Oncolytic Reovirus Research Articles

Stabilisation of p53 enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation

Background:Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored.Methods:Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit.Results:We show that despite similar reovirus replication in p53+/+ and p53−/− cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53−/− or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors.Conclusion:Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.

Synergistic effects of oncolytic reovirus and docetaxel chemotherapy in prostate cancer

BackgroundReovirus type 3 Dearing (T3D) has demonstrated oncolytic activity in vitro, in in vivo murine models and in early clinical trials. However the true potential of oncolytic viruses may only be realized fully in combination with other modalities such as chemotherapy, targeted therapy and radiotherapy. In this study, we examine the oncolytic activity of reovirus T3D and chemotherapeutic agents against human prostate cancer cell lines, with particular focus on the highly metastatic cell line PC3 and the chemotherapeutic agent docetaxel. Docetaxel is the standard of care for metastatic prostate cancer and acts by disrupting the normal process of microtubule assembly and disassembly. Reoviruses have been shown to associate with microtubules and may require this association for efficient viral replication.MethodsThe effects of reovirus and chemotherapy on in vitro cytotoxicity were investigated in PC3 and Du 145 cells and the interactions between agents were assessed by combination index analysis. An Annexin V/propidium iodide fluorescence-activated cell sorting-based assay was used to determine mode of cell death. The effects of reovirus and docetaxel administered as single agent or combination therapy were tested in vivo in a murine model. The effects of docetaxel and reovirus, alone and together, on microtubule stabilisation were investigated by Western blot analysis.ResultsVariable degrees of synergistic cytotoxicity were observed in PC3 and Du 145 cells exposed to live reovirus and several chemotherapy agents. Combination of reovirus infection with docetaxel exposure led to increased late apoptotic/necrotic cell populations. Reovirus/docetaxel combined therapy led to reduced tumour growth and increased survival in a PC3 tumour bearing mouse model. Microtubule stabilization was enhanced in PC3 cells treated with reovirus/docetaxel combined therapy compared to other reovirus/chemotherapy combinations.ConclusionsThe co-administration of a variety of chemotherapeutic agents with live reovirus was able to enhance cytotoxicity synergistically in vitro. The combination of docetaxel with reovirus also delayed tumour growth and improved survival in vivo. Enhanced microtubule stabilisation following this combination treatment may, in part, explain the mechanism of synergy. These results provide evidence to support the ongoing clinical trials using these agents.

The combined effects of oncolytic reovirus plus Newcastle disease virus and reovirus plus parvovirus on U87 and U373 cells in vitro and in vivo

Previous results had documented oncolytic capacity of reovirus, parvovirus and Newcastle disease virus (NDV) on several tumor cell types. To test whether combinations of these viruses may increase this capacity, human U87- and U373-glioblastoma cells, invitro or xenografted into immuno-compromised mice, were subjected to simultaneous double infections and analyzed. Our results show that reovirus (serotype-3) plus NDV (Hitcher-B1) and reovirus plus parvovirus-H1 lead to a significant increase in tumor cell killing invitro in both cell lines (Kruskal-Wallis test, P<0.01) and invivo. Immunofluorescence and flow cytometry analyses demonstrated the simultaneous replication of the viruses in nearly all cells (>95%) after combined infection. These data thus indicate that a synergistic anti-tumor effect can be achieved by the combined infection with oncolytic viruses.

Internalization of Oncolytic Reovirus by Human Dendritic Cell Carriers Protects the Virus from Neutralization

Dendritic cells (DC) may be the most effective way of delivering oncolytic viruses to patients. Reovirus, a naturally occurring oncolytic virus, is currently undergoing early clinical trials; however, intravenous delivery of the virus is hampered by pre-existing antiviral immunity. Systemic delivery via cell carriage is a novel approach currently under investigation and initial studies have indicated its feasibility by using a variety of cell types and viruses. This study addressed the efficacy of human DC to transport virus in the presence of human neutralizing serum. Following reovirus-loading, DC or T cells were cocultured with melanoma cells with or without neutralizing serum; the melanoma cells were then analyzed for cell death. Following reovirus loading, cells were examined by electron microscopy to identify mechanisms of delivery. The phagocytic function of reovirus-loaded DC was investigated by using labeled tumor cells and the ability of reovirus-loaded DC to prime T cells was also investigated. In the presence of human neutralizing serum DC, but not T cells, were able to deliver reovirus for melanoma cell killing in vitro. Electron microscopy suggested that DC protected the virus by internalization, whereas with T cells it remained bound to the surface and hence accessible to neutralizing antibodies. Furthermore, DC loaded with reovirus were fully functional with regard to phagocytosis and priming of specific antitumor immune responses. The delivery of reovirus via DC could be a promising new approach offering the possibility of combining systemic viral therapy for metastatic disease with induction of an antitumor immune response.

Abstract 4336: Oncolytic reovirus as a novel therapy for neuroblastoma

Abstract Background: Neuroblastoma (NB), a tumour of neurocrest progenitor cells of the sympathetic nervous system, is the most common extracranial pediatric tumour and accounts for approximately 15 percent of childhood cancer mortality. Even with surgery, radiation therapy, aggressive cytotoxic chemotherapy including autologous stem cell transplantation and more recently immunotherapy, high risk neuroblastoma has only a 30 percent predicted survival rate. Reovirus, a double-stranded RNA virus, has been shown to be effective against a myriad of cancers through its ability to preferentially lyse cancer cells with aberrant Ras pathway signaling. In this study, we investigate the potential of reovirus (serotype 3, strain Dearing) as a novel treatment for neuroblastoma and neuroblastoma tumour initiating cells (nbTIC). Experimental Design/Results: Reovirus induces dramatic cytotoxic effects at a multiplicity of infection (MOI) of 40 within 48h as assessed by WST-1 viability assays for the IMR-32, IMR-5, SK-N-AS, SK-N-SH, LAN-1, LAN-5, and SHEP human neuroblastoma cell lines in vitro. Interestingly the human neuroblastoma tumor-initiating cell lines, NB-12, NB88 and NB122 (kind gift from Dr David Kaplan) were also found to be very sensitive to reovirus-induced cytotoxicity, suggesting a role for reovirus treatment of refractory neuroblastoma. In order to further study reovirus, immunotherapy and neuroblastoma, a syngeneic, immunocompetent murine model (Neuro2a, A/J neuroblastoma model) was utilized to test reovirus-induced cytotoxicity and RV-directed immunotherapy of Neuro2A in vitro and in vivo. Preliminary data suggest that the Neuro2a cells are exquisitely sensitive to reovirus in vitro. Updated results will be presented. Conclusion: These preclinical results suggest that reovirus holds promise as a novel therapeutic for neuroblastoma and that it warrants further investigation in early phase clinical trials. 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 4336. doi:10.1158/1538-7445.AM2011-4336

Abstract 1779: Selective oncolysis of multiple myeloma cells by reovirus is mediated through apoptosis

Abstract 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&amp;lt;0.00001) increases in DNA fragmentation in response to reovirus and this increase was more pronounced as time progressed. Similarly expression of Annexin V was significantly higher (all F tests, p&amp;lt;0.00001) in all 3 cell lines with live reovirus treatment but the kinetics were cell line dependent where a significant (p&amp;lt; 0.05) time effect was seen for U266 but not for RPMI 8226 and NCIH929. In contrast, the MM cell lines HUNS-1, MC/CAR, and OPM2 appeared resistant to reovirus infection and showed no evidence of apoptosis at 72 h. Supporting the apoptotic mechanism of cell death, RPMI 8226 showed down regulation of the anti-apoptotic signalling protein, Phospho-Akt, at 24 hours of incubation with reovirus. This was accompanied by a simultaneous expression of activated caspase 3. 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

A Phase I Study of the Combination of Intravenous Reovirus Type 3 Dearing and Gemcitabine in Patients with Advanced Cancer

This study combined systemic administration of the oncolytic reovirus type 3 Dearing (reovirus) with chemotherapy in human subjects. We aimed to determine the safety and feasibility of combining reovirus administration with gemcitabine and to describe the effects of gemcitabine on the antireoviral immune response. Patients received reovirus in various doses, initially we dosed for five consecutive days but this was poorly tolerated. We amended the protocol to administer a single dose and administered up to 3 × 10(10) TCID(50). Toxicity was assessed by monitoring of clinical and laboratory measurements. We assessed antibody response by cytotoxicity neutralization assay. Sixteen patients received 47 cycles of reovirus. The two initial patients and one patient in the final cohort experienced dose limiting toxicity (DLT). The DLTs consisted of two asymptomatic grade 3 liver enzyme rises and one asymptomatic grade 3 troponin I rise. Common toxicities consisted of known reovirus and gemcitabine associated side effects. Further analysis showed a potential interaction between reovirus and gemcitabine in causing liver enzyme rises. Grade 3 rises in liver enzymes were associated with concomitant aminocetophen use. Importantly, the duration of the liver enzyme rise was short and reversible. Neutralizing antibody responses to reovirus were attenuated both in time-to-occurrence and peak height of the response. Reovirus at the dose of 1 × 10(10) TCID(50) can be safely combined with full dose gemcitabine. Combination of reovirus with gemcitabine affects the neutralizing antibody response and this could impact both safety and efficacy of this treatment schedule.

The Effect of Cell Cycle Synchronization on Tumor Sensitivity to Reovirus Oncolysis

The potential for increased sensitivity of tumor cells to oncolytic reovirus by altering the normal cell cycle using clinically available pharmacological agents was investigated. B16.F10 mouse melanoma cells were partially synchronized with hydroxyurea, thymidine, or by mitotic shake-off. Cell survival was determined using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium)] survival assay and virus yield in tumors by plaque assay. An enhanced sensitivity to reovirus was observed following the removal of either hydroxyurea or thymidine from the culture medium (P < 0.0001). The greatest survival difference compared to normal cycling cells was noted when the majority of cells were in S and G2/M phases, and was associated with increased viral replication. Cells collected by mitotic shake-off were nearly devoid of cells in S phase and were less susceptible to reovirus-induced cell kill than their nonsynchronized counterparts (P < 0.0001). In vivo combination of hydroxyurea followed by intratumoral reovirus resulted in reduced tumor growth and increased survival compared to monotherapy (P = 0.0041) at 15 days. Increased amounts of virus were retrieved from tumors from mice treated with sequential hydroxyurea/reovirus compared to concomitant treatment or reovirus monotherapy. These data justify clinical evaluation of this approach supported by the extensive experience, low cost, simple administration, and availability of hydroxyurea.

REO-10: A Phase I Study of Intravenous Reovirus and Docetaxel in Patients with Advanced Cancer

REOLYSIN (Oncolytics Biotech) consists of a wild-type oncolytic reovirus, which has selective cytotoxicity for tumor cells while sparing normal cells. In a phase I study as a single agent, repeated infusions of reovirus were safe with evidence of antitumor activity. Preclinical studies indicate potential for synergy between reovirus and chemotherapeutic agents. A multicenter, phase I dose escalation study was designed to assess the safety of combining reovirus with docetaxel chemotherapy in patients with advanced cancer. Patients received 75 mg/m(2) docetaxel (day 1) and escalating doses of reovirus up to 3 × 10(10) TCID(50) (days 1-5) every 3 weeks. Twenty-five patients were enrolled, and 24 patients were exposed to treatment, with 23 completing at least one cycle and 16 suitable for response assessment. Dose-limiting toxicity of grade 4 neutropenia was seen in one patient, but the maximum tolerated dose was not reached. Antitumor activity was seen with one complete response and three partial responses. A disease control rate (combined complete response, partial response, and stable disease) of 88% was observed. Immunohistochemical analysis of reovirus protein expression was observed in posttreatment tumor biopsies from three patients. The combination of reovirus and docetaxel is safe, with evidence of objective disease response, and warrants further evaluation in a phase II study at a recommended schedule of docetaxel (75 mg/m(2), three times weekly) and reovirus (3 × 10(10) TCID(50), days 1-5, every 3 weeks).

Systemic administration of reovirus (Reolysin) inhibits growth of human sarcoma xenografts

Despite advancement in therapies, overall survival rates for relapsed pediatric sarcomas are dismal. Newer therapies are needed to effectively salvage these patients. Oncolytic viruses (such as reovirus) and other genetically altered viruses (such as herpes simplex viruses and adenoviruses) have shown efficacy in a variety of solid tumors including sarcomas. Reolysin is an unmodified oncolytic reovirus that selectively replicates in Ras-activated cancer cells while not causing any significant human illness in its wild form. By using a panel of pediatric sarcoma cell lines in vitro and flank xenografts in vivo, Reolysin was evaluated as a single agent and in combination with cisplatin and radiation therapy. Electron microscopy and immunohistochemistry was used to demonstrated a cytopathic effect in treated tumors. Reolysin inhibited the proliferation and viability of sarcoma cell lines at a dose of 1 to 10 virus particles per cell. In vivo, 5 × 10(9) plaque-forming units (PFU) administered via the tail vein every other day for 3 doses every 21 days inhibited the growth of tumor xenografts with improvement in event-free survival. In the SKES1 Ewing sarcoma line, there was therapeutic enhancement when reovirus was administered in combination with radiation or cisplatin. In the RH30 line and the OS33 line, therapeutic enhancement was demonstrated with radiation and cisplatin, respectively. These results suggest that Reolysin alone or in combination with other cytotoxic agents may be effective therapy in pediatric sarcomas.

Efficacy and Safety/Toxicity Study of Recombinant Vaccinia Virus JX-594 in Two Immunocompetent Animal Models of Glioma

The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered “cured” (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSVΔM51. Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).

Efficacy of oncolytic reovirus against human gastric cancer with peritoneal metastasis in experimental animal model

The prognosis of gastric cancer patients with peritoneal dissemination is extremely poor, and the development of an effective treatment is necessary. The aim of this study was to investigate the efficacy of oncolytic reovirus against peritoneal metastasis in human gastric cancer using an experimental animal model. Four human gastric cancer cell lines, including MKN45p, NUGC4, MKN7 and KatoIII, a normal NIH3T3 cell line as a control, and reovirus serotype 3, were used in this study. We evaluated the cytopathic effect of reovirus and the Ras activity in each gastric cancer cell line in vitro. To evaluate oncolytic efficacy in vivo, reovirus (1x10(8) PFU) was administered into the peritoneal cavity of nude mice on days 7, 8 and 9 after inoculation with MKN45p cells. Mean volume of ascites and the total number and weight of the peritoneal tumors were measured after sacrifice. After reovirus infection, cytopathic effect was observed in all four gastric cancer cell lines, but not in the control cells. Ras activation assay showed that Ras activity in all four gastric cancer cell lines increased to a higher level than that in the control cells. In the animal model experiments, mean volume of ascites and the total number and weight of the peritoneal tumors in the reovirus treatment group were significantly lower than those in the control group. In conclusions, intraperitoneal administration of reovirus could be useful as a new modality against peritoneal metastasis in gastric cancer.

Z-FA-FMK as a novel potent inhibitor of reovirus pathogenesis and oncolysis in vivo

Respiratory enteric orphan (reo)virus is a promising oncolytic viral candidate. Reoviral anticancer therapy is currently undergoing multiple clinical trials targeting various human cancers; however, there is no effective reoviral inhibitor that can be used to block unwanted reovirus replication during reoviral anticancer therapy. Studies were conducted with transformed or normal cells in vitro and in vivo to characterize viral replication in the presence or absence of chemical inhibitors. We have identified a protease inhibitor that is very effective in the inhibition of viral replication. The dipeptide benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-FA-FMK) effectively inhibited reovirus replication in a susceptible host and cured cells of a persistent infection with reovirus in vitro. Electron microscopic analysis of Z-FA-FMK-treated cells revealed that internalized reovirus virions, retained in a perinuclear localization, no longer undergo further processing into viral factories following Z-FA-FMK treatment, suggesting that Z-FA-FMK specifically affects a reovirus virion maturation step. Animal studies showed that reovirus infection of Ras oncogenic tumours and host heart tissues is completely blocked by Z-FA-FMK treatment in severe combined immunodeficiency mice. Z-FA-FMK is a very effective viral inhibitor that can prevent reovirus replication in vitro and reovirus-mediated myocarditis, as well as reovirus-mediated oncolysis, in vivo. A potential application of this drug for inhibition of reovirus infection is suggested.

A phase I/II study of oncolytic reovirus plus carboplatin/paclitaxel in patients with advanced solid cancers with emphasis on squamous cell carcinoma of the head and neck (SCCHN).

3080 Background: Reolysin is an isolate of reovirus, a RNA virus which replicates in cells with activated Ras signaling pathway while sparing normal cells. We conducted a phase I/II dose escalation study of intravenous Reolysin in combination with chemotherapy. The study has completed enrollment. Methods: Reolysin was administered to cohorts of 3 patients in escalating doses (3x109, 1x1010, 3x1010 TCID50) on days 1-5 while carboplatin AUC 5 and paclitaxel 175 mg/m2 were administered on day 1 of a 3-weekly schedule. Eligible patients had good performance status (ECOG PS: 0-2) with advanced cancers which were not amenable to curative treatment or were refractory to standard therapy for which paclitaxel/carboplatin was appropriate palliative chemotherapy. Results: Thirty-one patients (24 males; median age 59 years) with head and neck cancer (n=24), melanoma (n=4), peritoneal/endometrial cancer (n=2) or sarcoma (n=1) received 147 cycles (median 5, range 1-8) of treatment. In the dose-escalation phase of the study, there were no dose-limiting toxicities. Grade 3/4 toxicities included anaemia, leucopenia, neutropenia, lymphopenia, thrombocytopenia, infection and hypotension. Neutralising anti-reovirus antibody responses will be presented. In the phase 1 study, partial responses (PR) were noted in 2 of 5 patients with head and neck cancer. The Phase 2 study treated head and neck cancer patients at the maximum dose level (3 × 1010 TCID50) in order to further assess tumour response. In total, 19 patients with head and neck cancer received at least 2 cycles and are evaluable for response. Most were SCCHN refractory to previous platinum-based chemotherapy for recurrent/metastatic disease. PR was seen in 8 patients (42%) and SD in 6 (32%). One additional PR and 1 SD were observed among 4 patients with malignant melanoma. Final results for median OS and PFS will be presented. Conclusions: Intravenous administration of reovirus in combination with carboplatin/paclitaxel is a safe and well-tolerated combination with promising anticancer activity in SCCHN. Further evaluation of this combination in a randomized phase III trial in SCCHN is underway. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Oncolytics Biotech Oncolytics Biotech Oncolytics Biotech Oncolytics Biotech Oncolytics Biotech

The viral tropism of two distinct oncolytic viruses, reovirus and myxoma virus, is modulated by cellular tumor suppressor gene status

Replication-competent oncolytic viruses hold great potential for the clinical treatment of many cancers. Importantly, many oncolytic virus candidates, such as reovirus and myxoma virus, preferentially infect cancer cells bearing abnormal cellular signaling pathways. Reovirus and myxoma virus are highly responsive to activated Ras and Akt signaling pathways, respectively, for their specificity for viral oncolysis. However, considering the complexity of cancer cell populations, it is possible that other tumor-specific signaling pathways may also contribute to viral discrimination between normal versus cancer cells. Because carcinogenesis is a multistep process involving the accumulation of both oncogene activations and the inactivation of tumor suppressor genes, we speculated that not only oncogenes but also tumor suppressor genes may have an important role in determining the tropism of these viruses for cancer cells. It has been previously shown that many cellular tumor suppressor genes, such as p53, ATM and Rb, are important for maintaining genomic stability; dysfunction of these tumor suppressors may disrupt intact cellular antiviral activity due to the accumulation of genomic instability or due to interference with apoptotic signaling. Therefore, we speculated that cells with dysfunctional tumor suppressors may display enhanced susceptibility to challenge with these oncolytic viruses, as previously seen with adenovirus. We report here that both reovirus and myxoma virus preferentially infect cancer cells bearing dysfunctional or deleted p53, ATM and Rb tumor suppressor genes compared to cells retaining normal counterparts of these genes. Thus, oncolysis by these viruses may be influenced by both oncogenic activation and tumor suppressor status.

Abstract 1773: The addition of Reolysin, an oncolytic reovirus, to irinotecan shows synergistic anticancer activity in colorectal cancer cell lines

Abstract Recent clinical data suggests that the anti EGFR antibodies, cetuximab and panitumumab, are ineffective in patients with colorectal cancer whose tumors harbor a mutation in the kras oncogene. Currently, for these patients, the only option after failure of front line therapy is irinotecan, and there is no drug that can be combined with it to improve clinical outcomes. We studied in vitro, the combination of Reolysin and irinotecan to develop a novel therapeutic approach for these patients. Reolysin (reovirus Serotype 3) has been shown to replicate specifically in tumors bearing an activated ras pathway. This specificity coupled with its relatively nonpathogenic nature in humans makes it an attractive anti-cancer therapy candidate. We have performed in vitro studies of Reo (multiplicity of infection 1 to 10), iri (0.5-10 µM), as single agents and in combination in human colorectal cancer cells to study the role of kras mutation. Reolysin was obtained from Oncolytics Biotech Inc. and irinotecan was obtained commercially. A panel of 8 colorectal cancer cell lines were infected with Reolysin (multiplicity of infection 1 to 10), irinotecan (0.5-10 µM), as a single agent or in combination. The cells were exposed to Reolysin for 6-8 hours and to irinotecan for 72 hours. Viable cells were determined 72 hours post treatment using MTT assay. The effect of Reolysin and irinotecan combination on cell viability was assessed using CalcuSyn software (Biosoft, UK) that generates combined cytotoxic effect by calculating combination indices (CIs). A CI of &amp;lt;1 indicates synergy, 1 denotes additive effect, and &amp;gt;1 denotes antagonism. We found that Reolysin and irinotecan as single agents were cytotoxic to all colon cell lines studied. Further, when Reolysin was combined with irinotecan, there was evidence of synergistic cytotoxicity in all (HT29, HCT116, DLD, KM12, SKCO-1, SW620, LIM2405) except one (SW948) cell line. There appears to be no association between kras mutation status and synergism. To further explore these findings and understand the mechanistic basis of drug synergism we are studying the effect of Reolysin and irinotecan in isogenic HCT116 (kras mutant) and Hke3 (kras WT) cell lines. In summary, the combination of Reolysin and irinotecan is synergistic in colorectal cancer cell lines including those with kras mutation and is worthy of exploration in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1773.

Down-regulation of HIF-1α by oncolytic reovirus infection independently of VHL and p53

Many oncolytic viruses are currently being tested as potential cancer therapeutic agents. To be effective, these viruses must replicate and propagate efficiently through the tumor mass. However, it is possible that the hypoxia that characterizes many tumors may be an obstacle to viral therapy because of its inhibition of viral replication and propagation. We, therefore, decided to test how oncolytic reovirus and its target cells respond to hypoxia. We found that reovirus infection suppresses hypoxia inducible factor (HIF)-1alpha protein levels (but not transcript abundance) in colon cancer HCT116 cells under CoCl(2) or hypoxia. Reovirus infection was able to reduce HIF-1alpha levels in both von Hippel Lindau (VHL)-/- renal carcinoma A498 and p53-/- HCT116 cells, indicating that the decrease of HIF-1alpha mediated by reovirus requires neither VHL nor p53 proteins. However, treatment with the inhibitor MG132 restored HIF-1alpha levels, suggesting that reovirus-induced HIF-1alpha decrease needs proteosomal activity. A498 VHL-/- cells with constitutive expression of HIF-1alpha were relatively resistant to reovirus-induced apoptosis when compared with A498 VHL+/+ cells. However, we found that the use of YC-1 to target HIF-1alpha promoted reovirus-induced apoptosis in A498 VHL-/- cells. Accordingly, we propose that reovirus may be used together with YC-1 as a potential therapeutic agent against chemoresistant or radioresistant tumors that are hypoxic and show increased levels of HIF-1alpha.

Abstract B242: A phase I/II study of oncolytic reovirus plus chemotherapy (carboplatin/paclitaxel) in patients with advanced solid tumors (with emphasis on patients with squamous cell carcinoma of the head and neck (SCCHN)

Abstract Background: Reovirus is a double-stranded RNA virus which replicates in cells with activated Ras signaling pathway, while sparing normal cells. Following our recent study of intravenous (i.v.) reovirus in patients with advanced malignancies1, we have conducted a Phase I/II dose escalation study of i.v. reovirus in combination with chemotherapy. Methods: REOLYSIN® was administered to cohorts of 3 patients in escalating doses (3 × 109, 1 × 1010, 3 × 1010 TCID50) on days 1–5 while carboplatin AUC 5 and paclitaxel 175 mg/m2 were administered on day 1 of a 3-weekly schedule. Eligible patients had good performance status (ECOG PS: 0–2) with advanced cancers which were not amenable to curative treatment or were refractory to standard therapy for which paclitaxel/carboplatin was appropriate palliative chemotherapy. Results: Twenty-three patients (15 males, median age 56.9 years) with head and neck cancer (n=17), melanoma (n=4) or peritoneal/endometrial cancer (n=2) received 106 cycles (median 6, range 1–8) of combination treatment. In the dose-escalation phase of the study, there were no dose-limiting toxicities. Grade 3/4 toxicities included anaemia, leucopenia, neutropenia, lymphopenia, thrombocytopenia, infection and hypotension. Viral shedding (as assayed by reverse transcription PCR) has been documented in 2 patients. Neutralising anti-reovirus antibody responses have been measured using a previously reported technique2 and will be presented. In the Phase 1 study partial responses (PR) were noted in one of four patients with melanoma and in two of five patients with head and neck cancer. An expanded cohort of head and neck cancer patients was therefore treated at the maximum dose level (3 × 1010 TCID50) in order to further assess tumor response in this setting. To date, 14 patients with head and neck cancer have received at least two cycles and are evaluable for response. Most of them were refractory to previous platinum-based chemotherapy for recurrent/metastatic disease. PR was seen in 6 patients (43%), SD in 5 (36%) and PD in 3 (21%). The study is ongoing. Conclusion: Intravenous administration of reovirus in combination with carboplatin/paclitaxel is a safe and well-tolerated combination with promising anticancer activity in SCCHN. Further evaluation of this combination in a randomized Phase III trial in SCCHN is planned. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B242.

Synergistic Effects of Oncolytic Reovirus and Cisplatin Chemotherapy in Murine Malignant Melanoma

To test combination treatment schedules of reovirus and cisplatin chemotherapy in human and murine melanoma cell lines and murine models of melanoma and to investigate the possible mechanisms of synergistic antitumor effects. The effects of reovirus +/- chemotherapy on in vitro cytotoxicity and viral replication were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and plaque assay. Interactions between agents were assessed by combination index analysis. Mode of cell death was assessed by Annexin V/propidium iodide fluorescence-activated cell sorting-based assays; gene expression profiling of single versus combination treatments was completed using the Agilent microarray system. Single agent and combination therapy effects were tested in vivo in two immunocompetent models of murine melanoma. Variable degrees of synergistic cytotoxicity between live reovirus and several chemotherapy agents were observed in B16.F10 mouse melanoma cells, most significantly with cisplatin (combination index of 0.42 +/- 0.03 at ED(50)). Combination of cisplatin and reovirus exposure led to increased late apoptotic/necrotic cell populations. Cisplatin almost completely abrogated the inflammatory cytokine gene up-regulation induced by reovirus. Combination therapy led to significantly delayed tumor growth and improved survival in vivo (P < 0.0001 and P = 0.0003, respectively). Cisplatin had no effect on the humoral response to reovirus in mice. However, cisplatin treatment suppressed the cytokine and chemokine response to reovirus in vitro and in vivo. The combination of reovirus and several chemotherapeutic agents synergistically enhanced cytotoxicity in human and murine melanoma cell lines in vitro and murine tumors in vivo. The data support the current reovirus/chemotherapy combination phase I clinical studies currently ongoing in the clinic.

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.