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In vitro characterization of the KLN205 cell line to reovirus, sunitinib and combination therapy.

CD8+ and CD4+ cell depletion confirmation study.

Chemokine expression in reovirus infected RENCA cells at 12 hours.

Purpose: In addition to their direct cytopathic effects, oncolytic viruses are capable of priming antitumor immune responses. However, strategies to enhance the immunotherapeutic potential of these agents are lacking. Here, we investigated the ability of the multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, sunitinib, to augment the antitumor immune response generated by oncolytic reovirus.Experimental Design:In vitro, oncolysis and chemokine production were assessed in a panel of human and murine RCC cell lines after exposure to reovirus, sunitinib, or their combination. In vivo, the RENCA syngeneic murine model of RCC was employed to determine therapeutic and tumor-specific immune responses after treatment with reovirus (intratumoral), sunitinib, or their combination. Parallel investigations employing the KLN205 syngeneic murine model of lung squamous cell carcinoma (NSCLC) were conducted for further validation.Results: Reovirus-mediated oncolysis and chemokine production was observed following RCC infection. Reovirus monotherapy reduced tumor burden and was capable of generating a systemic adaptive antitumor immune response evidenced by increased numbers of tumor-specific CD8+ IFNγ-producing cells. Coadministration of sunitinib with reovirus further reduced tumor burden resulting in improved survival, decreased accumulation of immune suppressor cells, and the establishment of protective immunity upon tumor rechallenge. Similar results were observed for KLN205 tumor–bearing mice, highlighting the potential broad applicability of this approach.Conclusions: The ability to repurpose sunitinib for augmentation of reovirus' immunotherapeutic efficacy positions this novel combination therapy as an attractive strategy ready for clinical testing against a range of histologies, including RCC and NSCLC. Clin Cancer Res; 22(23); 5839–50. ©2016 AACR.

In vitro and in vivo sunitinib dose response.

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Mouse body weights during treatment.

PDF file, 17K, Reovirus sensitivity and genetic lesions detected in multiple myeloma and MGUS patient tumor.

PDF file, 21K, Reovirus sensitivity and prevalnet genetic lesions of myeloma cell lines used in the current study.

Approximately 15% of newly diagnosed breast cancers (BC) are classified as triple negative (TNBC), which have a worse prognosis than hormonal receptor-positive BCs. Standard treatment for TNBC includes cytotoxic chemotherapies that are associated with significant toxicities. Currently the only targeted therapy (PARP inhibition) exists for the 10% of TNBC patients who have germline BRCA mutations. There is an ongoing unmet need for TNBC that may include targeted strategies to improve outcomes. The alkylating agent temozolomide (TMZ) is an oral chemotherapy drug used in the treatment of glioblastomas. The therapeutic benefit of TMZ largely depends on cells not being able to fully repair the DNA damage it causes. O6-methylguanine-DNA methyltransferase (MGMT) is normally expressed and able to repair damage caused by TMZ; however, MGMT is often silenced by promoter methylation in many tumours (mMGMT), and such tumours show a better response to TMZ than those with unmethylated promoters (uMGMT). MGMT has been shown to be methylated in up to 50% of TNBC, suggesting TMZ may be a targeted treatment option. Additionally, studies in other tumour sites suggest that adding an HDAC inhibitor (HDACi) to TMZ can increase response and early phase trials have shown this combination is well tolerated. We have previously shown cytotoxicity with TMZ in mMGMT TNBC cell lines and now hypothesize that the addition of HDAC inhibition to TMZ will have synergistic effects. Using in vitro models, we explored the efficacy of Vorinostat (HDACi) alone and in combination with TMZ and/or cisplatin utilizing TNBC cell lines: HTB132 (uMGMT) and HTB26 (mMGMT). Cytotoxicity assays were utilized to determine median effective dose (EC50) for each treatment and then we performed EC50 synergy testing. Percent viability was calculated as the absorbance ratio of treated/untreated cells X 100. The EC50 was then determined by calculating the intercept of the log effect vs log RV dose. We found that Cisplatin and HDAC inhibition showed similar cytotoxicities in both cell lines. The addition of cisplatin to either TMZ or HDACi increased cytotoxicity compared to either treatment alone in the mMGMT cell line HTB26; however, the addition of cisplatin to TMZ in the uMGMT cell line showed the greatest cytotoxicity. Interestingly, we also saw an increase in cytotoxicity when cells were treated with the combination TMZ and HDACi, regardless of methylation status of the MGMT promotor. Finally, the triple combination therapy did not increase cytotoxicity. Although HDAC inhibition showed cytotoxicity regardless of methylation status of the MGMT promotor; it was increased by the combination with TMZ in both cell lines evaluated. Further, the addition of cisplatin to TMZ did not improve cytotoxicity in the mMGMT cell line but did increase cytotoxicity in the uMGMT cell line. There appears to be no benefit to triple therapy in either cell line. We are currently evaluating these combinations in a xenograft murine model to provide pre-clinical support for a Phase II funded clinical trial in TNBC patients. Citation Format: Elizabeth N Kornaga, Ailian Yang, Kathy Gratton, Zhong Qiao Shi, Nancy A Nixon, Gloria Roldan Urgoiti, Don G Morris. Temozolomide & vorinostat as a targeted therapy strategy for triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-10.

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.

Purpose: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. Experimental Design: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. Results: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus’ antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. Conclusions: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

Brain and leptomeningeal metastases are common in breast cancer patients and our current treatments are ineffective. Reovirus type 3 is a replication competent, naturally occurring virus that usurps the activated Ras-signaling pathway (or an element thereof) of tumor cells and lyses them but leaves normal cells relatively unaffected. In this study we evaluated reovirus as an experimental therapeutic in models of central nervous system (CNS) metastasis from breast cancer. We found all breast cancer cell lines tested were susceptible to reovirus, with > 50% of these cells lysed within 72 h of infection. In vivo neurotoxicity studies showed only mild local inflammation at the injection site and mild communicating hydrocephalus with neither diffuse encephalitis nor behavioral abnormalities at the therapeutically effective dose of reovirus (intracranial) (ie 10(7) plaque-forming units) or one dose level higher. In vivo, a single intratumoral administration of reovirus significantly reduced the size of tumors established from two human breast cancer cell lines and significantly prolonged survival. Intrathecal administration of reovirus also remarkably prolonged survival in an immunocompetent racine model of leptomeningeal metastases. These data suggest that the evaluation of reovirus as an experimental therapeutic for CNS metastases from breast cancer is warranted.

In order to disclose the mechanisms that regulate synapse development we compared the distributions of agrin, acetylcholine receptors (AChR) and a basal lamina heparan sulfate proteoglycan (HSPG) in sections and cultures prepared from Xenopus laevis and Ambystoma mexicanum embryos. While agrin, AChR and HSPG may accumulate almost synchronously at synapses in vivo , agrin deposition usually lagged well behind the other synaptic markers during development in culture, and was not detectable at many differentiated junctions. Agrin deposition at nerve-muscle contacts in culture also appeared to require the presence of other synaptic components. A similarly variable deposition occurred on noninnervated myocytes, where agrin again collected near sites of HSPG and AChR accumulation on some cells. Profuse agrin accretion occurred consistently, however, within the extracellular matrices of surrounding epithelial cells derived from both myotomes and neural tubes. In cocultures of Ambystoma neurons and Xenopus myocytes Ambystoma agrin collected at some chimeric neuromuscular junctions, but also accumulated on noninnervated myocytes and in the extracellular matrices of salamander neuroendothelial cells. Based upon these observations we conclude that (a) focal agrin deposition is not required for synaptic differentiation on Xenopus myocytes and (b) agrin may be one of several muscle basal lamina components that stem mainly from the secreted products of nearby epithelial cells.

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.

Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates.Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys.Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus' antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates.These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

Medulloblastoma (MB), the most common pediatric brain tumor, is a highly malignant disease with a 5-year survival rate of only 60%. Tumor cells invade surrounding tissue and disseminate through cerebral spinal fluid, making treatment difficult. Human reovirus type 3 exploits an activated Ras pathway in tumor cells to support productive infection as an oncolytic virus. Here, we examined the ability of human reovirus to kill MB cells lines and surgical specimens in vitro and inhibit tumor growth/metastases in vivo. Most human MB cell lines tested (five of seven = 71.4%), two MB cell lines derived from spontaneously arising tumors in Patched-1(+/-) mice (two of two = 100%) and three MB primary cultures derived from surgical specimens, were susceptible to reovirus infection. Reovirus was internalized and transcribed in both susceptible and resistant cell lines. However, viral protein synthesis was restricted to cell lines with higher levels of activated Ras, suggesting that Ras plays a critical role in reovirus oncolysis in MB. Using an in vivo Daoy orthotopic animal model, we found that a single i.t. injection of reovirus dramatically prolonged survival compared with controls (160 versus 70 days, respectively; P = 0.0003). Repeating this experiment with GFP-labeled Daoy cells and multiple i.t. administrations of reovirus, we again found prolonged survival and a dramatic reduction in spinal and leptomeningeal metastases (66.7% in control injections versus 0.0% in the live virus group). These data suggest that this oncolytic virus may be a potentially effective novel therapy against human MB. Its ability to reduce metastases to the spinal cord could allow a reduction in the dose/field of total neuroaxis cerebral-spinal radiotherapy currently used to treat/prevent cerebral spinal fluid dissemination.

Introduction: Multiple myeloma (MM) is a hematological malignancy of plasma cells and despite recent therapeutic advances that have increased the median survival rates of patients, is still currently felt to be incurable. We recently demonstrated the potential of reovirus as a novel therapeutic agent for several MM cell lines and ex vivo patient tumors. Therapy resistant disease however, still remains a problem. In the present study we demonstrate that reovirus acts synergistically with carfilzomib and/or perifisone 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 as well as reovirus sensitive RPMI8226 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 of toxicity was determined. Using Calcusyn software, combination index (CI) values were generated and synergism determined as per Chou-Talalay method. Results: Synergistic cytotoxicity was observed with reovirus and carfilzomib and perifisone in both reovirus resistant OPM2, KMS-11 or sensitive RPMI8226 cell lines. Interestingly, reovirus resistant cell lines demonstrated enhanced synergism in comparison to reovirus sensitive RPMI8226. Conclusion: Not all MM cell lines are amenable to reovirus mediated cell death. As phase II clinical trials for MM are currently underway this has important implications for the future use of reovirus as a therapeutic agent for cancer. The present study highlights the significance of preclinical studies in evaluating effective reovirus drug combinations for future use that could be extrapolated to a clinical setting. Currently we are also evaluating the effects of these drug combinations in vivo in a mouse model that represents the clinical scenario of MM. The mechanisms underlying the cytotoxicity mediated by reovirus and drug combinations 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: Chandini M. Thirukkumaran, Zhong Qiao Shi, Paola Neri, Nizar Bahlis, Don Morris. Reovirus synergy with proteosome inhibitor carfilzomib and Akt inhibitor perifisone overcomes therapy resistance of multiple myeloma: promising preclinical activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1709. doi:10.1158/1538-7445.AM2014-1709

To explore whether a neural modulation of muscle integrins' extracellular ligand interactions contributes to synapse induction, we compared the distributions of beta1-integrins and basal lamina proteins on Xenopus myotomal myocytes developing in culture. beta1-Integrins formed numerous organized aggregates scattered over the entire muscle surface, with particularly dense accumulations at specialized sites resembling myotendinous and neuromuscular junctions. Integrin aggregates on muscle cells differed from those on surrounding fibroblasts and epithelial cells, both in their lack of response to cross-linking by multivalent ligands and in their consistent association with the cells' own extracellular matrices. Muscle integrin clusters were usually associated with congruent basal lamina accumulations containing laminin and a heparan sulfate proteoglycan (HSPG), sometimes including fibronectin and vitronectin acquired from the surrounding medium. Immediately prior to synaptic differentiation, any existing laminin and HSPG accumulations along the path of cell contact were eliminated, disrupting otherwise stable laminin-integrin complexes. This apparently proteolytic modulation of integrins' extracellular ligand interactions was soon followed by the accumulation of new congruent accumulations of laminin and HSPG in the developing synaptic basal lamina. Combining these results with earlier findings, we consider the possibility that postsynaptic differentiation is induced, at least in part, by the proteolytic disruption of integrin-ligand complexes at sites of nerve-muscle contact.

Background Inhibitor of Growth (ING) proteins are epigenetic “readers” that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes to chromatin. Methods and Principal Findings Here we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo. Conclusions These data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.

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

Introduction: Multiple Myeloma (MM) is an incurable cancer that accounts for 10% of hematological malignancies. Stem cell rescue following high-dose chemotherapy with autologous or allogenic transplantation has become standard therapy for a subset of good performance patients. Previously we have shown that reovirus, a double stranded RNA virus can successfully purge the RPMI 8226 MM cell line, as well as a patient tumor specimen with no effect on stem cells (Thirukkumaran et al. 2004 : Blood. 102:377-387). In the present study we demonstrate 1) Efficient reovirus ex vivo purging of MM in a murine model of transplantation. 2) Reovirus purging does not affect human CD34+ stem cell repopulation or lineage differentiation. Methods: RPMI 8226-GFP MM cell line was admixed with human apheresis cells (AP) to represent 1% tumor burden. Admixed products were purged with live (LV) or UV-inactivated (DV) reovirus at a multiplicity of infection (MOI) of 40 for 72 hours. To assess purging efficacy and stem cell repopulating ability, SCID/NOD mice were sublethally irradiated and intraperitoneally injected with anti-asialo antibody to eradicate NK cells. Mice were injected intravenously with either live or dead virus purged admixed apheresis cells. Mice showing symptoms of disease were sacrificed and subjected to whole body fluorescence imaging using a GFP Imaging System. Bone marrow cells were collected from mouse femurs and analyzed for CD38+/138+/GFP+ MM cells. Human CD34+ stem cells were isolated from AP cells using Robosep (Stem Cell technologies) and exposed to LV or DV for 72 hours. LV (n=8) and DV (n=5) virus purged stem cells were mixed with sublethally irradiated (2Gy) AP cells (5X 105) and injected into irradiated SCID/NOD mice. Mouse blood and bone marrow was harvested at days 25 or 35 and assessed for human stem cells (CD34+/ 45+), T cells (45+/3+), B cells (19+/45+), NK cells(45+/3-/16+/33+) and myeloid (45+/13+/33+) cells. Results: 100% of LV purged animals (unlike DV purged) were devoid of MM and survived until being sacrificed arbitrarily at day 110 (Kaplan-Meier analysis: p< 0.0012). Both LV and DV purged human stem cells showed re-population in mice and lineage differentiation illustrating a lack of viral effect. Conclusions: The sensitivity of reovirus towards MM and its lack of effect on human stem cells highlight the potential of reovirus as an ex vivo purging agent during autologous hematopoietic stem cell transplants for MM. Currently, we are in the process of studying reovirus purging efficacy of ex vivo MM patient tumor in order to generate data for a future phase I clinical trial. 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 396.