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.

Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system.

Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions.

PRSS3 promotes tumour growth and metastasis of human pancreatic cancer.

BACKGROUND AND AIMS: Metastasis accounts for the poor outcome of patients with pancreatic cancer. We recently discovered PRSS3 to be over-expressed in metastatic human pancreatic cancer cells. This study aimed to elucidate the role of PRSS3 in the growth and metastasis of human pancreatic cancer. METHODS: PRSS3 expression in human pancreatic cancer cell lines was detected by qPCR and immunoblotting. The effect of PRSS3 on cancer cell proliferation, migration and invasion in vitro, tumour growth and metastasis in vivo were investigated by manipulation of PRSS3 expression in human pancreatic cancer cell lines. VEGF expression was detected by ELISA, and the pathway through which PRSS3 regulates VEGF expression was investigated. The therapeutic effect of targeting this pathway on metastasis was assessed in vivo. Immunohistochemistry was employed to detect PRSS3 expression in human pancreatic cancer tissues. RESULTS: PRSS3 was over-expressed in the metastatic PaTu8988s cell line, but not in the non-metastatic PaTu8988t cell line. Over-expression of PRSS3 promoted pancreatic cancer cell proliferation as well as invasion in vitro, and tumour progression and metastasis in vivo. Stepwise investigations demonstrated that PRSS3 upregulates VEGF expression via the PAR1-mediated ERK pathway. ERK inhibitor significantly delayed the progression of metastases of pancreatic cancer and prolonged the survival of animals bearing metastatic pancreatic cancer (p<0.05). 40.54% of human pancreatic cancers (n=74) were positive for PRSS3 protein. A significant correlation was observed between PRSS3 expression and metastasis (p<0.01). Multivariate Cox regression analysis indicated that patients with PRSS3 expression in their tumours had a shorter survival time compared to those without PRSS3 expression (p<0.05). CONCLUSION: PRSS3 plays an important role in the progression, metastasis and prognosis of human pancreatic cancer. Targeting the PRSS3 signalling pathway may be an effective and feasible approach for treatment of this lethal cancer.

Oncolytic adenoviral mutants with E1B19K gene deletions enhance gemcitabine-induced apoptosis in pancreatic carcinoma cells and anti-tumor efficacy in vivo.

PURPOSE: Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal. We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. EXPERIMENTAL DESIGN: Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdDeltaE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. RESULTS: The DeltaE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdDeltaE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. CONCLUSIONS: Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways. These findings imply that less toxic doses than currently practiced in the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants.

A Virus-Infected, Reprogrammed Somatic Cell-Derived Tumor Cell (VIReST) Vaccination Regime Can Prevent Initiation and Progression of Pancreatic Cancer.

PURPOSE: Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses. EXPERIMENTAL DESIGN: We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using in situ gene editing via stable provision of KRas G12D and p53 R172H tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development. RESULTS: Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic. CONCLUSIONS: These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals.

Treatment and Prevention of Lung Cancer Using a Virus-Infected Reprogrammed Somatic Cell-Derived Tumor Cell Vaccination (VIReST) Regime.

Lung cancer is one of the most commonly diagnosed cancer and despite therapeutic advances, mortality remains high. The long period of clinical latency associated with lung cancer provides an ideal window of opportunity to administer vaccines to at-risk individuals that can prevent tumor progression and initiate long-term anti-tumor immune surveillance. Here we describe a personalized vaccination regime that could be applied for both therapeutic and prophylactic prevention of lung cancer, based on the derivation of lung cancer cells from induced pluripotent stem cells. Stem cells from healthy mice were modified to express Cre-dependent KRASG12D and Trp53R172H prior to differentiation to lung progenitor cells. Subsequent viral delivery of Cre caused activation of exogenous driver mutations, resulting in transformation and development of lung cancer cells. iPSC-derived lung cancer cells were highly antigenically related to lung cancer cells induced in LSL-KRASG12D/+; Trp53R172H/+ transgenic mice and were antigenically unrelated to original pluripotent stem cells or pancreatic cancer cells derived using the same technological platform. For vaccination, induced lung cancer cells were infected with oncolytic Adenovirus or Vaccinia virus, to act as vaccine adjuvants, prior to delivery of vaccines sequentially to a murine inducible transgenic model of lung cancer. Application of this Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime primed tumor-specific T cell responses that significantly prolonged survival in both subcutaneous post-vaccine challenge models and induced transgenic models of lung cancer, demonstrating that stem cell-derived prophylactic vaccines may be a feasible intervention for treatment or prevention of lung cancer development in at-risk individuals.

E3 gene manipulations affect oncolytic adenovirus activity in immunocompetent tumor models.

Oncolytic replication-selective adenoviruses constitute a rapidly growing therapeutic platform for cancer. However, the role of the host immune response and the E3 immunoregulatory genes of the human adenovirus were unknown until now. We identified four mouse carcinoma lines of variable permissivity for adenoviral gene expression, cytopathic effects and/or burst size. To determine E3 gene effects in immunocompetent tumor-bearing hosts, we injected tumors with one of three adenoviruses: Ad5 (E3 wild type), dl309 (del. E3 10.4/14.5, 14.7 kDa) or dl704 (del. E3 gp19 kDa). Compared with Ad5 and dl704, dl309 was cleared much more rapidly and/or its activity was lower in all four models. Intratumoral injection with dl309 resulted in markedly greater macrophage infiltration and expression of both tumor necrosis factor and interferon-gamma. Adenovirus replication, CD8(+) lymphocyte infiltration and efficacy were similar upon intratumoral injection with either dl704 or Ad5. E3-dependent differences were not evident in athymic mice. These findings have important implications for the design of oncolytic adenoviruses and may explain the rapid clearance of E3-10.4/14.5,14.7-deleted adenoviruses in patients.

Virus-associated RNA I-deleted adenovirus, a potential oncolytic agent targeting EBV-associated tumors.

Given the growing number of tumor types recognizably associated with EBV infection, it is critically important that therapeutic strategies are developed to treat such tumors. Replication-selective oncolytic adenoviruses represent a promising new platform for anticancer therapy. Virus-associated I (VAI) RNAs of adenoviruses are required for efficient translation of viral mRNAs. When the VAI gene is deleted, adenovirus replication is impeded in most cells (including HEK 293 cells). EBV-encoded small RNA1 is uniformly expressed in most EBV-associated human tumors and can functionally substitute for the VAI RNAs of adenovirus. It enables replication to proceed through complementation of VAI-deletion mutants. We hypothesized that VAI-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to the presence of EBV-encoded small RNA1 with no (or poor) replication in normal or EBV-negative tumor cells. In this report, we show that high levels of replication occurred in the VAI-deleted mutant in the EBV-positive tumor cells compared with low (or negligible) levels in EBV-negative and normal human primary cells. Correspondingly, high toxicity levels were observed in EBV-positive tumor cells but not in EBV-negative tumor or normal human primary cells. In vivo, VAI-deleted adenovirus showed superior antitumoral efficacy to wild-type adenovirus in EBV-positive tumor xenografts, with lower hepatotoxicity than wild-type adenovirus. Our data suggest that VAI-deleted adenovirus is a promising replication-selective oncolytic virus with targeting specificity for EBV-associated tumors.

Gene therapy developments for pancreatic cancer.

Treatment options for pancreatic cancer have limited success and it is therefore an appropriate target for the development of new strategies, including gene therapy. Gene therapy approaches include inhibition of activated oncogenes (KRAS, LSM1) with antisense and RNA interference strategies, replacement of inactivated tumour suppressor genes (TP53, CDKN2A, CDKN1A), targeting of cell signalling pathways, gene-directed prodrug-activation therapies and the use of replication-competent oncolytic viruses. Angiogenesis and apoptosis have also been targeted for gene therapy. Clinical trials of gene therapy have shown only moderate anti-tumour effects. As there are many genetic abnormalities in pancreatic cancer, strategies combining different targets or indeed different modalities of treatment, may be more successful. Identification of new targets and improvements in delivery and targeting may further improve the efficacy of gene therapy in pancreatic cancer.

Caspase-1 as a radio- and chemo-sensitiser in vitro and in vivo.

The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. This observation raises the possibility that factors affecting caspase activation might be important determinants of anticancer drug sensitivity. Ectopic expression of caspase-1 has been shown to trigger apoptosis. However, the role of caspase-1 in apoptosis is now considered as minor compared to other caspases. In patients, high levels of caspase-1 expression may be associated with spontaneous regression in neuroblastomas and with a good clinical response to chemotherapy in acute myeloid leukemia and osteosarcoma. In experimental therapeutics for cancer, caspase-1 has been related to some anticancer activity. These observations led us to examine the effect of over-expression on the response to chemotherapy and radiotherapy in vitro and in vivo. Caspase-1 expression mediated by an adenoviral vector was able to kill directly cells and to sensitise the remaining cells to cisplatin or gamma-radiation in vitro. In HeLa cells stably transfected with caspase-1, sensitisation to cisplatin was due to an amplification of the cisplatin-induced mitochondrial apoptotic pathway activation. Caspase-1 mediated sensitisation to cisplatin and gamma-radiation was also observed in vivo. Altogether, we conclude that caspase-1 can act as a radio- and chemo-sensitiser, in vitro and in vivo.

A novel assay to assess primary human cancer infectibility by replication-selective oncolytic adenoviruses.

PURPOSE: Replication-selective oncolytic adenoviruses hold promise for cancer treatment, but the predictive use of cell lines, dissociated tumor tissue, and animal models for efficacy against primary cancers are unclear. To further evaluate cytotoxicity and the potential for efficacy of replication-competent adenoviruses we therefore developed a novel methodology using primary human cancer specimens ex vivo; ovarian, colon, rectal, and breast carcinomas were included. EXPERIMENTAL DESIGN: Tissue culture conditions were developed to maintain viability of adenocarcinomas ex vivo for 48 hours postsurgery. Explants were infected by replication-competent (wild type 5 and E1A mutant dl922-947) and replication-defective (dl312) adenoviruses; early (E1A) and late (hexon) viral gene expression, alphav integrins, coxsackievirus and adenovirus receptor (CAR) and tissue viability were assessed by immunohistochemistry and histopathology. Viral replication was verified by replication assays on selected samples. RESULTS: Viral gene expression varied dramatically among cancer specimens (n = 41). With Ad5, hexon expression was high in 8 of 11 tested specimens, whereas E1A levels were detectable in 16 of 27 tumor explants. Viral gene expression, distribution, and cytopathic effects were greater postinfection with dl922-947. Specimens that supported early gene expression (E1A) also supported viral replication in 13 of 14 tested cases, determined by recovery of infectious units. As predicted, the replication-defective adenovirus dl312 was not associated with viral gene expression. CONCLUSIONS: Primary human tumor tissue remained viable when cultured ex vivo enabling evaluation of viral mutants in tissue with intact morphology. This assay may have great use in determining treatment-sensitive cancers and assess specific oncolytic mutants in individual cases.

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.

A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9.

The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.

Identification of a Three-Biomarker Panel in Urine for Early Detection of Pancreatic Adenocarcinoma.

PURPOSE: Noninvasive biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC) are currently not available. Here, we aimed to identify a set of urine proteins able to distinguish patients with early-stage PDAC from healthy individuals. EXPERIMENTAL DESIGN: Proteomes of 18 urine samples from healthy controls, chronic pancreatitis, and patients with PDAC (six/group) were assayed using GeLC/MS/MS analysis. The selected biomarkers were subsequently validated with ELISA assays using multiple logistic regression applied to a training dataset in a multicenter cohort comprising 488 urine samples. RESULTS: LYVE-1, REG1A, and TFF1 were selected as candidate biomarkers. When comparing PDAC (n = 192) with healthy (n = 87) urine specimens, the resulting areas under the receiver-operating characteristic curves (AUC) of the panel were 0.89 [95% confidence interval (CI), 0.84-0.94] in the training (70% of the data) and 0.92 (95% CI, 0.86-0.98) in the validation (30% of the data) datasets. When comparing PDAC stage I-II (n = 71) with healthy urine specimens, the panel achieved AUCs of 0.90 (95% CI, 0.84-0.96) and 0.93 (95% CI, 0.84-1.00) in the training and validation datasets, respectively. In PDAC stage I-II and healthy samples with matching plasma CA19.9, the panel achieved a higher AUC of 0.97 (95% CI, 0.94-0.99) than CA19.9 (AUC = 0.88; 95% CI, 0.81-0.95, P = 0.005). Adding plasma CA19.9 to the panel increased the AUC from 0.97 (95% CI, 0.94-0.99) to 0.99 (95% CI, 0.97-1.00, P = 0.04), but did not improve the comparison of stage I-IIA PDAC (n = 17) with healthy urine. CONCLUSIONS: We have established a novel, three-protein biomarker panel that is able to detect patients with early-stage pancreatic cancer in urine specimens.

Suicide gene therapy: conversion of ethanol to acetaldehyde mediated by human beta 2 alcohol dehydrogenase.

Acetaldehyde (AcH) produced in the physiological metabolism of ethanol can be potentially toxic and immunomodulating. The antitumour activity of a suicide gene system using adenovirus delivered alcohol dehydrogenase (ADH) to convert ethanol to acetaldehyde inside cancer cells has been investigated in vitro and in vivo. In vitro experiments confirmed the toxicity of acetaldehyde to a number of tumour cell lines. Daudi lymphoma cells grown in normal media increased by Day 4 to 650% of their starting number, while those exposed to 250 microM, 500 microM and 1 mM acetaldehyde reached 138, 30 and 5% respectively. Adenocarcinoma cells appeared to be less sensitive with CMT-64 cells and HeLa cells numbering 105 and 53% of their starting number by Day 4 with 1 mM acetaldehyde. After transduction with an adenovirus containing the human ADH beta 2 cDNA, CMT-64 cells exposed to 20 mM ethanol had a reduction in number to 74% by Day 2 and to 36% by Day 4. In a preclinical model with Ad-ADH CMT-64 cells, mice exposed to daily pulses of ethanol for 5 days formed tumours only 30% on Day 6 and 42% on Day 13 of the volume of those in mice exposed to water. The ability of this easily administered suicide gene system to produce significant effects on cell proliferation in vivo suggests that further optimized development is warranted.

10th International Conference on Gene Therapy of Cancer. 13-15 December, 2001, Sidney Kimmel Cancer Center, San Diego, CA, USA.

This annual meeting highlighted some incremental advances in the development of gene therapeutics, with genetic prodrug activation, novel tumor suppressor genes and replicating viral vectors at the leading edge for clinical application. Imaging technologies are proving to be very useful for pharmacokinetic and pharmacodynamic studies in preclinical models and should be translated into systems that can help in the development of surrogate markers in clinical trials.

Update on oncolytic viral therapy - targeting angiogenesis.

Oncolytic viruses (OVs) have the ability to selectively replicate in and lyse cancer cells. Angiogenesis is an essential requirement for tumor growth. Like OVs, the therapeutic effect of many angiogenesis inhibitors has been limited, leading to the development of more effective approaches to combine antiangiogenic therapy with OVs. Angiogenesis can be targeted either directly by OV infection of vascular endothelial cells, or by arming OVs with antiangiogenic transgenes, which are subsequently expressed locally in the tumor microenvironment. In this review, we describe the development and targeting of OVs, the role of angiogenesis in cancer, and the progress made in arming viruses with antiangiogenic transgenes. Future developments required to optimize this approach are addressed.

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.

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.