Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.

A role for macrophages under cytokine control in mediating resistance to ADI-PEG20 (pegargiminase) in ASS1-deficient mesothelioma.

BACKGROUND: Pegylated arginine deiminase (ADI-PEG20; pegargiminase) depletes arginine and improves survival outcomes for patients with argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). Optimisation of ADI-PEG20-based therapy will require a deeper understanding of resistance mechanisms, including those mediated by the tumor microenvironment. Here, we sought to reverse translate increased tumoral macrophage infiltration in patients with ASS1-deficient MPM relapsing on pegargiminase therapy. METHODS: Macrophage-MPM tumor cell line (2591, MSTO, JU77) co-cultures treated with ADI-PEG20 were analyzed by flow cytometry. Microarray experiments of gene expression profiling were performed in ADI-PEG20-treated MPM tumor cells, and macrophage-relevant genetic "hits" were validated by qPCR, ELISA, and LC/MS. Cytokine and argininosuccinate analyses were performed using plasma from pegargiminase-treated patients with MPM. RESULTS: We identified that ASS1-expressing macrophages promoted viability of ADI-PEG20-treated ASS1-negative MPM cell lines. Microarray gene expression data revealed a dominant CXCR2-dependent chemotactic signature and co-expression of VEGF-A and IL-1α in ADI-PEG20-treated MPM cell lines. We confirmed that ASS1 in macrophages was IL-1α-inducible and that the argininosuccinate concentration doubled in the cell supernatant sufficient to restore MPM cell viability under co-culture conditions with ADI-PEG20. For further validation, we detected elevated plasma VEGF-A and CXCR2-dependent cytokines, and increased argininosuccinate in patients with MPM progressing on ADI-PEG20. Finally, liposomal clodronate depleted ADI-PEG20-driven macrophage infiltration and suppressed growth significantly in the MSTO xenograft murine model. CONCLUSIONS: Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers.

Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

Oncolytic adenoviruses (OAd) can be employed to efficiently eliminate cancer cells through multiple mechanisms of action including cell lysis and immune activation. Our OAds, AdΔΔ and Ad-3∆-A20T, selectively infect, replicate in, and kill adenocarcinoma cells with the added benefit of re-sensitising drug-resistant cells in preclinical models. Further modifications are required to enable systemic delivery in patients due to the rapid hepatic elimination and neutralisation by blood factors and antibodies. Here, we show data that support the use of coating OAds with gold nanoparticles (AuNPs) as a possible new method of virus modification to help augment tumour uptake. The pre-incubation of cationic AuNPs with AdΔΔ, Ad-3∆-A20T and wild type adenovirus (Ad5wt) was performed prior to infection of prostate/pancreatic cancer cell lines (22Rv, PC3, Panc04.03, PT45) and a pancreatic stellate cell line (PS1). Levels of viral infection, replication and cell viability were quantified 24-72 h post-infection in the presence and absence of AuNPs. Viral spread was assessed in organotypic cultures. The presence of AuNPs significantly increased the uptake of Ad∆∆, Ad-3∆-A20T and Ad5wt in all the cell lines tested (ranging from 1.5-fold to 40-fold), compared to virus alone, with the greatest uptake observed in PS1, a usually adenovirus-resistant cell line. Pre-coating the AdΔΔ and Ad-3∆-A20T with AuNPs also increased viral replication, leading to enhanced cell killing, with maximal effect in the most virus-insensitive cells (from 1.4-fold to 5-fold). To conclude, the electrostatic association of virus with cationic agents provides a new avenue to increase the dose in tumour lesions and potentially protect the virus from detrimental blood factor binding. Such an approach warrants further investigation for clinical translation.

Targeting Triple Negative Breast Cancer With Oncolytic Adenoviruses.

Breast cancer (BC) is the most common cancer globally, accounting for 685,000 deaths in 2020. Triple-negative breast cancers (TNBC) lack oestrogen (ER) and progesterone (PR) hormone receptor expression and HER2 overexpression. TNBC represent 10-15% of all BC with high incidence in women under 50-years old that have BRCA mutations, and have a dismal prognosis. African American and Hispanic women are at higher risk partly due to the common occurrence of BRCA mutations. The standard treatment for TNBC includes surgery, radiotherapy, and chemotherapy although, resistance to all standard-of-care therapies eventually develops. It is crucial to identify and develop more efficacious therapeutics with different mechanisms of action to improve on survival in these women. Recent findings with oncolytic adenoviruses (OAds) may generate a new strategy to improve on the outcomes for women afflicted by TNBC and other types of BC. OAds are genetically engineered to selectively lyse, eliminate and recruit the host antitumour immune responses, leaving normal cells unharmed. The most common modifications are deletions in the early gene products including the E1B55 KDa protein, specific regions of the E1A protein, or insertion of tumour-specific promoters. Clinical trials using OAds for various adenocarcinomas have not yet been sufficiently evaluated in BC patients. Preclinical studies demonstrated efficacy in BC cell lines, including TNBC cells, with promising novel adenoviral mutants. Here we review the results reported for the most promising OAds in preclinical studies and clinical trials administered alone and in combination with current standard of care or with novel therapeutics. Combinations of OAds with small molecule drugs targeting the epidermal growth factor receptor (EGFR), androgen receptor (AR), and DNA damage repair by the novel PARP inhibitors are currently under investigation with reported enhanced efficacy. The combination of the PARP-inhibitor Olaparib with OAds showed an impressive anti-tumour effect. The most promising findings to date are with OAds in combination with antibodies towards the immune checkpoints or expression of cytokines from the viral backbone. Although safety and efficacy have been demonstrated in numerous clinical trials and preclinical studies with cancer-selective OAds, further developments are needed to eliminate metastatic lesions, increase immune activation and intratumoural viral spread. We discuss shortcomings of the OAds and potential solutions for improving on patient outcomes.

HDAC Inhibitors Enhance Efficacy of the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T in Pancreatic and Triple-Negative Breast Cancer Models.

The prognosis for triple-negative breast cancer (TNBC) and pancreatic ductal adenocarcinoma (PDAC) is dismal. TNBC and PDAC are highly aggressive cancers with few treatment options and a potential for rapid resistance to standard-of-care chemotherapeutics. Oncolytic adenoviruses (OAds) represent a promising tumour-selective strategy that can overcome treatment resistance and eliminate cancer cells by lysis and host immune activation. We demonstrate that histone deacetylase inhibitors (HDACi) potently enhanced the cancer-cell killing of our OAds, Ad∆∆ and Ad-3∆-A20T in TNBC and PDAC preclinical models. In the TNBC cell lines MDA-MB-436, SUM159 and CAL51, cell killing, viral uptake and replication were increased when treated with sublethal doses of the Class-I-selective HDACis Scriptaid, Romidepsin and MS-275. The pan-HDACi, TSA efficiently improved OAd efficacy, both in vitro and in SUM159 xenograft models in vivo. Cell killing and Ad∆∆ replication was also significantly increased in five PDAC cell lines when pre-treated with TSA. Efficacy was dependent on treatment time and dose, and on the specific genetic alterations in each cell line. Expression of the cancer specific αvß6-integrin supported higher viral uptake of the integrin-retargeted Ad-3∆-A20T in combination with Scriptaid. In conclusion, we demonstrate that inhibition of specific HDACs is a potential means to enhance OAd activity, supporting clinical translation.

Coating an adenovirus with functionalized gold nanoparticles favors uptake, intracellular trafficking and anti-cancer therapeutic efficacy.

Adenoviral (Ad) vectors have proven to be important tools for gene and cell therapy, although some issues still need to be addressed, such as undesired interactions with blood components and off-target sequestration that ultimately hamper efficacy. In the past years, several organic and inorganic materials have been developed to reduce immunogenicity and improve biodistribution of Ad vectors. Here we investigated the influence of the functionalization of 14 nm PEGylated gold nanoparticles (AuNPs) with quaternary ammonium groups and an arginine-glycine-aspartic acid (RGD)-motif on the uptake and biodistribution of Ad vectors. We report the formation of Ad@AuNPs complexes that promote cell attachment and uptake, independently of the presence of the coxsackievirus and adenovirus receptor (CAR) and αvß3 and αvß5 integrins, significantly improving transduction without limiting Ad bioactivity. Besides, the presence of the RGD peptide favors tumor targeting and decreases Ad sequestration in the liver. Additionally, tumor delivery of a coated Ad vector expressing the human sodium iodide symporter (hNIS) by mesenchymal stem cells induces increased accumulation of radioactive iodine (131I) and tumor volume reduction compared to naked Ad-hNIS, highlighting the promising potential of our coating formulation in cancer gene therapy. STATEMENT OF SIGNIFICANCE: Modification of adenoviral vectors with lipids and polymers can reduce interactions with blood components and increase tumor accumulation; however, increased toxicity and reduced transduction efficiency were indicated. Coating with gold nanoparticles has proven to be a successful strategy for increasing the efficiency of transduction of receptor-defective cell lines. Here we explore the contribution of cell surface receptors on the mechanisms of entry of Ad vectors coated with gold nanoparticles in cell lines with varying degrees of resistance to infection. The enhancement of the anti-tumoral effect shown in this work provides new evidence for the potential of our formulation.

Systemic delivery and SPECT/CT in vivo imaging of 125I-labelled oncolytic adenoviral mutants in models of pancreatic cancer.

Early phase clinical trials have demonstrated good therapeutic index for oncolytic adenoviruses in patients with solid tumours when administered intratumorally, resulting in local tumour elimination. Entrapment and binding of adenovirus to erythrocytes, blood factors, and neutralising antibodies have prevented efficient systemic delivery and targeting of distant lesions in the clinic. We previously generated the novel replication-selective Ad-3∆-A20T to improve tumour targeting by increasing the viral dose at distant sites. Here, we developed a protocol to directly radiolabel the virus for rapid and sensitive detection by single-photon emitted computed tomography (SPECT/CT) providing a convenient method for determining biodistribution following intravenous administration in murine models. Longitudinal whole-body scans, demonstrated efficient viral uptake in pancreatic Suit-2 and Panc04.03 xenografts with trace amounts of 125I-Ad-3∆-A20T up to 48 h after tail vein delivery. Hepatic and splenic radioactivity decreased over time. Analysis of tissues harvested at the end of the study, confirmed potency and selectivity of mutant viruses. Ad-3∆-A20T-treated animals showed higher viral genome copy numbers and E1A gene expression in tumors than in liver and spleen compared to Ad5wt. Our direct radiolabeling approach, allows for immediate screening of novel oncolytic adenoviruses and selection of optimal viral genome alterations to generate improved mutants.

Advances in oncolytic adenovirus therapy for pancreatic cancer.

Survival rates for pancreatic cancer patients have remained unchanged for the last four decades. The most aggressive, and most common, type of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), which has the lowest 5-year survival rate of all cancers globally. The poor prognosis is typically due to late presentation of often non-specific symptoms and rapid development of resistance to all current therapeutics, including the standard-of-care cytotoxic drug gemcitabine. While early surgical intervention can significantly prolong patient survival, there are few treatment options for late-stage non-resectable metastatic disease, resulting in mostly palliative care. In addition, a defining feature of pancreatic cancer is the immunosuppressive and impenetrable desmoplastic stroma that blocks access to tumour cells by therapeutic drugs. The limited effectiveness of conventional chemotherapeutics reveals an urgent need to develop novel therapies with different mechanisms of action for this malignancy. An emerging alternative to current therapeutics is oncolytic adenoviruses; these engineered biological agents have proven efficacy and tumour-selectivity in preclinical pancreatic cancer models, including models of drug-resistant cancer. Safety of oncolytic adenoviral mutants has been extensively assessed in clinical trials with only limited toxicity to normal healthy tissue being reported. Promising efficacy in combination with gemcitabine was demonstrated in preclinical and clinical studies. A recent surge in novel adenoviral mutants entering clinical trials for pancreatic cancer indicates improved efficacy through activation of the host anti-tumour responses. The potential for adenoviruses to synergise with chemotherapeutics, activate anti-tumour immune responses, and contribute to stromal dissemination render these mutants highly attractive candidates for improved patient outcomes. Currently, momentum is gathering towards the development of systemically-deliverable mutants that are able to overcome anti-viral host immune responses, erythrocyte binding and hepatic uptake, to promote elimination of primary and metastatic lesions. This review will cover the key components of pancreatic cancer oncogenesis; novel oncolytic adenoviruses; clinical trials; and the current progress in overcoming the challenges of systemic delivery.

Designer Oncolytic Adenovirus: Coming of Age.

The licensing of talimogene laherparepvec (T-Vec) represented a landmark moment for oncolytic virotherapy, since it provided unequivocal evidence for the long-touted potential of genetically modified replicating viruses as anti-cancer agents. Whilst T-Vec is promising as a locally delivered virotherapy, especially in combination with immune-checkpoint inhibitors, the quest continues for a virus capable of specific tumour cell killing via systemic administration. One candidate is oncolytic adenovirus (Ad); it’s double stranded DNA genome is easily manipulated and a wide range of strategies and technologies have been employed to empower the vector with improved pharmacokinetics and tumour targeting ability. As well characterised clinical and experimental agents, we have detailed knowledge of adenoviruses’ mechanisms of pathogenicity, supported by detailed virological studies and in vivo interactions. In this review we highlight the strides made in the engineering of bespoke adenoviral vectors to specifically infect, replicate within, and destroy tumour cells. We discuss how mutations in genes regulating adenoviral replication after cell entry can be used to restrict replication to the tumour, and summarise how detailed knowledge of viral capsid interactions enable rational modification to eliminate native tropisms, and simultaneously promote active uptake by cancerous tissues. We argue that these designer-viruses, exploiting the viruses natural mechanisms and regulated at every level of replication, represent the ideal platforms for local overexpression of therapeutic transgenes such as immunomodulatory agents. Where T-Vec has paved the way, Ad-based vectors now follow. The era of designer oncolytic virotherapies looks decidedly as though it will soon become a reality.

The Novel Oncolytic Adenoviral Mutant Ad5-3Δ-A20T Retargeted to αvß6 Integrins Efficiently Eliminates Pancreatic Cancer Cells.

Metastatic pancreatic ductal adenocarcinomas (PDAC) are incurable due to the rapid development of resistance to all current therapeutics. Oncolytic adenoviral mutants have emerged as a promising new strategy that negates such resistance. In contrast to normal tissue, the majority of PDACs express the αvß6 integrin receptor. To exploit this feature, we modified our previously reported oncolytic adenovirus, AdΔΔ, to selectively target αvß6 integrins to facilitate systemic delivery. Structural modifications to AdΔΔ include the expression of the small but potent αvß6-binding peptide, A20FMDV2, and ablation of binding to the native coxsackie and adenovirus receptor (CAR) within the fiber knob region. The resultant mutant, Ad5-3Δ-A20T, infected and killed αvß6 integrin-expressing cells more effectively than the parental wild-type (Ad5wt) virus and AdΔΔ. Viral uptake through αvß6 integrins rather than native viral receptors (CAR, αvß3 and αvß5 integrins) promoted viral propagation and spread. Superior efficacy of Ad5-3Δ-A20T compared with Ad5wt was demonstrated in 3D organotypic cocultures, and similar potency between the two viruses was observed in Suit-2 in vivo models. Importantly, Ad5-3Δ-A20T infected pancreatic stellate cells at low levels, which may further facilitate viral spread and cancer cell elimination either as a single agent or in combination with the chemotherapy drug, gemcitabine. We demonstrate that Ad5-3Δ-A20T is highly selective for αvß6 integrin-expressing pancreatic cancer cells, and with further development, this new and exciting strategy can potentially be extended to improve the systemic delivery of adenoviruses to pancreatic cancer patients. Mol Cancer Ther; 17(2); 575-87. ©2018 AACR.

Sensitisation to mitoxantrone-induced apoptosis by the oncolytic adenovirus Ad∆∆ through Bcl-2-dependent attenuation of autophagy.

Anti-apoptotic Bcl-2 is frequently activated in human malignant cells to promote cell survival and inhibit cell death. Replication-selective oncolytic adenoviruses deleted in the functional Bcl-2 homologue E1B19K potently synergise with apoptosis-inducing chemotherapeutic drugs, including mitoxantrone for prostate cancer. Here, we demonstrate that our previously generated oncolytic mutant Ad∆∆ (E1B19K- and E1ACR2-deleted) caused potent synergistic apoptotic cell death in both drug-sensitive 22Rv1, and drug-insensitive PC3 and PC3M prostate cancer cells. The synergistic cell killing was dependent on Bcl-2 expression and was prevented by Bcl-2 knockdown, which led to activation of the autophagy pathway. Mitoxantrone-induced autophagy, which was decreased in combination with Ad∆∆-infection resulting in increased apoptosis. Expression of the viral E1A12S protein alone mimicked the synergistic effects with Ad∆∆ in combination with mitoxantrone while intact wild-type virus (Ad5) had no effect. Early and late-stage inhibition of autophagy by Atg7 knockdown and chloroquine respectively, promoted apoptotic cell killing with mitoxantrone similar to Ad∆∆. These findings revealed currently unexplored actions of E1B19K-deleted oncolytic adenoviruses and the central role of Bcl-2 in the synergistic cell killing. This study suggests that cancers with functional Bcl-2 expression may be selectively re-sensitised to drugs by Ad∆∆.

Oncolytic adenovirus-mediated therapy for prostate cancer.

Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen-androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses.

The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11.

Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics.

Ionizing radiation enhances dl922-947-mediated cell death of anaplastic thyroid carcinoma cells.

dl922-947 is an oncolytic adenovirus potentially suitable for the treatment of aggressive localized tumors, such as anaplastic thyroid carcinoma (ATC). In this study, we have analyzed the effects of dl922-947 in combination with ionizing radiations, testing different schedules of administration and observing synergistic effects only when ATC cells were irradiated 24 h prior to viral infection. Cells undergoing combined treatment exhibited a marked increase in cell death and viral replication, suggesting that irradiation blocks cells in a more permissive state for viral life cycle. We also show that dl922-947 triggers a DNA damage response, characterized by mobilization of the MRN complex (composed by Mre11-Rad50-Nbs1), accumulation of γH2AX, and activation of the checkpoint kinases ataxia telangiectasia mutated (ATM) and Chk1. Based on these observations, we speculate that the DNA damage response acts as a cellular protective mechanism to hinder viral infection and replication. To confirm this hypothesis, we demonstrate that the ATM inhibitor KU55933 increased the oncolytic activity of dl922-947 and its replication. Finally, we validate the potential therapeutic use of this approach by showing in vivo that the combined treatment slows tumor xenograft growth more potently than either irradiation or infection alone.

Adenovirus-mediated sensitization to the cytotoxic drugs docetaxel and mitoxantrone is dependent on regulatory domains in the E1ACR1 gene-region.

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922-947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922-947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.

Oncolytic virotherapy with modified adenoviruses and novel therapeutic targets.

INTRODUCTION: Numerous oncolytic viral mutants derived from a variety of strains have antitumor efficacy with limited or no toxicity to normal tissue. While all modes of administration were determined to be safe in patients with solid cancers refractory to current standard of care, this therapeutic approach requires further improvements to achieve definite efficacy. AREAS COVERED: We review the most promising clinical developments with several oncolytic viruses. The focus is on preclinical and clinical findings with replication-selective adenoviral mutants including ONYX-015, H101 and Ad5ΔCR mutants that, to date, are the most studied oncolytic viruses. Cellular pathways reported to play a role in virus-induced cell killing are reviewed as potential targets for the development of more effective combinatorial therapies. EXPERT OPINION: The most promising clinical outcomes for metastatic cancers have been reported for oncolytic vaccinia and herpes virus mutants expressing the cytokine GMCSF. However, highly efficacious and selective adenoviral mutants have been developed that interact synergistically with cytotoxic drugs in model systems. We anticipate that by delineating the cellular targets for synergistic cancer cell killing in response to adenoviral mutants and drugs such as apoptosis and autophagy signaling, greatly improved anticancer therapies will result in the near future.

Synergistic and Selective Cancer Cell Killing Mediated by the Oncolytic Adenoviral Mutant AdΔΔ and Dietary Phytochemicals in Prostate Cancer Models.

AdΔΔ is an oncolytic adenoviral mutant that has been engineered to selectively target tumors with deregulated cell cycle and apoptosis pathways. AdΔΔ potentiates apoptotic cell death induced by drugs, including mitoxantrone and docetaxel, which are commonly used to treat prostate cancer. Here, we demonstrate that AdΔΔ can also interact synergistically with dietary phytochemicals known to have anti-cancer activities, without incurring the toxic side effects of chemodrugs. Curcumin, genistein, epigallocatechin-gallate, equol, and resveratrol efficiently killed both androgen-receptor positive (22Rv1) and negative cell lines (PC-3, DU145) in combination with adenoviral mutants. Synergistic cell killing was demonstrated with wild-type virus (Ad5) and AdΔΔ in combination with equol and resveratrol. EC(50) values for both phytochemicals and viruses were reduced three- to eightfold in all three combination-treated cell lines. The most potent efficacy was achieved in the cytotoxic drug- and virus-insensitive PC-3 cells, both in vitro and in vivo, while cell killing in normal bronchial epithelial cells was not enhanced. Although equol and resveratrol induced only low levels of apoptosis when administered alone, in combination with wild-type virus or AdΔΔ, the level of apoptotic cell death was significantly increased in PC-3 and DU145 cells. In vivo studies using suboptimal doses of AdΔΔ and equol or resveratrol, showed reduced tumor growth without toxicity to normal tissue. These findings identify novel functions for AdΔΔ and phytochemicals in promoting cancer cell killing and apoptosis, suggesting the use of these natural nontoxic compounds might be a feasible and currently unexploited anti-cancer strategy.

Inhibition of autophagy enhances the effects of E1A-defective oncolytic adenovirus dl922-947 against glioma cells in vitro and in vivo.

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.

Adenoviral gene expression and replication in human tumor explant models.

Promising results have been reported from numerous studies with replication-selective oncolytic adenoviral mutants as novel treatments for a variety of cancers. Most of these studies were performed in cancer cell lines, dissociated tumor tissue, or animal models, and the predictive utility for efficacy and safety in the clinical setting is unclear. Indeed, the outcome of many clinical trials with viral mutants that demonstrated high efficacy preclinically has so far been disappointing, necessitating better test models. To this end, we developed a methodology using primary human cancer specimens for evaluation of cytotoxicity ex vivo including colorectal liver metastasis, ovarian, breast, colon, and prostate carcinomas. Under optimized culture conditions, primary human tumor tissue remained viable for up to 48 h, enabling evaluation of viral mutants in tissue with intact morphology. This assay may have great utility to investigate novel viral mutants and to identify treatment sensitive cancers by assessing specific oncolytic mutants in individual cases.