Biology subjects

Butera, F.

Publications and source records attributed to Butera, F..

3 recordsLinked to original sources

bioRxiv2023

PARP inhibitors enhance reovirus-mediated cell killing through the death-inducing signaling complex (DISC) with an associated NF-kappa B-regulated immune response.

Oncolytic Reovirus type 3 Dearing (RT3D), is a naturally occurring double-stranded (ds) RNA virus that is under development as an oncolytic immunotherapy We used an unbiased high-throughput cytotoxicity screen of different targeted therapeutic agents with the aim of identifying potential drug-viral sensitizers to enhance RT3D tumour killing. Talazoparib, a clinical poly(ADP)-ribose polymerase 1 (PARP-1) inhibitor, was identified as a top hit and found to cause profound sensitisation to RT3D. This effect was not seen with other classes of oncolytic virus and was not mediated by enhanced viral replication or PARP inhibitor-related effects on the DNA damage response. RT3D interacts with retinoic acid-induced gene-1 (RIG-I) and activates PARP-1, with consequent PARylation of components of the extrinsic apoptosis pathway. Pharmacological and genetic inhibition of PARP-1 abrogates this PARylation and increases levels of extrinsic apoptosis, NF-kB signalling and pro-inflammatory cell death. Direct interaction between PARP-1 and RIG-I following RT3D/talazoparib treatment is a key factor in activating downstream signaling pathways that lead to IFN-{beta} and TNF-/TRAIL production which, in turn, amplify the therapeutic effect through positive feedback. Critically, it was possible to phenocopy the effect of RT3D through the use of non-viral ds-RNA therapy and RIG-I agonism. In in vivo studies, we demonstrated profound combinatorial efficacy of RT3D and talazoparib in human A375 melanoma in immunodeficient mice. More impressively, in immunocompetent mouse models of 4434 murine melanoma, we achieved 100% tumour control and protection from subsequent tumour rechallenge with the combination regimen. Correlative immunophenotyping confirmed significant innate and adaptive immune activation with the combination of RT3D and PARP inhibition. Taken together, these data provide a clear line of sight to clinical translation of combined regimens of PARP inhibition or ds-RNA agonism, with either viral or non-viral agents, in tumour types beyond the relatively narrow confines of current licensed indications for PARP inhibition.

cancer biology↗

bioRxiv2023

Re-engineering of TNFα-NF-κB signalling dynamics in cancer cells using pathogenic E. coli effectors

Re-engineering NF-{kappa}B signalling towards enhancing beneficial outcomes such as tumour cell elimination, while minimising inflammatory damage, is a potential therapeutic avenue. In this study, we explored the ability of bacterial effectors injected into host cells by the type III secretion system to regulate NF-{kappa}B translocation dynamics. We used the enteropathogenic Escherichia coli effectors Tir (NF-{kappa}B activator), NleC (NF-{kappa}B protease) and NleE (TAB2/3 methyltransferase), to manipulate NF-{kappa}B translocation and cancer cell survival. We discovered that while these effectors have either limited or no cytotoxicity alone, they greatly enhanced caspase-8-dependent pancreatic cancer cell death in the presence of TNF. Single cell analysis revealed that the sub-population of cells showing high NF-{kappa}B activation is less susceptible to cell death caused by NleC or NleE but instead is more susceptible to Tir. A combination of Tir, NleE and TNF eliminated 95% cancer cells with limited NF-{kappa}B activation, potentially due to NleE-dependent blockage of the immediate pro-survival NF-{kappa}B activation without inhibiting Tirs long-term NF-{kappa}B activation that promotes cell death. This work demonstrates that effector combinations could be used to re-engineer stress responses towards favourable outcomes.

cell biology↗

bioRxiv2022

Actin networks modulate heterogenous NF-κB dynamics in response to TNFα

The canonical NF-{kappa}B transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. Here, we characterised previously unknown endogenous RELA-GFP dynamics in PDAC cell lines by live single cell imaging, which revealed rapid, sustained and non-oscillatory nuclear RELA following TNF stimulation. Using Bayesian analysis of single cell datasets with variation in nuclear RELA, we computationally predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. By RNA-seq, we identified the actin regulators NUAK2 and ARHGAP31 as transcriptionally regulated by RELA. In turn, NUAK2 or ARHGAP31 siRNA depletion downregulates TNF-stimulated RELA nuclear localisation in PDAC cells, establishing a novel negative feedback loop regulating RELA activation by TNF. We identify an additional actin-independent feedback loop involving RELB, which suppresses TNF-mediated RELA nuclear localisation following RELA mediated upregulation of RELB. Taken together, we provide computational and experimental support for interdependence between the F-actin network and RELA translocation dynamics in PDAC.

cancer biology↗