Characterisation of RELA dynamics in pancreatic ductal adenocarcinoma

Abstract

The NF-kB transcription factor RELA is a master regulator of the immune response and cell survival. Numerous studies have determined the parameters and configuration of single cell RELA translocation dynamics, with various cell types displaying oscillations in RELA localisation. Nonetheless, single cell RELA dynamics in Pancreatic Ductal Adenocarcinoma (PDAC) cells are uncharacterised, even though RELA is constitutively active in PDAC and RELA is a prognostic marker for poor survival. additionally, The Bakal lab has shown that RELA localisation is regulated by cell shape, but the mechanistic basis is unknown. Using live imaging of CRISPR-CAS9 fluorescently tagged RELA, I characterised single cell endogenous RELA responses in two human PDAC cell lines in response to TNFa levels physiologically relevant to PDAC. PDAC cells responded atypically and with high sensitivity to TNFa, as RELA dynamics are sustained, non-oscillatory and cell cycle independent. Remarkably, single cells with higher RELA nuclear localisation are more likely to undergo TNF-a induced cell death, adding complexity to prior understanding of RELA as a pro-survival factor. To pinpoint the molecular mechanism of cell shape control of RELA, I incorporated measures of geometry and cytoskeletal proteins into Bayesian network models and identified actin dynamics as a source of heterogeneity in RELA translocation with TNFa. Using small molecule inhibitors, I discovered that the nucleation of actin stressed fibres and branched actin function in RELA responses to TNFa. I the used RNA sequencing and Co-IP with protein mass spectrometry to identify effectors and interactors of RELA in PDAC. Notably, RELA significantly upregulates the non-canonical NF-kB pathway, as well as the actin regulators ARHGAP31 and NUAK2. Furthermore, I found evidence that negative feedback by IkBb and e govern RELA dynamics in PDAc cells. Finally, I characterised RELA responses to reovirus infection, in combination with PARP inhibition based on a screen result, providing the first temporal resolution of single cell RELA responses to oncolytic viruses. PDAC cells responded to the reovirus T3D with continuous RELA dynamics while melanoma cells displayed a switch-like response. Moreover, cells with high RELA nuclear localisation with reovirus infection underwent cell death. Notably, PARP inhibition enhanced reovirus-induced RELA nuclear translocation and cell death. These discoveries confirm RELA hyperactivity in PDAC on the single cell level and identify positive feedback loops between RELA and actin, in addition to perturbation of negative feedback, as underlying mechanisms. I also provide evidence that elevated RELA nuclear localisation is associated with cell death in multiple immune-related settings.