Pharmacological Inhibition of ß3 Integrin Reduces the Inflammatory Toxicities Caused by Oncolytic Adenovirus without Compromising Anticancer Activity.

Adenoviruses have been clinically tested as anticancer therapies but their utility has been severely limited by rapid, systemic cytokine release and consequent inflammatory toxicity. Here, we describe a new approach to tackling these dangerous side effects. Using human ovarian cancer cell lines as well as malignant epithelial cells harvested from the ascites of women with ovarian cancer, we show that tumor cells do not produce cytokines in the first 24 hours following in vitro infection with the oncolytic adenovirus dl922-947. In contrast, dl922-947 does induce inflammatory cytokines at early time points following intraperitoneal delivery in mice with human ovarian cancer intraperitoneal xenografts. In these animals, cytokines originate predominantly in murine tissues, especially in macrophage-rich organs such as the spleen. We use a nonreplicating adenovirus to confirm that early cytokine production is independent of adenoviral replication. Using ß3 integrin knockout mice injected intraperitoneally with dl922-947 and ß3 null murine peritoneal macrophages, we confirm a role for macrophage cell surface ß3 integrin in this dl922-947-induced inflammation. We present new evidence that co-administration of a cyclic RGD-mimetic-specific inhibitor of ß3 integrin significantly attenuates the cytokine release and inflammatory hepatic toxicity induced by dl922-947 in an intraperitoneal murine model of ovarian cancer. Importantly, we find no evidence that ß3 inhibition compromises viral infectivity and oncolysis in vitro or anticancer efficacy in vivo. By enabling safe, systemic delivery of replicating adenoviruses, this novel approach could have a major impact on the future development of these effective anticancer agents.

Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control.

Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the link between paclitaxel resistance and oncolytic adenovirus activity using established ovarian cancer cell line models, xenografts with de novo paclitaxel resistance and tumour samples from two separate trials. The activity of multiple Ad5 vectors, including dl922-947 (E1A CR2-deleted), dl1520 (E1B-55K deleted) and Ad5 WT, was significantly increased in paclitaxel resistant ovarian cancer in vitro and in vivo. This was associated with greater infectivity resulting from increased expression of the primary receptor for Ad5, CAR (coxsackie adenovirus receptor). This, in turn, resulted from increased CAR transcription secondary to histone modification in resistant cells. There was increased CAR expression in intraperitoneal tumours with de novo paclitaxel resistance and in tumours from patients with clinical resistance to paclitaxel. Increased CAR expression did not cause paclitaxel resistance, but did increase inflammatory cytokine expression. Finally, we identified dysregulated cell cycle control as a second mechanism of increased adenovirus efficacy in paclitaxel-resistant ovarian cancer. Ad11 and Ad35, both group B adenoviruses that utilise non-CAR receptors to infect cells, are also significantly more effective in paclitaxel-resistant ovarian cell models. Inhibition of CDK4/6 using PD-0332991 was able both to reverse paclitaxel resistance and reduce adenovirus efficacy. Thus, paclitaxel resistance increases oncolytic adenovirus efficacy via at least two separate mechanisms - if validated further, this information could have future clinical utility to aid patient selection for clinical trials.

Failure of translation of human adenovirus mRNA in murine cancer cells can be partially overcome by L4-100K expression in vitro and in vivo.

Adaptive immune responses may be vital in the overall efficacy of oncolytic viruses in human malignancies. However, immune responses to oncolytic adenoviruses are poorly understood because these viruses lack activity in murine cells, which precludes evaluation in immunocompetent murine cancer models. We have evaluated human adenovirus activity in murine cells. We show that a panel of murine carcinoma cells, including CMT64, MOVCAR7, and MOSEC/ID8, can readily be infected with human adenovirus. These cells also support viral gene transcription, messenger RNA (mRNA) processing, and genome replication. However, there is a profound failure of adenovirus protein synthesis, especially late structural proteins, both in vitro and in vivo, with reduced loading of late mRNA onto ribosomes. Our data also show that in trans expression of the nonstructural late protein L4-100K increases both the amount of viral mRNA on ribosomes and the synthesis of late proteins, accompanied by reduced phosphorylation of eIF2α and improved anticancer efficacy. These results suggest that murine models that support human adenovirus replication could be generated, thus allowing evaluation of human adenoviruses in immunocompetent mice.

Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells.

Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.

Inhibition of the inflammatory cytokine TNF-α increases adenovirus activity in ovarian cancer via modulation of cIAP1/2 expression.

Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-α (TNF-α). We investigated whether inhibition of TNF-α augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-α and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-α reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-α antibody infliximab. In vivo, stable knockdown of TNF-α in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-α using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-α acts as a survival factor in adenovirus-infected cells. Combining TNF-α inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.

p21 Promotes oncolytic adenoviral activity in ovarian cancer and is a potential biomarker.

The oncolytic adenovirus dl922-947 replicates selectively within and lyses cells with a dysregulated Rb pathway, a finding seen in > 90% human cancers. dl922-947 is more potent than wild type adenovirus and the E1B-deletion mutant dl1520 (Onyx-015). We wished to determine which host cell factors influence cytotoxicity. SV40 large T-transformed MRC5-VA cells are 3-logs more sensitive to dl922-947 than isogenic parental MRC5 cells, confirming that an abnormal G1/S checkpoint increases viral efficacy. The sensitivity of ovarian cancer cells to dl922-947 varied widely: IC50 values ranged from 51 (SKOV3ip1) to 0.03 pfu/cell (TOV21G). Cells sensitive to dl922-947 had higher S phase populations and supported earlier E1A expression. Cytotoxicity correlated poorly with both infectivity and replication, but well with expression of p21 by microarray and western blot analyses. Matched p21+/+ and -/- Hct116 cells confirmed that p21 influences dl922-947 activity in vitro and in vivo. siRNA-mediated p21 knockdown in sensitive TOV21G cells decreases E1A expression and viral cytotoxicity, whilst expression of p21 in resistant A2780CP cells increases virus activity in vitro and in intraperitoneal xenografts. These results highlight that host cell factors beyond simple infectivity can influence the efficacy of oncolytic adenoviruses. p21 expression may be an important biomarker of response in clinical trials.

Genome-wide pharmacogenetics of antidepressant response in the GENDEP project.

OBJECTIVE: The purpose of this study was to identify genetic variants underlying the considerable individual differences in response to antidepressant treatment. The authors performed a genome-wide association analysis of improvement of depression severity with two antidepressant drugs. METHOD: High-quality Illumina Human610-quad chip genotyping data were available for 706 unrelated participants of European ancestry treated for major depression with escitalopram (N=394) or nortriptyline (N=312) over a 12-week period in the Genome-Based Therapeutic Drugs for Depression (GENDEP) project, a partially randomized open-label pharmacogenetic trial. RESULTS: Single nucleotide polymorphisms in two intergenic regions containing copy number variants on chromosomes 1 and 10 were associated with the outcome of treatment with escitalopram or nortriptyline at suggestive levels of significance and with a high posterior likelihood of true association. Drug-specific analyses revealed a genome-wide significant association between marker rs2500535 in the uronyl 2-sulphotransferase gene and response to nortriptyline. Response to escitalopram was best predicted by a marker in the interleukin-11 (IL11) gene. A set of 72 a priori-selected candidate genes did not show pharmacogenetic associations above a chance level, but an association with response to escitalopram was detected in the interleukin-6 gene, which is a close homologue of IL11. CONCLUSIONS: While limited statistical power means that a number of true associations may have been missed, these results suggest that efficacy of antidepressants may be predicted by genetic markers other than traditional candidates. Genome-wide studies, if properly replicated, may thus be important steps in the elucidation of the genetic basis of pharmacological response.