Carvedilol targets ß-arrestins to rewire innate immunity and improve oncolytic adenoviral therapy.

Oncolytic viruses are being tested in clinical trials, including in women with ovarian cancer. We use a drug-repurposing approach to identify existing drugs that enhance the activity of oncolytic adenoviruses. This reveals that carvedilol, a ß-arrestin-biased ß-blocker, synergises with both wild-type adenovirus and the E1A-CR2-deleted oncolytic adenovirus, dl922-947. Synergy is not due to ß-adrenergic blockade but is dependent on ß-arrestins and is reversed by ß-arrestin CRISPR gene editing. Co-treatment with dl922-947 and carvedilol causes increased viral DNA replication, greater viral protein expression and higher titres of infectious viral particles. Carvedilol also enhances viral efficacy in orthotopic, intraperitoneal murine models, achieving more rapid tumour clearance than virus alone. Increased anti-cancer activity is associated with an intratumoural inflammatory cell infiltrate and systemic cytokine release. In summary, carvedilol augments the activity of oncolytic adenoviruses via ß-arrestins to re-wire cytokine networks and innate immunity and could therefore improve oncolytic viruses for cancer patient treatment.

Chloroxine overrides DNA damage tolerance to restore platinum sensitivity in high-grade serous ovarian cancer.

High-grade serous cancer (HGSC) accounts for ~67% of all ovarian cancer deaths. Although initially sensitive to platinum chemotherapy, resistance is inevitable and there is an unmet clinical need for novel therapies that can circumvent this event. We performed a drug screen with 1177 FDA-approved drugs and identified the hydroxyquinoline drug, chloroxine. In extensive validation experiments, chloroxine restored sensitivity to both cisplatin and carboplatin, demonstrating broad synergy in our range of experimental models of platinum-resistant HGSC. Synergy was independent of chloroxine's predicted ionophore activity and did not relate to platinum uptake as measured by atomic absorption spectroscopy. Further mechanistic investigation revealed that chloroxine overrides DNA damage tolerance in platinum-resistant HGSC. Co-treatment with carboplatin and chloroxine (but not either drug alone) caused an increase in γH2AX expression, followed by a reduction in platinum-induced RAD51 foci. Moreover, this unrepaired DNA damage was associated with p53 stabilisation, cell cycle re-entry and triggering of caspase 3/7-mediated cell death. Finally, in our platinum-resistant, intraperitoneal in vivo model, treatment with carboplatin alone resulted in a transient tumour response followed by tumour regrowth. In contrast, treatment with chloroxine and carboplatin combined, was able to maintain tumour volume at baseline for over 4 months. In conclusion, our novel results show that chloroxine facilitates platinum-induced DNA damage to restore platinum sensitivity in HGSC. Since chloroxine is already licensed, this exciting combination therapy could now be rapidly translated for patient benefit.

Physiological strength electric fields modulate human T cell activation and polarisation.

The factors and signals driving T cell activation and polarisation during immune responses have been studied mainly at the level of cells and chemical mediators. Here we describe a physical driver of these processes in the form of physiological-strength electric fields (EFs). EFs are generated at sites where epithelium is disrupted (e.g. wounded skin/bronchial epithelia) and where T cells frequently are present. Using live-cell imaging, we show human primary T cells migrate directionally to the cathode in low strength (50/150 mV/mm) EFs. Strikingly, we show for the first time that EFs significantly downregulate T cell activation following stimulation with antigen-activated APCs or anti-CD3/CD28 antibodies, as demonstrated by decreased IL-2 secretion and proliferation. These EF-induced functional changes were accompanied by a significant dampening of CD4+ T cell polarisation. Expression of critical markers of the Th17 lineage, RORγt and IL-17, and the Th17 polarisation mediator phospho-STAT3 were reduced significantly, while STAT1, ERK and c-Jun phosphorylation were comparatively unaffected suggesting STAT3 modulation by EFs as one mechanism driving effects. Overall, we identify electrical signals as important contributors to the co-ordination and regulation of human T cell functions, paving the way for a new research area into effects of naturally occurring and clinically-applied EFs in conditions where control of T cell activity is paramount.

SOCS3 is a modulator of human macrophage phagocytosis.

Suppressor of cytokine signaling (SOCS) proteins are recognized as key feedback inhibitors modulating the inflammatory activities of macrophages, but comparatively little is known about whether and how they affect phagocytosis. Here, we evaluated the role of SOCS3 in driving the inflammatory phenotype and phagocytic uptake of apoptotic cells by human macrophages and the signaling pathways that are necessary for efficient phagocytosis. In M1-activated human monocyte-derived macrophages, SOCS3 silencing, using short interfering RNA technology, resulted in a decreased expression of proinflammatory markers and an increased expression of M2 macrophage markers. Strikingly, we demonstrated for the first time that SOCS3 knockdown significantly enhances the phagocytic capacity of M1 macrophages for carboxylate-modified beads and apoptotic neutrophils. With the use of live-cell video microscopy, we showed that SOCS3 knockdown radically affects the temporal dynamics of particle engulfment, enabling more rapid uptake of a second target and delaying postengulfment processing, as evidenced by deferred acquisition of phagosome maturation markers. SOCS3 knockdown impacts on phagocytosis through increased PI3K and Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, pathways essential for engulfment and clearance of apoptotic cells. Enhanced phagocytosis in SOCS3-silenced cells was reversed by pharmacological PI3K inhibition. Furthermore, we revealed that actin polymerization, downstream of PI3K/Rac1 activation, was significantly altered in SOCS3-silenced cells, providing a mechanism for their greater phagocytic activity. The findings support a new model, whereby SOCS3 not only plays an important role in driving macrophage inflammatory responses but modulates key signaling pathways organizing the actin cytoskeleton to regulate the efficiency of phagocytic processes.

Electric fields are novel determinants of human macrophage functions.

Macrophages are key cells in inflammation and repair, and their activity requires close regulation. The characterization of cues coordinating macrophage function has focused on biologic and soluble mediators, with little known about their responses to physical stimuli, such as the electrical fields that are generated naturally in injured tissue and which accelerate wound healing. To address this gap in understanding, we tested how properties of human monocyte-derived macrophages are regulated by applied electrical fields, similar in strengths to those established naturally. With the use of live-cell video microscopy, we show that macrophage migration is directed anodally by electrical fields as low as 5 mV/mm and is electrical field strength dependent, with effects peaking ∼300 mV/mm. Monocytes, as macrophage precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that electrical fields significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils, and the nominal opportunist pathogen Candida albicans, which engage different classes of surface receptors. These electrical field-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium, and actin polarization. Electrical fields also modulate cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the coordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically applied electrical fields in conditions where macrophage activity is critical.