Eric Bartee

The Addition of Tumor Necrosis Factor plus Beta Interferon Induces a Novel Synergistic Antiviral State against Poxviruses in Primary Human Fibroblasts

ABSTRACT Tumor necrosis factor (TNF) and members of the interferon (IFN) family have been shown to independently inhibit the replication of a variety of viruses. In addition, previous reports have shown that treatment with various combinations of these antiviral cytokines induces a synergistic antiviral state that can be significantly more potent than addition of any of these cytokines alone. The mechanism of this cytokine synergy and its effects on global gene expression, however, are not well characterized. Here, we use DNA microarray analysis to demonstrate that treatment of uninfected primary human fibroblasts with TNF plus IFN-β induces a distinct synergistic state characterized by significant perturbations of several hundred genes which are coinduced by the individual cytokines alone, as well as the induction of more than 850 novel host cell genes. This synergy is mediated directly by the two ligands, not by intermediate secreted factors, and is necessary and sufficient to completely block the productive replication and spread of myxoma virus in human fibroblasts. In contrast, the replication of two other poxviruses, vaccinia virus and tanapox virus, are only partially inhibited in these cells by the synergistic antiviral state, whereas the spread of both of these viruses to neighboring cells was efficiently blocked. Taken together, our data indicate that the combination of TNF and IFN-β induces a novel synergistic antiviral state that is highly distinct from that induced by either cytokine alone.

Oncolytic Virotherapy for Hematological Malignancies

Hematological malignancies such as leukemias, lymphomas, multiple myeloma (MM), and the myelodysplastic syndromes (MDSs) primarily affect adults and are difficult to treat. For high-risk disease, hematopoietic stem cell transplant (HCT) can be used. However, in the setting of autologous HCT, relapse due to contamination of the autograft with cancer cells remains a major challenge. Ex vivo manipulations of the autograft to purge cancer cells using chemotherapies and toxins have been attempted. Because these past strategies lack specificity for malignant cells and often impair the normal hematopoietic stem and progenitor cells, prior efforts to ex vivo purge autografts have resulted in prolonged cytopenias and graft failure. The ideal ex vivo purging agent would selectively target the contaminating cancer cells while spare normal stem and progenitor cells and would be applied quickly without toxicities to the recipient. One agent which meets these criteria is oncolytic viruses. This paper details experimental progress with reovirus, myxoma virus, measles virus, vesicular stomatitis virus, coxsackievirus, and vaccinia virus as well as requirements for translation of these results to the clinic.

Myxoma and vaccinia viruses bind differentially to human leukocytes.

ABSTRACT Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34+ hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes.

Tumor-Localized Secretion of Soluble PD1 Enhances Oncolytic Virotherapy

Oncolytic virotherapy represents an attractive option for the treatment of a variety of aggressive or refractory tumors. While this therapy is effective at rapidly debulking directly injected tumor masses, achieving complete eradication of established disease has proven difficult. One method to overcome this challenge is to use oncolytic viruses to induce secondary antitumor immune responses. Unfortunately, while the initial induction of these immune responses is typically robust, their subsequent efficacy is often inhibited through a variety of immunoregulatory mechanisms, including the PD1/PDL1 T-cell checkpoint pathway. To overcome this inhibition, we generated a novel recombinant myxoma virus (vPD1), which inhibits the PD1/PDL1 pathway specifically within the tumor microenvironment by secreting a soluble form of PD1 from infected cells. This virus both induced and maintained antitumor CD8+ T-cell responses within directly treated tumors and proved safer and more effective than combination therapy using unmodified myxoma and systemic αPD1 antibodies. Localized vPD1 treatment combined with systemic elimination of regulatory T cells had potent synergistic effects against metastatic disease that was already established in secondary solid organs. These results demonstrate that tumor-localized inhibition of the PD1/PDL1 pathway can significantly improve outcomes during oncolytic virotherapy. Furthermore, they establish a feasible path to translate these findings against clinically relevant disease. Cancer Res; 77(11); 2952-63. ©2017 AACR.

Myxoma Virus Induces Ligand Independent Extrinsic Apoptosis in Human Myeloma Cells.

INTRODUCTION Multiple myeloma is a clonal malignancy of plasma B cells. Although recent advances have improved overall prognosis, virtually all myeloma patients still succumb to relapsing disease. Therefore, novel therapies to treat this disease remain urgently needed. We have recently shown that treatment of human multiple myeloma cells with an oncolytic virus known as myxoma results in rapid cell death even in the absence of viral replication; however, the specific mechanisms and pathways involved remain unknown. MATERIALS AND METHODS To determine how myxoma virus eliminates human multiple myeloma cells, we queried the apoptotic pathways that were activated after viral infection using immunoblot analysis and other cell biology approaches. RESULTS Our results indicate that myxoma virus infection initiates apoptosis in multiple myeloma cells through activation of the extrinsic initiator caspase-8. Caspase-8 activation subsequently results in cleavage of BH3 interacting-domain death agonist and loss of mitochondrial membrane potential causing secondary activation of caspase-9. Activation of caspase-8 appears to be independent of extrinsic death ligands and instead correlates with depletion of cellular inhibitors of apoptosis. We hypothesize that this depletion results from virally mediated host-protein shutoff because a myxoma construct that overexpresses the viral decapping enzymes displays improved oncolytic potential. CONCLUSION Taken together, these results suggest that myxoma virus eliminates human multiple myeloma cells through a pathway unique to oncolytic poxviruses, making it an excellent therapeutic option for the treatment of relapsed or refractory patients.

Systemic therapy with oncolytic myxoma virus cures established residual multiple myeloma in mice

Multiple myeloma is an incurable malignancy of plasma B-cells. Traditional chemotherapeutic regimes often induce initial tumor regression; however, virtually all patients eventually succumb to relapse caused by either reintroduction of disease during autologous transplant or expansion of chemotherapy resistant minimal residual disease. It has been previously demonstrated that an oncolytic virus known as myxoma can completely prevent myeloma relapse caused by reintroduction of malignant cells during autologous transplant. The ability of this virus to treat established residual disease in vivo, however, remained unknown. Here we demonstrate that intravenous administration of myxoma virus into mice bearing disseminated myeloma results in the elimination of 70–90% of malignant cells within 24 hours. This rapid debulking was dependent on direct contact of myxoma virus with residual myeloma and did not occur through destruction of the hematopoietic bone marrow niche. Importantly, systemic myxoma therapy also induced potent antimyeloma CD8+ T cell responses which localized to the bone marrow and were capable of completely eradicating established myeloma in some animals. These results demonstrate that oncolytic myxoma virus is not only effective at preventing relapse caused by reinfusion of tumor cells during stem cell transplant, but is also potentially curative for patients bearing established minimal residual disease.

Myxoma virus attenuates expression of activating transcription factor 4 (ATF4) which has implications for the treatment of proteasome inhibitor-resistant multiple myeloma.

The recent development of chemotherapeutic proteasome inhibitors, such as bortezomib, has improved the outcomes of patients suffering from the plasma cell malignancy multiple myeloma. Unfortunately, many patients treated with these drugs still suffer relapsing disease due to treatment-induced upregulation of the antiapoptotic protein Mcl1. We have recently demonstrated that an oncolytic poxvirus, known as myxoma, can rapidly eliminate primary myeloma cells by inducing cellular apoptosis. The efficacy of myxoma treatment on proteasome inhibitor–relapsed or –refractory myeloma, however, remains unknown. We now demonstrate that myxoma-based elimination of myeloma is not affected by cellular resistance to proteasome inhibitors. Additionally, myxoma virus infection specifically prevents expression of Mcl1 following induction of the unfolded protein response, by blocking translation of the unfolded protein response activating transcription factor (ATF)4. These results suggest that myxoma-based oncolytic therapy represents an attractive option for myeloma patients whose disease is refractory to chemotherapeutic proteasome inhibitors due to upregulation of Mcl1.

Potential of oncolytic viruses in the treatment of multiple myeloma

Multiple myeloma (MM) is a clonal malignancy of plasma cells that is newly diagnosed in ~30,000 patients in the US each year. While recently developed therapies have improved the prognosis for MM patients, relapse rates remain unacceptably high. To overcome this challenge, researchers have begun to investigate the therapeutic potential of oncolytic viruses as a novel treatment option for MM. Preclinical work with these viruses has demonstrated that their infection can be highly specific for MM cells and results in impressive therapeutic efficacy in a variety of preclinical models. This has led to the recent initiation of several human trials. This review summarizes the current state of oncolytic therapy as a therapeutic option for MM and highlights a variety of areas that need to be addressed as the field moves forward.

In vivo and in situ programming of tumor immunity by combining oncolytics and PD-1 immune checkpoint blockade

Blockade of the programmed cell death protein 1 (PD1) pathway is clinically effective against human cancers. Although multiple types of malignancies have been shown to respond to PD1 agents, only a small percentage of patients typically benefit from this treatment. In addition, PD1 therapy often causes serious immune-related adverse events. A recent study demonstrated that local, intra-tumoral, administration of modified oncolytic myxoma virus which expresses a truncated version of the PD1 protein resulted in both increased efficacy and reduced toxicity in a clinically relevant melanoma model.