Jessica G. Tong

Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

BackgroundThe identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5) that blocks a unique late stage of the HIV-1 life cycle.ResultsHERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV) Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus.ConclusionsHERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.

BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Tumor cells have unstable genomes relative to non‐tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti‐cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2‐targeting second‐generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non‐tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti‐cancer agent.

Myxoma virus-mediated oncolysis of ascites-derived human ovarian cancer cells and spheroids is impacted by differential AKT activity

OBJECTIVE We propose that metastatic epithelial ovarian cancer (EOC) is a potential therapeutic target for the oncolytic agent, Myxoma virus (MYXV). METHODS Primary EOC cells were isolated from patient ascites and cultured as adherent cells or in suspension using Ultra Low-Attachment dishes. MYXV expressing green fluorescent protein was used to infect cells and spheroids. Infection was monitored by fluorescence microscopy, viral titering and immunoblotting for M-T7 and M130 virus protein expression, and cell viability by alamarBlue assay. Akti-1/2 (5 μM) and rapamycin (20 nM) were used to assay the role of PI3K-AKT signaling in mediating MYXV infection. RESULTS Ascites-derived EOC cells grown in adherent culture are effectively killed by MYXV infection. EOC cells grown in suspension to form three-dimensional EOC spheroids readily permit MYXV entry into cells, yet are protected from the cytopathic effects of late MYXV infection. Upon reattachment (to model secondary metastasis), EOC spheroids are re-sensitized to MYXV-mediated oncolysis. The critical determinant that facilitates efficient MYXV infection is the presence of an activated PI3K-AKT signaling pathway. Treatment with the specific AKT inhibitor Akti-1/2 reduces infection of monolayer EOC cells and spheroids. Direct infection of freshly-collected ascites demonstrated that 54.5% of patient samples were sensitive to MYXV-mediated oncolytic cell killing. We also demonstrate that factor(s) present in ascites may negatively impact MYXV infection and oncolysis of EOC cells, which may be due to a down-regulation in endogenous AKT activity. CONCLUSIONS Differential activity of AKT serves as the mechanistic basis for regulating MYXV-mediated oncolysis of EOC spheroids during key steps of the metastatic program. In addition, we provide the first evidence that MYXV oncolytic therapy may be efficacious for a significant proportion of ovarian cancer patients with metastatic disease.

Evidence for Differential Viral Oncolytic Efficacy in an In Vitro Model of Epithelial Ovarian Cancer Metastasis

Epithelial ovarian cancer is unique among most carcinomas in that metastasis occurs by direct dissemination of malignant cells traversing throughout the intraperitoneal fluid. Accordingly, we test new therapeutic strategies using an in vitro three-dimensional spheroid suspension culture model that mimics key steps of this metastatic process. In the present study, we sought to uncover the differential oncolytic efficacy among three different viruses—Myxoma virus, double-deleted vaccinia virus, and Maraba virus—using three ovarian cancer cell lines in our metastasis model system. Herein, we demonstrate that Maraba virus effectively infects, replicates, and kills epithelial ovarian cancer (EOC) cells in proliferating adherent cells and with slightly slower kinetics in tumor spheroids. Myxoma virus and vaccinia viruses infect and kill adherent cells to a much lesser extent than Maraba virus, and their oncolytic potential is almost completely attenuated in spheroids. Myxoma virus and vaccinia are able to infect and spread throughout spheroids, but are blocked in the final stages of the lytic cycle, and oncolytic-mediated cell killing is reactivated upon spheroid reattachment. Alternatively, Maraba virus has a remarkably reduced ability to initially enter spheroid cells, yet rapidly infects and spreads throughout spheroids generating significant cell killing effects. We show that low-density lipoprotein receptor expression in ovarian cancer spheroids is reduced and this controls efficient Maraba virus binding and entry into infected cells. Taken together, these results are the first to implicate the potential impact of differential viral oncolytic properties at key steps of ovarian cancer metastasis.

Interferon-induced HERC5 is evolving under positive selection and inhibits HIV-1 particle production by a novel mechanism targeting Rev/RRE-dependent RNA nuclear export

BackgroundType I interferon (IFN) inhibits virus replication by activating multiple antiviral mechanisms and pathways. It has long been recognized that type I IFNs can potently block HIV-1 replication in vitro; as such, HIV-1 has been used as a system to identify and characterize IFN-induced antiviral proteins responsible for this block. IFN-induced HERC5 contains an amino-terminal Regulator of Chromosome Condensation 1 (RCC1)-like domain and a carboxyl-terminal Homologous to the E6-AP Carboxyl Terminus (HECT) domain. HERC5 is the main cellular E3 ligase that conjugates the IFN-induced protein ISG15 to proteins. This E3 ligase activity was previously shown to inhibit the replication of evolutionarily diverse viruses, including HIV-1. The contribution of the RCC1-like domain to the antiviral activity of HERC5 was previously unknown.ResultsIn this study, we showed that HERC5 inhibits HIV-1 particle production by a second distinct mechanism that targets the nuclear export of Rev/RRE-dependent RNA. Unexpectedly, the E3 ligase activity of HERC5 was not required for this inhibition. Instead, this activity required the amino-terminal RCC1-like domain of HERC5. Inhibition correlated with a reduction in intracellular RanGTP protein levels and/or the ability of RanGTP to interact with RanBP1. Inhibition also correlated with altered subcellular localization of HIV-1 Rev. In addition, we demonstrated that positive evolutionary selection is operating on HERC5. We identified a region in the RCC1-like domain that exhibits an exceptionally high probability of having evolved under positive selection and showed that this region is required for HERC5-mediated inhibition of nuclear export.ConclusionsWe have identified a second distinct mechanism by which HERC5 inhibits HIV-1 replication and demonstrate that HERC5 is evolving under strong positive selection. Together, our findings contribute to a growing body of evidence suggesting that HERC5 is a novel host restriction factor.

Oncolytic virotherapy for ovarian cancer

In the past two decades, more than 20 viruses with selective tropism for tumor cells have been developed as oncolytic viruses (OVs) for treatments of a variety of malignancies. Of these viruses, eleven have been tested in human ovarian cancer models in preclinical studies. So far, nine phase I or II clinical trials have been conducted or initiated using four different types of OVs in patients with recurrent ovarian cancers. In this article, we summarize the different OVs that are being assessed as therapeutics for ovarian cancer. We also present an overview of recent advances in identification of key genetic or immune-response pathways involved in tumorigenesis of ovarian cancer, which provides a better understanding of the tumor specificities and oncolytic properties of OVs. In addition, we discuss how next-generation OVs could be genetically modified or integrated into multimodality regimens to improve clinical outcomes based on recent advances in ovarian cancer biology.

Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential

BackgroundEpithelial ovarian cancer exhibits extensive interpatient and intratumoral heterogeneity, which can hinder successful treatment strategies. Herein, we investigated the efficacy of an emerging oncolytic, Maraba virus (MRBV), in an in vitro model of ovarian tumour heterogeneity.MethodsFour ovarian high-grade serous cancer (HGSC) cell lines were isolated and established from a single patient at four points during disease progression. Limiting-dilution subcloning generated seven additional subclone lines to assess intratumoral heterogeneity. MRBV entry and oncolytic efficacy were assessed among all 11 cell lines. Low-density receptor (LDLR) expression, conditioned media treatments and co-cultures were performed to determine factors impacting MRBV oncolysis.ResultsTemporal and intratumoral heterogeneity identified two subpopulations of cells: one that was highly sensitive to MRBV, and another set which exhibited 1000-fold reduced susceptibility to MRBV-mediated oncolysis. We explored both intracellular and extracellular mechanisms influencing sensitivity to MRBV and identified that LDLR can partially mediate MRBV infection. LDLR expression, however, was not the singular determinant of sensitivity to MRBV among the HGSC cell lines and subclones. We verified that there were no apparent extracellular factors, such as type I interferon responses, contributing to MRBV resistance. However, direct cell-cell contact by co-culture of MRBV-resistant subclones with sensitive cells restored virus infection and oncolytic killing of mixed population.ConclusionsOur data is the first to demonstrate differential efficacy of an oncolytic virus in the context of both spatial and temporal heterogeneity of HGSC cells and to evaluate whether it will constitute a barrier to effective viral oncolytic therapy.

Abstract 1687: A BRCA2-targeting antisense oligodeoxynucleotide enhances cisplatin effectiveness by decreasing human tumor cell proliferation, metastatic frequency, and metabolic response

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BRCA2 is involved in homologous recombination repair of double-stranded DNA breaks in human cells. Inactivating mutations in BRCA2 predispose to early onset cancer of the breast, ovary and other tissues. However, patients with tumors that harbor BRCA2 mutations respond better to cancer therapy. Therefore, reducing BRCA2 in cancer cells capable of homologous recombination repair may sensitize otherwise resistant tumors to DNA-damaging anti-cancer treatment. We developed a second generation antisense oligodeoxynucleotide (BR-1) that specifically targets BRCA2. BR-1 decreased BRCA2 mRNA and protein and inhibited BRCA2-mediated RAD51 repair focus formation. BR-1 potently sensitized A549 (lung), SKOV-3 (ovarian), and MDA-MB-231 (breast) cells to cisplatin as evidenced by decreased cell proliferation. However, non-cancerous HK-2 kidney cells were not sensitized to cisplatin by BR-1. In A549 cells BR-1 enhanced cisplatin or ionizing radiation-induced inhibition of in vitro colony formation. Cisplatin-resistant head and neck squamous cancer cells (HN-15a) were rendered as sensitive to cisplatin following BR-1 treatment as the parent population. In addition, BR-1 plus cisplatin treatment decreased A549 cell metastatic frequency in an in vivo chicken chorio-allantoic membrane (CAM) model by over 70% when compared with drug treatment alone. Treatment with BR-1 and cisplatin decreased cellular respiration (decreased oxygen consumption) compared to control oligonucleotide plus cisplatin treatment, suggesting that downregulation of BRCA2 in the context of platinating drug treatment alters cellular metabolism. This change in respiration occurred independently of changes in mitochondrial number. We are continuing to develop and test BR-1 with the aim of applying it in an adjuvant setting with cisplatin in human clinical trials in the future. Supported by grants from the Ontario Centres of Excellence (OCE) Citation Format: Mateusz Rytelewski, Jessica Tong, Adrian Buensuceso, Hon Leong, Peter Ferguson, Saman Maleki Vareki, Christine Di Cresce, Larissa Romanow, Trevor Shepherd, Bonnie Deroo, Ann Chambers, Mark Vincent, James Koropatnick. A BRCA2-targeting antisense oligodeoxynucleotide enhances cisplatin effectiveness by decreasing human tumor cell proliferation, metastatic frequency, and metabolic response. [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 1687. doi:10.1158/1538-7445.AM2014-1687

Abstract C77: A novel BRCA2 targeting antisense oligonucleotide sensitizes human tumor cells to chemotherapy and radiotherapy - the induction of ‘complementary lethality’ by targeting DNA repair.

BRCA2 is a protein involved in homologous recombination repair of double-stranded DNA breaks in human cells. Inactivating mutations in BRCA2 predispose to early onset cancer of the breast, ovary and other tissues. However, patients with tumors that harbour BRCA2 mutations respond more favourably to cancer therapy. Therefore, inhibiting BRCA2 function in cancer cells capable of homologous recombination repair may sensitize otherwise resistant tumors to distinct types of anti-cancer treatment. We have reported that BRCA2 siRNA sensitizes human tumor cells to specific DNA-damaging agents (e.g., cisplatin), but not anti-folate drugs (e.g., pemetrexed), a phenomenon we termed ‘complementary lethality’. Based on these results, we developed and are testing a second generation antisense oligodeoxynucleotide (BR-1) that specifically targets BRCA2. BR-1 decreased BRCA2 mRNA and protein levels, and also inhibited BRCA2-mediated RAD51 repair focus formation. BR-1 potently sensitized human non-small cell lung cancer (A549) cells to cisplatin, melphalan, and carboplatin as evidenced by reduced overall proliferation. In addition, A549 cells exhibited reduced ability to form colonies after concomitant treatment with BR-1 and cisplatin, melphalan, or ionizing radiation. Furthermore, cisplatin-resistant, patient-derived head and neck squamous cancer cells (HN-5a) were rendered sensitive to cisplatin following BR-1 treatment, and the level of cisplatin sensitivity was comparable to that of the parent population. Interestingly, treatment with BR-1 and cisplatin significantly decreased cellular respiration compared to control oligonucleotide and cisplatin treatment, suggesting that downregulation of BRCA2 in the context of alkylating drug treatment alters cellular metabolism. However, this change in respiration occurred independently of mitochondrial integrity. BR-1 is currently being tested in vivo to determine its ability to downregulate BRCA2 in human xenografts and sensitize solid tumors to chemotherapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C77. Citation Format: Mateusz Rytelewski, Jessica Tong, Adrian Buensucesco, Saman Maleki Vareki, Peter J. Ferguson, Rene Figueredo, Mark Vincent, Trevor Shepherd, Bonnie J. Deroo, James D. Koropatnick. A novel BRCA2 targeting antisense oligonucleotide sensitizes human tumor cells to chemotherapy and radiotherapy - the induction of ‘complementary lethality’ by targeting DNA repair. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C77.

Cellular Restriction Factors: Exploiting the Body’s Antiviral Proteins to Combat HIV-1/AIDS

1.1 Overview of HIV-1/AIDS therapies In 1983, when researchers first isolated HIV-1 from an AIDS patient, few imagined that it foretold a worldwide pandemic (Broder & Gallo, 1984, Barre-Sinoussi et al., 1983). More than 25 years later, 65 million people have been infected with HIV-1; nearly half of these people have died of AIDS, and despite many scientific advances we are still without an efficacious vaccine (Merson, 2006). The majority of HIV-1 infections and deaths have occurred in developing countries, with sub-Saharan Africa accounting for over 38 million HIV-1 infections alone. Sadly, the number of new infections currently exceeds our ability to treat everyone infected with the virus, and in the hardest-hit countries the social and economic backlash has been profound. After HIV-1 was isolated, a blood test to screen patients and the blood supply quickly followed, as did research on its structure and pathogenesis. Many assumed that a vaccine would be available in a few years, and excitement increased further with the licensing of the first effective drug against HIV-1, zidovudine (AZT) (Fischl et al., 1987). However, researchers soon discovered that the virus was highly resilient, and HIV-1 quickly developed resistance to AZT (Poli et al., 1989, Richman et al., 1994). Over the next few years, a number of new antiretroviral drugs were developed that attacked the virus in different ways, and it was at this time that a new approach to therapy ensued. Highly active antiretroviral therapy (HAART) combined three or more different drugs to reduce HIV-1 replication, and significantly improved the prognosis of HIV-1-infected individuals (Richman et al., 2009). However, HAART was not a cure, and many patients were resistant to at least one of the antiretroviral drugs. In addition, the drugs were highly toxic, making adherence to treatment difficult. During this time, the vaccine field was also hard at work, trying to develop a safe and effective HIV-1 vaccine. Most initial vaccine approaches focused on the HIV-1 envelope protein (gp120), and aimed to induce an antibody response to gp120. AIDSVAX was the first vaccine candidate of this type, and was developed by a U.S. pharmaceutical company called VaxGen (Flynn et al., 2005, Pitisuttithum et al., 2006). An alternative approach, developed by Merck, aimed to induce a T-cell response to HIV-1 using a recombinant adenovirus vector expressing HIV-1 Gag, Pol and Nef proteins (Shiver et al., 2002). Unfortunately, results from