Viral Replication In Normal Cells Research Articles

Ruxolitinib Rescues Mice from Lethal Interferon Beta Toxicity and TLS

Introduction. Oncolytic Vesicular Stomatitis Virus expressing human interferon beta and the sodium iodide symporter reporter gene (VSV-hIFNβ-NIS) is in Phase 1 clinical testing for systemic administration in patients with relapsed multiple myeloma, acute myeloid leukemia and T cell lymphoma (NCT03017820). Local production of interferon β enhances virotherapy by activating the immune system, and restricts viral replication in normal cells by activating IFN induced antiviral responses. Thus, the virus propagates efficiently in IFN non-responsive cancer cells but is constrained in IFN responsive cells. Cancer cells that are defective in their viral sensing and IFN response pathways are exquisitely sensitive to VSV-IFNβ-NIS oncolysis, cumulating in tumor lysis syndrome and IFNβ induced toxicity in the MPC-11 mouse plasmacytoma model. Here, we evaluated the feasibility of using ruxolitinib, a clinically approved Jak1/2 inhibitor, to rescue mice from virotherapy induced TLS and IFNβ toxicity.Methods. To characterize IFNβ toxicity in the absence of TLS , varying levels of mIFNβ were achieved in the blood of mice by intramuscular injection of an AAV1 vector encoding mIFNβ. For the virotherapy-associated TLS and IFNβ toxicity model, Balb/c mice with subcutaneous MPC-11 murine plasmacytoma were given one intravenous administration of VSV-mIFNβ-NIS. To determine whether the Jak/STAT inhibitor ruxolitonib could impact IFNβ toxicity, mice were treated with ruxolitinib (2 mg/dose by oral gavage) given twice a day for 10 days. All mice were monitored for adverse clinical signs andtumor size. Plasma IFNβ levels, CBC and comprehensive clinical chemistry panels were analyzed at necropsy.Results. There was a dose dependent increase in plasma IFNβ levels with increasing doses of AAV1-mIFNβ given intramuscularly to mice. Adverse clinical signs were seen in mice given high doses of AAV1-mIFNβ. There was a good correlation between IFNβ and AST or ALT levels. High circulating levels of IFNβ resulted in elevation in transaminases, thrombocytopenia, anemia and lymphopenia. IFN receptor defective mice were completely resistant to these toxic effects even with blood levels of >80,000 pg/ml IFNβ. Nontumor bearing Balb/c mice were shown to tolerate high IV doses of VSV-mIFNβ-NIS with transient lymphopenia and transaminases. In Balb/c mice with IFN nonresponsive MPC-11 tumors that support robust viral replication, VSV-mIFNβ-NIS therapy was highly effective for tumor control, but resulted in TLS and IFNβ toxicity, requiring early euthanasia of mice. Mice given VSV and a neutralizing anti-IFNR antibody did not show any adverse clinical signs due to IFNβ toxicity but control of tumor growth was significantly impaired in comparison to virus treatment alone. In contrast combination treatment of mice VSV and ruxolitinib resulted in reversal of lethal IFNβ toxicity without loss of effective tumor control.Conclusions. Our data demonstrate that ruxolitinib reverses the toxicity of IFNβ seen following highly efficacious VSV-IFNβ-NIS virotherapy in the MPC-11 plasmacytoma model. Clinical evaluation of this promising combination is warranted. [Display omitted] DisclosuresPeng:Vyriad: Equity Ownership; Imanis Life Sciences: Employment, Equity Ownership. Russell:Vyriad: Equity Ownership; Imanis Life Sciences: Equity Ownership.

Highly selective and potent p16-CDK-RB-E2F targeted oncolytic virus therapies.

e14543 Background: Growth factor and p16-CDK-RB-E2F pathway components are mutated in almost all cancers, resulting in oncogenic E2F transcriptional activity that drives uncontrolled proliferation. Chemotherapies indiscriminately kill all proliferating cells, resulting in limiting toxicities. CDK inhibitors are more targeted but cytostatic as opposed to tumoricidal. E2F was discovered as a factor that transcribes adenovirus (Ad) E2 and cellular genes to drive viral replication. Ad E1A binds to RB, which activates E2F. On this basis, viruses with E1A-RB binding mutations are being evaluated in the clinic as oncolytic viruses (OVs) that selectively replicate in and kill RB mutant tumor cells. However, E1A-RB mutations alone are not sufficient to prevent viral replication in normal cells. The addition of ‘tumor specific promoters’ improves tumor selectivity but drastically impacts replication and lytic activity. Therefore, a critical unmet need is the engineering of E2F addicted OVs that replicate like wildtype viruses in tumor cells, but leave normal cells unharmed. Methods: We have developed a proprietary platform that enables Ad therapeutics to be assembled from libraries of functional genomic parts that confer desirable properties. Here we describe E1 and E4 genomic modules that confer E2F dependent tumor selective replication. We assembled a panel of ̃50 viruses with combinations of mutations that ablate i) E1A interactions with RB/p107/p130 and/or EP300 ii) E4-ORF1 activation of PI3-Kinase and/or MYC iii) E4-ORF6/7 induced E2F1/DP1 dimerization. These viruses were screened in panels of primary, tumor, and CRISPR-engineered cells. Results: These studies reveal that E4-ORF6/7 functions independently of E1A to activate E2F transcriptional targets, S phase entry and viral replication. We show that viruses with both E1A and E4-ORF6/7 mutations (AdSyn-181) have the ideal tumor selectivity/efficacy profiles and outperform existing OVs. We also show that the deletion of p16 in primary cells rescues AdSyn-181 replication by ̃15 fold (̃20% wildtype virus), demonstrating the E2F dependence and tumor selectivity mechanism. Furthermore, we show that in tumor cells, which have multiple p16-CDK-RB pathway mutations, AdSyn-181 replication is rescued to 100% wildtype virus levels. ICVB-1042, which incorporates these selectivity mutations – together with other modules that enhance lytic cell death and spread, tumor tropisms and IV delivery – is in IND-enabling studies. Conclusions: Leveraging extensive research into the overlapping logic of viral and cancer pathways, we have engineered OVs, including ICVB-1042, that distinguish between tumor and normal cell proliferation. Furthermore, unlike therapies that target a single component, ICVB-1042 targets tumors with different p16-CDK-RB mutations, which all converge in activating oncogenic E2F transcription, driving exponential and tumoricidal viral replication.

Abstract 4576: Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects

Abstract The recombinant Vaccinia virus Copenhagen strain deleted in Thymidine Kinase and Ribonucleotide Reductase (VVCOPTK−RR−) is a potent and versatile oncolytic platform that has demonstrated strong activity in various preclinical models. The deletion of such VV genes inhibits viral replication in normal cells, while retaining therapeutic replication in tumor cells. TG6002, a VVCOPTK−RR− expressing the suicide gene FCU1, is under investigation in Phase I trials in patients with advanced gastrointestinal tumors. We have developed a new product based on the VVCOPTK−RR− vector, named TG6010, expressing the human cytidine deaminase (CDA) that efficiently catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. The tumor specific expression of CDA by the VV leads to a depletion of cytidine and deoxycytidine. This cytidine/deoxycytidine depletion resulting from CDA overexpression, activated a DNA damage response highlighted by an induction of H2AX phosphorylation. Next, to validate the potential therapeutic use of TG6010, we analyzed the effects of the virus on human xenograft tumors implanted in mice. We observed, after systemic injection of TG6010, high expression of CDA in the tumors with a significant increase in DNA damage in vivo as revealed by the γH2AX foci assay. In addition, intraperitoneal administration of TG6010 in a xenograft model of human ovarian carcinomatosis resulted in significantly prolonged animal survival. In conclusion, the oncolytic vaccinia virus expressing the cytidine deaminase (TG6010) has shown potent anti-tumor effects. Mechanistically, due to the cytidine deaminase overexpression, we observed induction of a DNA damage response. These are data encouraging of clinical development for this highly potent oncolytic Vaccinia virus TG6010 against ovarian cancer. Citation Format: Johann Foloppe, Juliette Kempf, Christelle Pichon, Annie Findeli, Isabelle Farine, Julie Hortelano, Baptiste Moreau, Michèle Klein, Sandrine Cochin, Renée Brandely, Véronique Koerper, Nathalie Silvestre, Christelle Machon, Jérôme Guitton, Eric Quéméneur, Philippe Erbs. Oncolytic vaccinia virus TG6010 expressing cytidine deaminase induces DNA damage and shows potent anti-tumor effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4576.

Abstract B23: microRNA attenuated oHSV-1 armed with multiple immunomodulatory payloads mediates robust and selective antitumor immune response in preclinical tumor models

Abstract Although oncolytic viruses have shown clinical efficacy for local treatment of cancerous tumors, the ability to induce immune-mediated regression to visceral lesions is not robust. Immune checkpoint modulation has been efficacious in a fraction of cancers associated with an inflamed microenvironment but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies has the exciting potential to be an effective cancer therapy. Oncolytic virus can be genetically modified to decrease pathogenicity to normal cells and to increase their lytic potential and ability to stimulate antitumor immunity, thus improving the risk-benefit ratio for cancer patients. Oncorus oHSV (ONCR) are novel oncolytic herpes simplex virus type-1 vectors developed for the treatment of solid tumors. ONCR utilizes a unique conditional-lethal strategy in which tissue-specific miRs binding sites are inserted into early genes essential for viral replication and in the neurovirulence gene ICP34.5. This strategy curtails viral replication in normal cells, including neurons, and limits the expression of g34.5 to tumor cells, thus promoting potent viral replication in presence of type I interferon. Fifteen immunostimulatory transgenes and combination thereof were systematically evaluated in dual flank syngeneic mouse tumor models to test both for oncolysis and abscopal efficacy on the contralateral noninjected tumor. Among the selected transgenes, IL-12 was found to elicit the most potent efficacy on the ipsilateral and contralateral tumors. We demonstrated that the replication of ONCR and the expression of IL-12 was limited to the injected tumors. By contrast, induction of systemic immunity as assessed by IFNg production in contralateral tumors and in plasma was observed. ONCR expressing IL-12 elicited efficacy in multiple subcutaneous and metastatic models. Additional payloads designed to stimulate antigen presentation, recruitment of immune cells and inhibition of tumor immune suppression were identified as further enhancing IL-12 activity. Data supporting the selection of a suite of payloads cloned into a single ONCR vector will be presented. ONCR represents a new class of oncolytic virus that promote antitumor responses through a multiprong mechanism of action dependent on selective tumor cell killing, the induction of systemic antitumor immunity and reversion of immune suppression. Citation Format: Lorena Lerner, Edward M. Kennedy, Terry Farkaly, Allison Colthart, Caity Montagna, Prajna Behera, Judith Jacques, Peter Grzesik, Jennifer Lee, Agnieszka Denslow, Jacqueline Gursha, Brian Haines, Michael Ball, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Michael Paglia, Laura Viggiano Salta, Lorenz Ponce, Caroline Webb, Mitchell Finer, Christophe Quéva. microRNA attenuated oHSV-1 armed with multiple immunomodulatory payloads mediates robust and selective antitumor immune response in preclinical tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B23.

JAK/STAT inhibition with ruxolitinib enhances oncolytic virotherapy in non-small cell lung cancer models.

The concept of using viruses to treat cancer has now shown proof of concept in several recent clinical trials, leading to the first FDA approval of virotherapy for melanoma last year. Vesicular stomatitis virus (VSV) is a promising oncolytic virus that has inhibitory effects on a number of cancer types including non-small cell lung cancer. One of the major mechanisms of resistance to VSV infection is the type I interferon (IFN) response, leading to the development of VSV expressing IFNβ which will lead to resistance of viral replication in normal cells which have intact IFN signaling but allow replication in cancer cells with defective IFNβ signaling. However, some cancer cells have intact or partially intact IFN signaling pathways leading to resistance to virotherapy. Here we utilized JAK/STAT inhibitor, ruxolitinib, in combination with VSV-IFNβ to see if inhibition of JAK/STAT signaling will enhance VSV-IFNβ therapy for lung cancer. We used five human and two murine NSCLC cell lines in vitro, and the combination treatment was assayed for cytotoxicity. We performed western blots on treated cells to see the effects of ruxolitinib on JAK/STAT signaling and PDL-1 expression in treated cells. Finally, the combination of VSV-IFNβ and ruxolitinib was tested in an immune competent murine model of NSCLC. Ruxolitinib enhanced virotherapy in resistant and sensitive NSCLC cells. The addition of ruxolitinib inhibited STAT1 phosphorylation and to a lesser extent STAT3 phosphorylation. Ruxolitinib treatment prevented NSCLC cells from enhancing PDL-1 expression in response to virotherapy. Combination ruxolitinib and VSV-IFNβ therapy resulted in a trend toward improved survival of mice without substantially effecting PDL-1 levels or levels of immune infiltration into the tumor. These results support further clinical evaluation of the combination of JAK/STAT inhibition with virotherapy.

Exploring the Possibility of Using Herpes Simplex Virus in Oncolytic Virotherapy of Multiple Myeloma

Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Approximately, 80,000 people have died of the disease in the United States and 25,000 new cases are registered every year. Majority of patients develop resistance to current therapeutic treatments and die within 5-10 years of diagnosis. Thus, need of novel therapeutic intervention is extremely urgent. Although the field of oncolytic virotherapy (OV) based on using viruses with natural or engineered tumor selective replication to intentionally infect and kill tumor cells has been extensively explored for the treatment of solid tumors, only few data are available for the treatment of hematopoietic malignancies. Our laboratory was one of the first to show that OV using Reovirus can be an effective therapeutic strategy for the treatment of MM in vitro and in MM patients. In this work we aim at exploring the possibility of using genetically engineered HSV1 (Herpes Simplex Virus) for the treatment of MM. HSV1 is an enveloped, double stranded DNA virus. Engineered HSV1 (HSVQ) has both copies of viral gene important in viral replication in normal cells viz., ICP 34.5 gene deleted and has one copy of GFP inserted into viral ICP6 gene. Such engineered virus has been used for cancer cell selective killing in preclinical and clinical studies for the treatment of several types of solid tumors including melanoma and glioblastoma multiforme. In this study, we investigated the biological and preclinical impact of HSVQ on MM cell in vitro and in vivo.Method: Recombinant HSVQ was amplified in African green monkey kidney epithelial Vero cells, purified by sucrose density gradient centrifugation and titrated by plaque assay on Vero cells. Several MM cell lines (MM1.S, U266, RPMI8226, L363, NIH-H929) were infected with HSVQ at Multiplicity of Infection (MOI) 0.01 to 5. Fluorescence microscopy and flow cytometry analysis were used to assess MM cell infectivity with the virus. RT-PCR was performed to detect presence of viral genome in MM cell lines. Viral replication assays were also performed. Cell proliferation and apoptotic assays including MTT Assay, Tryphan Blue exclusion test, LIVE/DEAD cell viability staining and Annexin/7-AAD assays were done to determine viability of virus infected MM cells. Western Blot analysis was carried out to determine endoplasmic reticulum (ER) stress response mediated by ERK, Hsp90, Bip/GRP78, Hsp40 and apoptosis in HSVQ treated MM cells. Total bone marrow (BM) cells obtained from MM patients were infected with HSVQ and multi parametric flow analysis was performed to determine infectivity and specific killing of CD138+ MM cells by the virus.To study in vivo anti-tumorigenic properties of HSVQ, 12.5 x106 GFP/Luc + MM1.S or NIH-H929 cells were subcutaneously injected into the right flank of 20 NOD-SCID mice. Two weeks after injection, mice with comparable size tumors were randomly divided (5 animal for each treatment group) and treated twice with 1x107PFU (Plaque Forming Unit) HSVQ for 2 weeks or with saline. Tumor growth was measured to determine anti tumorigenic effect of HSVQ on MM tumors.Results and Conclusion: Fluorescence microscopy and flow cytometry revealed that MM cell lines can be effectively infected with and killed by HSVQ even at MOI as low as 0.1. Under such conditions, Western Blot analysis revealed increased BAX expression, decreased BCL2 expression and cleavage of Caspase 3 and PARP indicating apoptosis of virus infected cells. Interestingly, multi parametric flow analysis revealed that HSVQ specifically infects and kills CD138+ MM plasma cells in a total population of BM cellular fraction isolated from MM patients. Moreover in vivo preclinical data show that HSVQ dramatically reduces tumor volume (p<0.001) in both MM.1S and NIH-H929 xenograft mouse models. Thus, from the preliminary observations, it can be concluded that HSVQ can selectively infect and induce apoptosis in myeloma cells. Mechanisms of HSVQ replication in MM cells and induced MM cell killing are being currently investigated. Here for the first time we are providing clear evidences that HSVQ can infect and specifically kill MM cells supporting the idea of the use of HSV for the treatment of MM. Moreover, since the backbone of HSVQ can be further engineered, it can be used to specifically deliver anti-angiogenic and anti-inflammatory genes to MM cells for the treatment of MM. DisclosuresHofmeister:Arno Therapeutics, Inc.: Research Funding; Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Incyte, Corp: Membership on an entity’s Board of Directors or advisory committees; Janssen: Pharmaceutical Companies of Johnson & Johnson: Research Funding; Signal Genetics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Takeda Pharmaceutical Company: Research Funding; Teva: Membership on an entity’s Board of Directors or advisory committees.

647. Evaluation of Dose, Scheduling, Combination with Chemotherapy and Specificity of Ad5/3 Fiber-Chimeric Oncolytic Adenoviruses for Treatment of Ovarian Cancer

Conditionally replicating adenoviruses (CRAds) represent a potent approach for treatment of neoplastic diseases. Replication of CRAds can be restricted to cancer cells in two different ways: limit the expression of viral E1A to tumor tissues with tumor-specific promoters or engineer deletions in genes critical for viral replication in normal cells but not in tumor cells. Gene therapy applications are usually based on Ad serotype 5, which binds to the coxsackie- adenovirus receptor (CAR). However, expression of CAR is frequently dysregulated in many types of advanced cancers. Substituting the knob domain of Ad5 with the corresponding domain of serotype 3 allows CAR-independent binding and entry through the Ad3 receptor, which is expressed to high degree on ovarian cancer cells.

Elucidation of the Molecular Mechanism Underlying Tumor-Selective Replication of the Oncolytic Adenovirus Mutant Onyx-015

Tumor-selective replicating viruses offer appealing advantages over conventional cancer therapy. ONYX-015 (dl1520) is the prototype for oncolytic adenoviral therapy. It has undergone extensive clinical testing with proven safety and evidence of promising clinical efficacy. The strategy underlying its tumor-selective cell killing is based on deletion of the viral E1B-55K gene, which is crucial for efficient viral replication in normal cells but dispensable in tumor cells. Originally, the successful replication of ONYX-015 was thought to strictly depend on deregulated p53 signaling in tumor cells. However, recent preclinical as well as clinical evidence questions this mechanism. The study by O'Shea and colleagues is of immense importance as it sheds new light into the molecular mechanism underlying the tumor-selective replication of ONYX-015. Based on these findings, modulation of the proposed molecular mechanism by pharmacologic agents or hyperthermia may largely enhance the therapeutic index of ONYX-015 for tumor cells versus normal tissue and improve clinical efficacy. Finally, new strategies to allow successful patient stratification for future clinical trials appear to be in reach, based on the reported results.

Adenoviral E1a expression levels affect virus-selective replication in human cancer cells

One of the promising strategies for targeting replication of oncolytic adenovirus in tumor cells is to regulate the expression of essential viral genes such as E1a by using tumor- or tissue-specific promoters that are preferentially active in cancer cells. However, this approach may lead to some degree of viral replication in normal cells other than in cancer cells if the viral gene also expresses in normal cells. In this study, we investigated the effect of E1a expression levels on the virus replication ability in human cells. Three vectors, all with mutated E1B55K, were created, one without any promoter controlling the E1a gene and two vectors with the E1a gene being controlled by either its endogenous promoter or a strong CMV promoter. We observed that the CMV promoter-mediated high levels of E1A expression could increase virus replication, resulting in the titers of the E1B55K-mutated virus being even higher than the wild-type virus in some cancer cells. However, the strong CMV promoter could not always enhance virus replication, such as in cancer cells OE33 and OsACL. The results suggest that whether increased E1A levels would enhance E1B55K-mutated virus replication may be also depended on cellular factors or pathways in cancer cells. We also observed that the virus without any promoter for the E1a gene could still express leaky levels of E1A which can lead to viral replication in normal and cancer cells. Future efforts in the development of transcription-controlled oncolytic adenoviruses should focus on how to completely block E1a expression in normal cells.

Survivin-Responsive Conditionally Replicating Adenovirus Exhibits Cancer-Specific and Efficient Viral Replication

Although a conditionally replicating adenovirus (CRA) exhibiting cancer-selective replication and induction of cell death is an innovative potential anticancer agent, current imperfections in cancer specificity and efficient viral replication limit the usefulness of this technique. Here, we constructed survivin-responsive CRAs (Surv.CRAs), in which expression of the wild-type or mutant adenoviral early region 1A (E1A) gene is regulated by the promoter of survivin, a new member of the inhibitor of apoptosis gene family. We explored the cancer specificity and effectiveness of viral replication of Surv.CRAs, evaluating their potential as a treatment for cancer. The survivin promoter was strongly activated in all cancers examined at levels similar to or even higher than those seen for representative strong promoters; in contrast, low activity was observed in normal cells. Surv.CRAs efficiently replicated and potently induced cell death in most types of cancer. In contrast, minimal viral replication in normal cells did not induce any detectable cytotoxicity. A single injection of Surv.CRAs into a preestablished tumor expressing survivin, even at relatively low levels, induced significant tumor death and inhibition of tumor growth. Furthermore, Surv.CRAs were superior to telomerase-dependent CRAs, one of the most effective CRAs that have been examined to date, both in terms of cancer specificity and efficiency. Thus, Surv.CRAs are an attractive potential anticancer agent that could effectively and specifically treat a variety of cancers.