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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.

Background and purpose Reovirus is a ubiquitous RNA virus that exploits aberrant signaling pathways for its replication. The oncolytic potential of reovirus against numerous cancers under pre-clinical/clinical conditions has been documented by us and others. Despite its proven clinical activity, the underlying mechanisms of reovirus oncolysis is still not well elucidated. If reovirus therapy is to be optimized for cancer, including breast cancer patients, it is imperative to understand the mechanisms of reovirus oncolysis, especially in treatment of resistant tumour. Experimental approach and results In the present study global gene expression profiling was utilized as a preliminary roadmap to tease-out pivotal molecules involved in reovirus induced apoptosis in breast cancer. Reovirus treated HTB133 and MCF7 breast cancer cells revealed transcriptional alteration of a defined subset of apoptotic genes and members of the nuclear factor-kappa B (NF-kB) family and p53 upregulated modulator of apoptosis (PUMA) were prominent. Since NF-kB can paradoxically suppress or promote apoptosis in cancer, the significance of NF-kB in reovirus oncolysis of breast cancer was investigated. Real time PCR analysis indicated a 2.9-4.3 fold increase in NF-kB p65 message levels following reovirus infection of MCF7 and HTB133, respectively. Nuclear translocation of NF-kB p65 protein was also dramatically augmented post reovirus treatment and correlated with enhanced DNA binding. Pharmacologic inhibition of NF-kB lead to oncolytic protection and significant down regulation of PUMA message levels. PUMA down regulation using siRNA suppressed reovirus oncolysis via significantly repressed apoptosis in p53 mutant HTB133 cells. Conclusions This study demonstrates for the first time that a prominent pathway of reovirus oncolysis of breast cancer is mediated through NF-kB and that PUMA upregulation is dependent on NF-kB activation. These findings represent potential therapeutic indicators of reovirus treatment in future clinical trials.

Autologous stem cell rescue (ASCT) following high-dose myeloablative chemotherapy is considered to be a therapeutic option for many multiple myeloma (MM) patients; however relapse post ASCT presents a major challenge. The oncolytic potential of reovirus has been previously demonstrated and is currently undergoing phase I monotherapy clinical trials for MM and phase II/III clinical trials for solid tumors. Here we tested the hypothesis that reovirus can successfully purge MM in a murine model that partially recapitulates human MM. RPMI 8226, MM1S, H929 and U266 human myeloma cell lines were exposed to reovirus and oncolysis was assessed. Apheresis product admixed with MM cells was purged with live reovirus (LV) or dead virus (DV) and purging efficacy was monitored via flow cytometry, reverse transcribed-PCR (RT-PCR) and disease relapse in non obese diabetic/severe combined immune deficient (NOD/SCID) mice. Significant LV purging was seen with MM1S, H929 and U266 and the complete ex vivo purging achieved with RPMI 8226 was confirmed by flow cytometry, RT-PCR and absence of disease relapse in vivo. Mice that received LV-purged autografts exhibited 100% survival in comparison to mice that received DV-purged controls. Reovirus's unique ability to kill MM while sparing hematopoietic stem cells places it as an attractive purging agent for MM during ASCT.

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) re-population/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 8226GFP+ 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 re-population/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 = 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. Clin Cancer Res; 18(18); 4962–72. ©2012 AACR.

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

Myxoma virus, a poxvirus previously considered rabbit specific, can replicate productively in a variety of human tumor cells in culture. The purpose of this study was to determine if there was efficacy or toxicities of this oncolytic virus against experimental models of human malignant gliomas in vitro, in vivo, and ex vivo in malignant glioma specimens. In vitro, the majority of glioma cell lines tested (7 of 8, 87.5%) were fully permissive for myxoma virus replication and killed by infection. In vivo, intracerebral (i.c.) myxoma virus inoculation was well tolerated and produced only minimal focal inflammatory changes at the site of viral inoculation. U87 and U251 orthotopic xenograft models were used to assess myxoma virus efficacy in vivo. A single intratumoral injection of myxoma virus dramatically prolonged median survival compared with treatment with UV-inactivated myxoma virus. Median survival was not reached in myxoma virus-treated groups versus 47.3 days (U87; P = 0.0002) and 50.7 days (U251; P = 0.0027) in UV-inactivated myxoma virus-treated groups. Most myxoma virus-treated animals (12 of 13, 92%) were alive and apparently “cured” when the experiment was finished (>130 days). Interestingly, we found a selective and long-lived myxoma virus infection in gliomas in vivo. This is the first demonstration of the oncolytic activity of myxoma virus in vivo. The nonpathogenic nature of myxoma virus outside of the rabbit host, its capacity to be genetically modified, its ability to produce a long-lived infection in human tumor cells, and the lack of preexisting antibodies in the human population suggest that myxoma virus may be an attractive oncolytic agent against human malignant glioma.

Introduction: Multiple myeloma (MM) is a malignancy of plasma cells that is still considered incurable due to the 90% relapse rate in patients. Despite the advent of new agents the majority of MM patients relapse secondary to therapy resistance. The potential of reovirus (RV) as a novel therapeutic agent for MM under in vitro, in vivo and ex vivo conditions has been demonstrated by us and a phase I clinical trial of MM with RV therapy has shown indications of efficacy. Recently we have shown that RV could synergize with several standard of care MM drugs such as bortezomib (BTZ), carfilzomib (CFZ) and dexamethasone and importantly, enhance its therapeutic potential in therapy resistant human MM cell lines in vitro. Utilizing the syngeneic Vk*MYC BTZ resistant transplantable MM mouse model, we demonstrate that mice harbouring BTZ insensitive MM tumors significantly respond to RV treatment. Our data indicate that this RV treatment sensitivity is manifested via, direct oncolysis in conjunction with immune modulatory effects. Methods: Vk*MYC myeloma cells (Vk12598) were injected via tail vein (IV) into 3 groups of C57BL/6 wt recipient mice. Seven days post tumor injection, mice were treated with PBS (vehicle) or live (LV) or dead reovirus (DV) (5×10⁁8 PFU) administered IV every 6 days in a total of 5 doses. Serum gamma globulins (M-spike) were assessed weekly by serum protein electrophoresis. Mice were sacrificed on day 35 post tumour injection and their spleens and bone marrow (BM) were harvested. Splenic and BM cells were stained for CD45+/CD45R-/CD138+ (MM tumors), CD4+/CD8+ (T cells), CD3+/NKG2D+ (NKT cells), CD11b+Ly6C+ (monocytic myeloid derived suppressor cells (Mo-MDSC) and CD11b+Ly6G+ (polymorphonuclear (PMN) - MDSCs) and analyzed by flow cytometry. Results: Mice treated with RV demonstrated highly significant (P Conclusions: Our results indicate reovirotherapy is successful in a validated syngeneic BTZ resistant MM model. These results have important implications for future clinical trials. Currently we are conducting further experiments to assess the immune modulatory (such as T - memory and T - regulatory cells) contributions of RV in this model. Citation Format: Chandini M. Thirukkumaran, Zhong Qiao Shi, Joanne Luider, Karen Kopciuk, Marta Chesi, Leif Bergsagel, Yuan Dong, Chunfen Zhang, Ahmed Mostafa, Kathy Gratton, Satbir Thakur, Don Morris. Successful oncolytic virotherapy in a bortezomib resistant syngeneic mouse model of multiple myeloma: Implications for translational significance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 541.

Purpose: Oncolytic viruses (OV) are attractive for use against multiple tumours given their ability to prime therapeutic immune responses in addition to their direct cytotoxic effects. We investigated the utility of reovirus, an OV currently in phase III clinical trials, as a novel immunotherapeutic against renal cell (RCC), non small cell lung (NSCLC) and breast carcinomas (BrCa) as both a monotherapy and in combination with sunitinib, a first line metastatic RCC agent. In addition the utility of adding an immune checkpoint inhibitor was investigated. Experimental Design: In-vitro, cytotoxicity, viral replication and chemokine production were assessed in a panel of human and murine cancer cell lines following exposure to reovirus, sunitinib or their combination. In-vivo, RENCA (murine renal cell carcinoma) cells were implanted subcutaneously into female balb/c mice to establish a syngeneic immunocompetent model of RCC. Tumor growth and overall survival were assessed following treatment with reovirus, sunitinib or their combination. IFN-γ, myeloid-derived suppressor cells (MDSC), and protective immunity were assessed by ELISA, flow cytometry (Gr1+/CD11b+) and adoptive transfer experiments, respectively. PDL-1 and PD-1 expression on both tumour and immune cells were assayed. Results: In vitro, exposure of RCC cell lines to reovirus resulted in an inflammatory cytotoxic response that was augmented through combination with sunitinib. In vivo, reovirus significantly reduced RENCA tumor burden and generated an anti-tumor immune response that was augmented by sunitinib. Combinations with sunitinib also lead to a decrease in myeloid-derived suppressor cells (MDSC) and resulted in improved overall survival and enhanced protective immunity. Preliminary results in NSCLC and BrCa are suggestive that this strategy maybe applicable to many cancer histologies. Both PDL-1 and PD-1 expression were up regulated by RV (similar to IFN gamma) in the majority of cell lines tested suggesting a potential role for checkpoint blockade (CB). Conclusions: Reovirus has both direct oncolytic and immunotherapeutic activity against RCC, which is augmented through combination with sunitinib. The role of immune CB is ongoing. These proof-of-principle investigations provide clear rationale to investigate RV/sunitinib/immune CB as a promising approach for early phase clinical trials as a novel treatment strategy for cancer. Citation Format: Ahmed Mostafa, Kathy Gratton, Keith A. Lawson, Zhong-Qiao Shi, Chandini Thirukkumaran, Don G. Morris. PDL-1 blockade and Sunitinib enhance the efficiency of oncolytic viral therapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A12.

Background: Lung cancer is the leading cause of cancer mortality worldwide. Non-small cell lung cancer (NSCLC) represents approximately 85% of these diagnoses and despite incremental improvements with cytotoxic chemotherapy and targeted therapeutics, the five-year overall survival is only 17%. Recently improved understanding of immune checkpoint pathways and immunosuppresive cellular populations such as myeloid-derived suppressor cells (MDSCs) have led to a resurgence in immunotherapeutic strategies. Reovirus (RV) is an oncolytic virus that only targets cancer cells with certain aberrant signaling pathways such as Ras while avoiding normal cells. Furthermore, its role in stimulating an anti-tumor immune response has been recently described. Additionally, Sunitinib (S), a multitargeted tyrosine kinase inhibitor (anti-VEGFR, C-Kit and PDGFRa) also affects tumor microenvironment Tregs and MDSC numerically, which could consequentially augment RV immunotherapeutic potential. Hypothesis: RV and S combination therapy is an effective treatment modality for NSCLC via direct oncolysis, tumor immunosuppression reversal, and host anti-tumor immune response stimulation. Methods: To determine the relative susceptibility of selected NSCLC cell lines to RV, S and combination therapy, a WST-1 cell viability assay was conducted. Four human NSCLC cell lines A549, H460, H1299, and H1975 and the mouse Lewis lung carcinoma (LLC) cell line LL2 were tested, and the combination index (CI) was calculated using Calcusyn software to determine in vitro synergy. The LLC syngeneic C57BL/6 immunocompetent mouse model bearing LL2 cells in the right hind-flank was treated with PBS, S, RV, UV-inactivated RV (DV), or combination therapies (N = 6 mice/group). Mice were sacrificed on day 27. Spleens, blood, and tumors were harvested for analysis. Splenocytes were subject to flow cytometry CD11b and Gr-1 detection for MDSC quantification. Splenocyte CD8+ enrichment was performed, co-cultured with LL2, RV, DV, S or combinations and subject to IFN-γ quantification via ELISA to determine immune recognition. Results: Treatment resulted in efficacy and synergy of RV/S in A549, H460, and H1299 in vitro as indicated by CI values < 1. LL2 showed additive to synergistic responses at varying treatment concentrations. When LLC tumors in a C57BL/6 immunocompetent murine model were treated with the combination of RV/S, a significant tumor reduction occurred compared to monotherapies and untreated controls (non-overlapping 95% confidence intervals). Furthermore, an increase in anti-tumor immunity stimulating IFN-γ was detected in mice samples upon RV and combination exposure at 0.13μg/mL and 0.09μg/mL compared to PBS treated mice at 0μg/mL. Both S and RV/S treated mice had a significant splenocyte MDSC reduction. These results indicate that Reovirus and Sunitinib combination therapy holds promise as a novel treatment strategy for NSCLC. Citation Format: Jianrui Liu, Jason Spurrel, Zhong Qiao Shi, WenQian Chen, Don G. Morris. Oncolytic viral therapy with immune modulation is an effective novel treatment strategy for non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5355. doi:10.1158/1538-7445.AM2015-5355

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.

Introduction: Multiple myeloma (MM) is an incurable hematologic malignancy that accounts for 10% of hematopoietic neoplasms. Recently we have demonstrated the potential of reovirus as novel therapeutic agent for a majority of MM cell lines and ex vivo patient tumor. However, therapy resistant disease still remains a problem. In the present study we demonstrate that reovirus acts synergistically with bortezomib and/or dexamethasone in oncolysis of therapy resistant MM cell lines. Methods: The MM cell lines RPMI 8226, U266, NCIH929, U266, INA-6, KMS11 and OPM2 were incubated with live (LV) or UV-inactivated (DV) reovirus at a multiplicity of infection (MOI) of 40 for 24, 48, and 72 hours respectively and cell death was assessed via the WST assay. Reovirus resistant OPM2 and moderately sensitive KMS-11 that are also resistant to bortezomib and dexamethasone were selected for combination therapy and treated with reovirus and drugs at various concentrations to evaluate Effective Dose 50% (ED50) values. ED50 values for each drug and reovirus was combined in various concentrations but with consistent ratios and percent toxicity was determined. Using Calcusyn software, combination index (CI) values were generated and synergism determined as per Chou-Talalay method. Results: Highly synergistic cytotoxicity was observed with reovirus and bortezomib in both reovirus and drug resistant cell lines OPM2 and KMS-11 at all drug combination ratios. Dexamethasone and reovirus treatment induced synergy in OPM2 cells. Conclusion: Not all MM cell lines are amenable to reovirus mediated cell death. This has important implications for the future use of reovirus as a therapeutic agent for cancer as phase II clinical trials for MM are underway. This study highlights the importance of preclinical studies in evaluating effective reovirus drug combinations for future use that could be extrapolated to a clinical setting. Currently we are evaluating the effects of these drug combinations under in vivo conditions in a mouse model that represents the clinical course of MM. The underlying mechanism of reovirus and drug induced cytotoxicity is also being investigated at present. Understanding the signalling pathways of resistant tumor will help develop a more personalized approach of reovirus therapy for cancer patients in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3718. doi:1538-7445.AM2012-3718

Introduction: Multiple myeloma (MM) is an incurable hematologic malignancy that accounts for 10% of hematopoietic neoplasms. Recently we have demonstrated the potential of reovirus as novel therapeutic agent for MM, however, the oncolytic mechanism has not been elucidated yet. Reovirus is known to utilize multiple signalling pathways for cancer cell destruction depending on histological origin. It is currently unknown if the apoptotic signalling pathways are involved in reovirus mediated cell death of multiple myeloma. Methods: The MM cell lines RPMI 8226, U266, NCIH929, HUNS-1, MC/CAR, and OPM2 were incubated with live or UV-inactivated reovirus at a multiplicity of infection of 40 for 24, 48, and 72 hours respectively. Apoptosis was assayed by Annexin V/7-AAD expression and DNA fragmentation using flow cytometry. RPMI 8226 was further investigated at 2, 6 12, 24 and 48 hours for the presence of Phospho -Akt and activated caspase 3 post reovirus infection. Results: RPMI 8226, U266, and NCIH929 demonstrated an apoptotic response to reovirus infection within 24 hours. All 3 cell lines showed statistically significant (all F tests, p Conclusion: Not all MM cell lines are amenable to reovirus mediated cell death. This has important implications for the future use of reovirus as a therapeutic agent for cancer and highlights the importance of understanding cell signalling mechanisms as Phase 3 clinical trials of solid tumours with reovirus are currently underway. Ongoing research utilizing pharmacologic and molecular inhibitors of Akt and caspase3 will enable us to confirm the importance of these molecules in reovirus oncolysis of haematological malignancies. Understanding these signalling pathways will help develop a more personalized approach of reovirus therapy for cancer patients in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1779. doi:10.1158/1538-7445.AM2011-1779