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.

PTEN loss shapes macrophage dynamics in high grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSC) remains a disease of poor prognosis that is unresponsive to current immune checkpoint inhibitors. Although PI3K pathway alterations, such as PTEN loss, are common in HGSC, attempts to target this pathway have been unsuccessful. We hypothesized that aberrant PI3K pathway activation may alter the HGSC immune microenvironment and present a targeting opportunity. Single-cell RNA sequencing identified populations of resident macrophages specific to Pten-null omental tumors in murine models, which were confirmed by flow cytometry. These macrophages derived from peritoneal fluid macrophages and had a unique gene expression program, marked by high expression of the enzyme heme oxygenase-1 (HMOX1). Targeting resident peritoneal macrophages prevented the appearance of HMOX1hi macrophages and reduced tumor growth. Furthermore, direct inhibition of HMOX1 extended survival in vivo. RNA sequencing identified IL33 in Pten-null tumor cells as a likely candidate driver leading to the appearance of HMOX1hi macrophages. Human HGSC tumors also contained HMOX1hi macrophages with a corresponding gene expression program. Moreover, the presence of these macrophages correlated with activated tumoral PI3K/mTOR signaling and poor overall survival in HGSC patients. In contrast, tumors with low numbers of HMOX1hi macrophages were marked by increased adaptive immune response gene expression. These data suggest targeting HMOX1hi macrophages as a potential therapeutic strategy for treating poor prognosis HGSC.

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.

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∆∆.