Nuclear Export Inhibitor Selinexor Enhances Oncolytic Myxoma Virus Therapy against Cancer.

Oncolytic viruses exploited for cancer therapy have been developed to selectively infect, replicate, and kill cancer cells to inhibit tumor growth. However, in some cancer cells, oncolytic viruses are often limited in completing their full replication cycle, forming progeny virions, and/or spreading in the tumor bed because of the heterogeneous cell types within the tumor bed. Here, we report that the nuclear export pathway regulates oncolytic myxoma virus (MYXV) infection and cytoplasmic viral replication in a subclass of human cancer cell types where viral replication is restricted. Inhibition of the XPO-1 (exportin 1) nuclear export pathway with nuclear export inhibitors can overcome this restriction by trapping restriction factors in the nucleus and allow significantly enhanced viral replication and killing of cancer cells. Furthermore, knockdown of XPO-1 significantly enhanced MYXV replication in restrictive human cancer cells and reduced the formation of antiviral granules associated with RNA helicase DHX9. Both in vitro and in vivo, we demonstrated that the approved XPO1 inhibitor drug selinexor enhances the replication of MYXV and kills diverse human cancer cells. In a xenograft tumor model in NSG mice, combination therapy with selinexor plus MYXV significantly reduced the tumor burden and enhanced the survival of animals. In addition, we performed global-scale proteomic analysis of nuclear and cytosolic proteins in human cancer cells to identify the host and viral proteins that were upregulated or downregulated by different treatments. These results indicate, for the first time, that selinexor in combination with oncolytic MYXV can be used as a potential new therapy. Significance: We demonstrated that a combination of nuclear export inhibitor selinexor and oncolytic MYXV significantly enhanced viral replication, reduced cancer cell proliferation, reduced tumor burden, and enhanced the overall survival of animals. Thus, selinexor and oncolytic MYXV can be used as potential new anticancer therapy.

Systemic Delivery of mLIGHT-Armed Myxoma Virus Is Therapeutic for Later-Stage Syngeneic Murine Lung Metastatic Osteosarcoma.

Cancers that metastasize to the lungs represent a major challenge in both basic and clinical cancer research. Oncolytic viruses are newly emerging options but successful delivery and choice of appropriate therapeutic armings are two critical issues. Using an immunocompetent murine K7M2-luc lung metastases model, the efficacy of MYXV armed with murine LIGHT (TNFSF14/CD258) expressed under virus-specific early/late promoter was tested in an advanced later-stage disease K7M2-luc model. Results in this model show that mLIGHT-armed MYXV, delivered systemically using ex vivo pre-loaded PBMCs as carrier cells, reduced tumor burden and increased median survival time. In vitro, when comparing direct infection of K7M2-luc cancer cells with free MYXV vs. PBMC-loaded virus, vMyx-mLIGHT/PBMCs also demonstrated greater cytotoxic capacity against the K7M2 cancer cell targets. In vivo, systemically delivered vMyx-mLIGHT/PBMCs increased viral reporter transgene expression levels both in the periphery and in lung tumors compared to unarmed MYXV, in a tumor- and transgene-dependent fashion. We conclude that vMyx-mLIGHT, especially when delivered using PBMC carrier cells, represents a new potential therapeutic strategy for solid cancers that metastasize to the lung.

Transplantation of autologous bone marrow pre-loaded ex vivo with oncolytic myxoma virus is efficacious against drug-resistant Vk*MYC mouse myeloma.

Multiple myeloma (MM) is a hematological malignancy of plasma cells that remains incurable despite significant progress with myeloablative regimens and autologous stem cell transplantation for eligible patients and, more recently with T cell redirected immunotherapy. Recently, we reported that ex vivo virotherapy with oncolytic myxoma virus (MYXV) improved MM-free survival in an autologous-transplant Balb/c mouse model. Here, we tested the Vk*MYC transplantable C57BL/6 mouse MM model that more closely recapitulates human disease. In vitro, the murine bortezomib-resistant Vk12598 cell line is fully susceptible to MYXV infection. In vivo results demonstrate: (i) autologous bone marrow (BM) leukocytes armed ex vivo with MYXV exhibit moderate therapeutic effects against MM cells pre-seeded into recipient mice; (ii) Cyclophosphamide in combination with BM/MYXV delays the onset of myeloma in mice seeded with Vk12598 cells; (iii) BM/MYXV synergizes with the Smac-mimetics LCL161 and with immune checkpoint inhibitor α-PD-1 to control the progression of established MM in vivo, resulting in significant improvement of survival rates and decreased of tumor burden; (iv) Survivor mice from (ii) and (iii), when re-challenged with fresh Vk12598 cells, developed acquired anti-MM immunity. These results highlight the utility of autologous BM grafts armed ex vivo with oncolytic MYXV alone or in combination with chemotherapy/immunotherapy to treat drug-resistant MM in vivo.

Virus-Derived Chemokine Modulating Protein Pre-Treatment Blocks Chemokine-Glycosaminoglycan Interactions and Significantly Reduces Transplant Immune Damage.

Immune cell invasion after the transplantation of solid organs is directed by chemokines binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal transplant is the preferred treatment for end stage renal failure, but organ supply is limited and allografts are often injured during transport, surgery or by cytokine storm in deceased donors. While treatment for adaptive immune responses during rejection is excellent, treatment for early inflammatory damage is less effective. Viruses have developed highly active chemokine inhibitors as a means to evade host responses. The myxoma virus-derived M-T7 protein blocks chemokine: GAG binding. We have investigated M-T7 and also antisense (ASO) as pre-treatments to modify chemokine: GAG interactions to reduce donor organ damage. Immediate pre-treatment of donor kidneys with M-T7 to block chemokine: GAG binding significantly reduced the inflammation and scarring in subcapsular and subcutaneous allografts. Antisense to N-deacetylase N-sulfotransferase1 (ASONdst1) that modifies heparan sulfate, was less effective with immediate pre-treatment, but reduced scarring and C4d staining with donor pre-treatment for 7 days before transplantation. Grafts with conditional Ndst1 deficiency had reduced inflammation. Local inhibition of chemokine: GAG binding in donor organs immediately prior to transplant provides a new approach to reduce transplant damage and graft loss.

Recombinant Myxoma Virus-Derived Immune Modulator M-T7 Accelerates Cutaneous Wound Healing and Improves Tissue Remodeling.

Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or "fine tune" the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.

Autologous Transplantation Using Donor Leukocytes Loaded Ex Vivo with Oncolytic Myxoma Virus Can Eliminate Residual Multiple Myeloma.

Multiple myeloma (MM) is a hematological malignancy of monoclonal plasma cells that remains incurable. Standard treatments for MM include myeloablative regimens and autologous cell transplantation for eligible patients. A major challenge of these treatments is the relapse of the disease due to residual MM in niches that become refractory to treatments. Therefore, novel therapies are needed in order to eliminate minimal residual disease (MRD). Recently, our laboratory reported that virotherapy with oncolytic myxoma virus (MYXV) improved MM-free survival in an allogeneic transplant mouse model. In this study, we demonstrate the capacity of donor autologous murine leukocytes, pre-armed with MYXV, to eliminate MRD in a BALB/c MM model. We report that MYXV-armed bone marrow (BM) carrier leukocytes are therapeutically superior to MYXV-armed peripheral blood mononuclear cells (PBMCs) or free virus. Importantly, when cured survivor mice were re-challenged with fresh myeloma cells, they developed immunity to the same MM that had comprised MRD. In vivo imaging demonstrated that autologous carrier cells armed with MYXV were very efficient at delivery of MYXV into the recipient tumor microenvironment. Finally, we demonstrate that treatment with MYXV activates the secretion of pro-immune molecules from the tumor bed. These results highlight the utility of exploiting autologous leukocytes to enhance tumor delivery of MYXV to treat MRD in vivo.