No abstract

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor. Cancer Res; 70(6); 2435-44. ©2010 AACR.

Hematologic stem cell rescue after highdose cytotoxic therapy is extensively used for the treatment of many hematopoietic and solid cancers. Gene marking studies suggest that occult tumor cells within the autograft may contribute to clinical relapse. To date purging of autografts contaminated with cancer cells has been unsuccessful. The selective oncolytic property of reovirus against myriad malignant histologies in in vitro, in vivo, and ex vivo systems has been previously demonstrated. In the present study we have shown that reovirus can successfully purge cancer cells within autografts. IntroductionAutologous hematopoietic progenitor stem cell (ASC) transplantations following high-dose chemotherapy has gained extensive application as a therapeutic modality in several malignancies. [1][2][3][4][5][6][7] Globally, the number of autologous blood and marrow transplantations now surpasses the number of allotransplantations. [6][7][8] Despite the significant increase in ASC transplantations, controversy still exists as to the contribution of minimal residual disease to the development of relapse after high-dose chemotherapy. Evidence supported by gene marking studies indicates relapse following high-dose ablative therapy followed by ASC transplantation may be due to contaminating cancer cells within the autograft. [9][10][11] It has been estimated that more than 30% of all autografts are contaminated with tumor cells, and this number likely will increase with better detection methodology and increasing use of ASC transplantations in patients with advanced disease.To minimize the number of contaminating tumor cells, a variety of purging techniques to rid the graft of residual tumor cells have been used. The ideal purging technique should preferentially destroy contaminating tumor cells while preserving the number and function of the collected stem cells. Widely cited purging methods of autografts include the use of ex vivo chemotherapy, tumor targeting monoclonal antibodies linked with toxins or selected on immunocolumns, and positive (CD34 ϩ ) selection. 12,13 More recently, photodynamic purging processes, [14][15][16] virus-directed enzyme prodrug therapy, 17 receptor-targeted ligand toxins, 18,19 and attenuated replication-competent virus-based purging techniques 20 have been reported. Yet, to date no method has proved 100% successful in depleting autografts of tumor cells in the clinical setting. Although several studies have suggested that graft manipulation is of clinical benefit, 21-24 until purging techniques lead to a complete eradication of contaminating tumor cells, it will be unclear whether the recurrence of the disease is the result of the contaminating tumor cells or a reflection of a resistant in vivo malignancy or both. In the present study we investigated the use of an unattenuated oncolytic virus, reovirus, as the basis for a novel purging strategy for ASC transplantation.Reovirus is an ubiquitous double-stranded RNA virus that can be isolated from the upper respiratory and gastrointestinal tract...

Purpose: Despite the recent advances made in the treatment of multiple myeloma, the disease still remains incurable. The oncolytic potential of reovirus has previously been shown and is currently in phase III clinical trials for solid tumors. We tested the hypothesis that reovirus can successfully target human multiple myeloma in vitro, ex vivo, and in vivo without affecting human hematopoietic stem cell (HHSC) repopulation/differentiation in a murine model that partially recapitulates human multiple myeloma.Experimental Design: Human myeloma cell lines and ex vivo tumor specimens were exposed to reovirus and oncolysis and mechanisms of cell death were assessed. RPMI 8226GFPþ cells were injected intravenously to non-obese diabetic/severe combined immune deficient (NOD/SCID) mice and treated with live reovirus (LV) or dead virus (DV). Multiple myeloma disease progression was evaluated via whole-body fluorescence and bone marrow infiltration. HHSCs exposed to LV/DV were injected to NOD/SCID mice and repopulation/differentiation was monitored.Results: A total of six of seven myeloma cell lines and five of seven patient tumor specimens exposed to reovirus showed significant in vitro sensitivity. Tumor response of multiple myeloma by LV, but not DV, was confirmed by comparison of total tumor weights (P ¼ 0.05), and bone marrow infiltration (1/6, LV; 5/6, DV). Mice injected with LV-or DV-exposed HHSCs maintained in vivo re-population/lineage differentiation showing a lack of viral effect on the stem cell compartment. Reovirus oncolysis was mediated primarily by activation of the apoptotic pathways.Conclusions: The unique ability of reovirus to selectively kill multiple myeloma while sparing HHSCs places it as a promising systemic multiple myeloma therapeutic for clinical testing.

Multiple myeloma (MM) is a clonal plasma cell malignancy that accounts for 10-15% of newly diagnosed hematological cancers. Although significant advances have been made in the treatment of MM the disease still remains incurable. The oncolytic potential of reovirus has previously been demonstrated by others and us and is currently in phase III clinical trials for solid tumors. In addition a phase I clinical trial has recently been initiated for MM. Despite the clinical activity, the mechanism(s) of cell death caused by reovirus in MM is yet not yet well elucidated. A comprehensive understanding of reovirus-mediated histology-specific cell death mechanisms is imperative if this therapeutic is to become a standard of care for patients. Previously we have shown that reovirus-mediated cell death of breast and prostate cancer is orchestrated via apoptosis. The present study demonstrates for the first time that in addition to inducing apoptosis reovirus also upregulates autophagy during oncolysis of MM.

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus (RV) for the treatment of breast cancer (BrCa). In vitro, oncolysis and cytokine production were assessed in human and murine BrCa cell lines following RV exposure. Furthermore, RV-induced upregulation of tumor cell PD-L1 was evaluated. In vivo, the immunocompetent, syngeneic EMT6 murine model of BrCa was employed to determine therapeutic and tumor-specific immune responses following treatment with RV, anti-PD-1 antibodies or in combination. RV-mediated oncolysis and cytokine production were observed following BrCa cell infection and RV upregulated tumor cell expression of PD-L1. In vivo, RV monotherapy significantly reduced disease burden and enhanced survival in treated mice, and was further enhanced by PD-1 blockade. RV therapy increased the number of intratumoral regulatory T cells, which was reversed by the addition of PD-1 blockade. Finally, dual treatment led to the generation of a systemic adaptive anti-tumor immune response evidenced by an increase in tumor-specific IFN-γ producing CD8+ T cells, and immunity from tumor re-challenge. The combination of PD-1 blockade and RV appears to be an efficacious immunotherapeutic strategy for the treatment of BrCa, and warrants further investigation in early-phase clinical trials.

Multiple Myeloma (MM), a clonal malignancy of antibody-producing plasma cells, is the second most common hematologic malignancy and results in significant patient morbidity and mortality. The high degree of immune dysregulation in MM, including T cell imbalances and up-regulation of immunosuppressive checkpoint proteins and myeloid derived suppressor cells, allows this malignancy to escape from host immune control. Despite advances in the therapeutic landscape of MM over the last decade, including the introduction of immunomodulatory drugs, the prognosis for this disease is poor, with less than 50% of patients surviving 5 years. Thus, novel treatment strategies are required. Oncolytic viruses (OV) are a promising new class of therapeutics that rely on tumour specific oncolysis and the generation of a potent adaptive anti-tumour immune response for efficacy. To date, a number of OV have shown efficacy in pre-clinical studies of MM with three reaching early phase clinical trials. OVs represent a rational therapeutic strategy for MM based on (1) their tumour tropism, (2) their ability to potentiate anti-tumour immunity and (3) their ability to be rationally combined with other immunotherapeutic agents to achieve a more robust clinical response.

The oncolytic reovirus (RV) has demonstrated clinical efficacy and minimal toxicity in a variety of cancers, including multiple myeloma (MM). MM is a malignancy of plasma cells that is considered treatable but incurable because of the 90% relapse rate that is primarily from drug resistance. The systemic nature of MM and the antitumor immunosuppression by its tumor microenvironment presents an ongoing therapeutic challenge. In the present study, we demonstrate that RV synergizes with the standard-of-care MM drug bortezomib (BTZ) and, importantly, enhances its therapeutic potential in therapy-resistant human MM cell lines in vitro. Using the syngeneic Vk*MYC BTZ-resistant immunocompetent transplantable MM murine model, we also demonstrate that mice harboring BTZ-insensitive MM tumors respond to the RV/BTZ combination treatment in terms of decreased tumor burden and improved overall survival (P < .00001). We demonstrate that BTZ augments RV replication in tumor-associated endothelial cells and myeloma cells, leading to enhanced viral delivery and thereby stimulating cytokine release, immune activity, apoptosis, and reduction of the MM-associated immune suppression. We conclude that combined RV/BTZ is an attractive therapeutic strategy with no safety signals for the treatment of MM.