nonrandomized biological end point study (trial registration: EudraCT number 2012-000704-15), which builds on the success of the presurgical intravenous delivery of oncolytic viruses to tumors. Nine patients with either colorectal cancer liver metastases or metastatic melanoma were treated with a single intravenous infusion of Pexa-Vec ahead of planned surgical resection of the metastases. Grade 3 and 4 Pexa-Vec-associated side effects were lymphopaenia and neutropaenia. Pexa-Vec was peripherally carried in plasma and was not associated with peripheral blood mononuclear cells. Upon surgical resection, Pexa-Vec was found in the majority of analyzed tumors. Pexa-Vec therapy associated with IFNα secretion, chemokine induction, and resulted in transient innate and long-lived adaptive anticancer immunity. In the 2 patients with significant and complete tumor necrosis, a reduction in the peripheral T-cell receptor diversity was observed at the time of surgery. These results support the development of presurgical oncolytic vaccinia virus-based therapies to stimulate anticancer immunity and increase the chances to cure patients with cancer.

Reciprocal human dendritic cell-natural killer cell interactions induce antitumor activity following tumor cell infection by oncolytic reovirus.

Oncolytic virotherapy may mediate antitumor effects via direct oncolysis or immune-mediated tumor regression. Although the ability of oncolytic viruses to generate adaptive antitumor immunity has been characterized, their interactions with the innate immune system are relatively unclear. Using a human in vitro system, this study investigates the innate immunological consequences of reovirus therapy and its potential to activate NK cell-mediated antitumor activity. Dendritic cells (DC) loaded with reovirus-infected human melanoma Mel888 cells (DC-MelReo), but not reovirus-infected tumor cells alone, induced IFN-gamma production within the NK cell population upon coculture with PBMC, in a cell-to-cell contact-dependent manner. DC-MelReo secreted the chemokines CCL2, 3, 4, 5, 7, 8, 11, and CXCL10; these culture supernatants induced NK cell chemotaxis. Coculture of DC-MelReo with purified NK cells induced reciprocal contact-dependent phenotypic DC maturation, while DC-MelReo elicited up-regulation of the activation marker CD69 on NK cells, in a partially contact and partially IL-12 dependent manner. Significantly, DC-MelReo induced NK cell cytotoxicity toward tumor cells by a type I IFN dependent mechanism. These data demonstrate that tumor infection by reovirus can act via DC to induce NK cell recruitment, activation, and cytotoxicity, along with reciprocal DC maturation. These findings suggest that reciprocal DC-NK cell interactions, following reovirus therapy, may play an important role in altering the immune milieu of the tumor microenvironment and mediating tumor regression.

Therapeutic effect of sodium iodide symporter gene therapy combined with external beam radiotherapy and targeted drugs that inhibit DNA repair.

Adenoviral (AdV) transfer of sodium iodide symporter (NIS) gene has translational potential, but relatively low levels of transduction and subsequent radioisotope uptake limit the efficacy of the approach. In previous studies, we showed that combining NIS gene delivery with external beam radiotherapy (EBRT) and DNA damage repair inhibitors increased viral gene expression and radioiodide uptake. Here, we report the therapeutic efficacy of this strategy. An adenovirus expressing NIS from a telomerase promoter (Ad-hTR-NIS) was cytotoxic combined with relatively high-dose (50 microCi) (131)I therapy and enhanced the efficacy of EBRT combined with low-dose (10 and 25 microCi) (131)I therapy in colorectal and head and neck cancer cells. Combining this approach with ataxia-telangiectasia mutated (ATM) or DNA-dependent protein kinase (DNA-PK) inhibition caused maintenance of double-stranded DNA breaks (DSBs) at 24 hours and increased cytotoxicity on clonogenic assay. When the triplet of NIS-mediated (131)I therapy, EBRT, and DNA-PKi was used in vivo, 90% of mice were tumor-free at 5 weeks. Acute radiation toxicity in the EBRT field was not exacerbated. In contrast, DNA-PKi did not enhance the therapeutic efficacy of EBRT plus adenovirus-mediated HSVtk/ganciclovir (GCV). Therefore, combining NIS gene therapy and EBRT represents an ideal strategy to exploit the therapeutic benefits of novel radiosensitizers.

Glycolysis and Fatty Acid Oxidation Inhibition Improves Survival in Glioblastoma.

Glioblastoma (GBM) is the most aggressive adult glioma with a median survival of 14 months. While standard treatments (safe maximal resection, radiation, and temozolomide chemotherapy) have increased the median survival in favorable O(6)-methylguanine-DNA methyltransferase (MGMT)-methylated GBM (~21 months), a large proportion of patients experience a highly debilitating and rapidly fatal disease. This study examined GBM cellular energetic pathways and blockade using repurposed drugs: the glycolytic inhibitor, namely dicholoroacetate (DCA), and the partial fatty acid oxidation (FAO) inhibitor, namely ranolazine (Rano). Gene expression data show that GBM subtypes have similar glucose and FAO pathways, and GBM tumors have significant upregulation of enzymes in both pathways, compared to normal brain tissue (p < 0.01). DCA and the DCA/Rano combination showed reduced colony-forming activity of GBM and increased oxidative stress, DNA damage, autophagy, and apoptosis in vitro. In the orthotopic Gl261 and CT2A syngeneic murine models of GBM, DCA, Rano, and DCA/Rano increased median survival and induced focal tumor necrosis and hemorrhage. In conclusion, dual targeting of glycolytic and FAO metabolic pathways provides a viable treatment that warrants further investigation concurrently or as an adjuvant to standard chemoradiation for GBM.

OK432-activated human dendritic cells kill tumor cells via CD40/CD40 ligand interactions.

In vivo, dendritic cells (DC) are programmed to orchestrate innate and adaptive immunity in response to pathogen-derived "danger" signals. Under particular circumstances, DC can also be directly cytotoxic against tumor cells, potentially allowing them to release tumor associated Ags from dying cells and then prime antitumor immunity against them. In this study, we describe the innate characteristics of DC (OK-DC) generated in vitro after exposure of immature human myeloid-derived DC to OK432, a penicillin-inactivated and lyophilized preparation of Streptococcus pyrogenes. OK-DC produced proinflammatory cytokines, stimulated autologous T cell proliferation and IFN-gamma secretion, expressed CCR7, and migrated in response to MIP-3beta. Moreover, OK-DC displayed strong, specific cytotoxicity toward tumor cell targets. This cytotoxicity was associated with novel, OK432-induced up-regulation of CD40L on the cell surface of OK-DC, and was absolutely dependent on expression of CD40 on the tumor targets. These data demonstrate that maturation of human DC with OK432, an adjuvant suitable for clinical use, induces direct tumor cell killing by DC, and describes a novel CD40/CD40L-mediated mechanism for specific DC antitumor cytotoxicity.

The Immunogenic Potential of Recurrent Cancer Drug Resistance Mutations: An In Silico Study.

Cancer somatic mutations have been identified as a source of antigens that can be targeted by cancer immunotherapy. In this work, expanding on previous studies, we analyze the HLA-presentation properties of mutations that are known to drive resistance to cancer targeted-therapies. We survey a large dataset of mutations that confer resistance to different drugs and occur in numerous genes and tumor types. We show that a significant number of them are predicted in silico to be potentially immunogenic across a large proportion of the human population. Further, by analyzing a cohort of patients carrying a small subset of these resistance mutations, we provide evidence that what is observed in the general population may be indicative of the mutations' immunogenic potential in resistant patients. Two of the mutations in our dataset had previously been experimentally validated by others and it was confirmed that some of their associated neopeptides elicit T-cell responses in vitro. The identification of potent cancer-specific antigens can be instrumental for developing more effective immunotherapies. In this work, we propose a novel list of drug-resistance mutations, several of which are recurrent, that could be of particular interest in the context of off-the-shelf precision immunotherapies such as therapeutic cancer vaccines.

Inflammatory microenvironment remodelling by tumour cells after radiotherapy.

The development of immune checkpoint inhibitors (ICIs) is revolutionizing the way we think about cancer treatment. Even so, for most types of cancer, only a minority of patients currently benefit from ICI therapies. Intrinsic and acquired resistance to ICIs has focused research towards new combination therapy approaches that seek to increase response rates, the depth of remission and the durability of benefit. In this Review, we describe how radiotherapy, through its immunomodulating effects, represents a promising combination partner with ICIs. We describe how recent research on DNA damage response (DDR) inhibitors in combination with radiotherapy may be used to augment this approach. Radiotherapy can kill cancer cells while simultaneously triggering the release of pro-inflammatory mediators and increasing tumour-infiltrating immune cells - phenomena often described colloquially as turning immunologically 'cold' tumours 'hot'. Here, we focus on new developments illustrating the key role of tumour cell-autonomous signalling after radiotherapy. Radiotherapy-induced tumour cell micronuclei activate cytosolic nucleic acid sensor pathways, such as cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), and propagation of the resulting inflammatory signals remodels the immune contexture of the tumour microenvironment. In parallel, radiation can impact immunosurveillance by modulating neoantigen expression. Finally, we highlight how tumour cell-autonomous mechanisms might be exploited by combining DDR inhibitors, ICIs and radiotherapy.

The PERK Inhibitor GSK2606414 Enhances Reovirus Infection in Head and Neck Squamous Cell Carcinoma via an ATF4-Dependent Mechanism.

Reovirus type 3 Dearing (reovirus) is a tumor-selective oncolytic virus currently under evaluation in clinical trials. Here, we report that the therapeutic efficacy of reovirus in head and neck squamous cell cancer can be enhanced by targeting the unfolded protein response (UPR) kinase, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). PERK inhibition by GSK2606414 increased reovirus efficacy in both 2D and 3D models in vitro, while perturbing the normal host cell response to reovirus-induced endoplasmic reticulum (ER) stress. UPR reporter constructs were used for live-cell 3D spheroid imaging. Profiling of eIF2a-ATF4, IRE1a-XBP1, and ATF6 pathway activity revealed a context-dependent increase in eIF2a-ATF4 signaling due to GSK2606414. GSK2606414 blocked eIF2a-ATF4 signaling because of the canonical ER stress agent thapsigargin. In the context of reovirus infection, GSK2606414 induced eIF2a-ATF4 signaling. Knockdown of eIF2a kinases PERK, GCN2, and PKR revealed eIF2a-ATF4 reporter activity was dependent on either PERK or GCN2. Knockdown of ATF4 abrogated the GSK2606414-induced increase in reovirus protein levels, confirming eIF2a-ATF signaling as key to the observed phenotype. Our work identifies a novel approach to enhance the efficacy and replication of reovirus in a therapeutic setting.

Combining BRAF inhibition with oncolytic herpes simplex virus enhances the immune-mediated antitumor therapy of BRAF-mutant thyroid cancer.

BACKGROUND: The aggressive clinical behavior of poorly differentiated and anaplastic thyroid cancers (PDTC and ATC) has proven challenging to treat, and survival beyond a few months from diagnosis is rare. Although 30%-60% of these tumors contain mutations in the BRAF gene, inhibitors designed specifically to target oncogenic BRAF have shown limited and only short-lasting therapeutic benefits as single agents, thus highlighting the need for improved treatment strategies, including novel combinations. METHODS: Using a BRAFV600E-driven mouse model of ATC, we investigated the therapeutic efficacy of the combination of BRAF inhibition and oncolytic herpes simplex virus (oHSV). Analyses of samples from tumor-bearing mice were performed to immunologically characterize the effects of different treatments. These immune data were used to inform the incorporation of immune checkpoint inhibitors into triple combination therapies. RESULTS: We characterized the immune landscape in vivo following BRAF inhibitor treatment and detected only modest immune changes. We, therefore, hypothesized that the addition of oncolytic virotherapy to BRAF inhibition in thyroid cancer would create a more favorable tumor immune microenvironment, boost the inflammatory status of tumors and improve BRAF inhibitor therapy. First, we showed that thyroid cancer cells were susceptible to infection with oHSV and that this process was associated with activation of the immune tumor microenvironment in vivo. Next, we showed improved therapeutic responses when combining oHSV and BRAF inhibition in vivo, although no synergistic effects were seen in vitro, further confirming that the dominant effect of oHSV in this context was likely immune-mediated. Importantly, both gene and protein expression data revealed an increase in activation of T cells and natural killer (NK) cells in the tumor in combination-treated samples. The benefit of combination oHSV and BRAF inhibitor therapy was abrogated when T cells or NK cells were depleted in vivo. In addition, we showed upregulation of PD-L1 and CTLA-4 following combined treatment and demonstrated that blockade of the PD-1/PD-L1 axis or CTLA-4 further improved combination therapy. CONCLUSIONS: The combination of oHSV and BRAF inhibition significantly improved survival in a mouse model of ATC by enhancing immune-mediated antitumor effects, and triple combination therapies, including either PD-1 or CTLA-4 blockade, further improved therapy.

RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma.

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti-tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre-clinical application of an in vivo treatment protocol for soft-tissue sarcoma that directly engages RIPK1-mediated immunogenic cell death. We find that RIPK1-mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1-induced cell death in patients with advanced disease at limb extremities.

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation.