TRAIN-ERS Research Areas

WP4 (ER-BIO) will focus on acquiring a comprehensive mechanistic understanding of the ER stress response and its pathway interplays using state of the art “omics” technologies, bioinformatics, quantitative molecular and cell biology, and systems biological methodologies. There are 5 ESR projects in WP4.

WP5 (ER-DISEASE) will provide the rationale for targeting specific UPR components as a therapeutic strategy in three disease areas by demonstrating that modulation of key UPR regulators using genetic, chemical and biological approaches can modify disease development or progression in disease models. There are 4 ESR projects in Work Package 5(WP5.)

WP6 (ER-DRUG) will focus on developing/identifying compounds that modulate the function of validated UPR targets through a combination of molecular/computational modelling and drug design, virtual screening, chemical synthesis and structure activity relationship analysis. WP6 will also develop serum-based multiplexed UPR activation diagnostics assays.There are 5 ESR projects in Work Package 6 (WP6).

The research programme is organised into 3 complementary multidisciplinary Work packages.

Work Package 4

ESR1: Omics technologies to understand the ER stress response

Studies in ER stress to date have largely used traditional single gene and protein centred approaches in diverse disease models, and there has been no comprehensive study to understand the global response to ER stress in normal cells.

ESR1 will use cutting edge “omic” technologies to comprehensively detect the global cellular changes in RNA, Protein, glycosylation and metabolites due to ER stress in normal epithelial cell lines.

ESR2: Contribution of RIDD activity to cell survival/death

To uncover key RIDD substrates that influence cell survival and cell death ESR2 will use recombinant IRE1α cytosolic domain and shRNA technology to assess the contribution of RIDD substrates to normal cellular processes and on cancer cell characteristics (e.g., chemoresistance and cell growth, adhesion/migration and invasion

ESR3: ER stress regulated autophagy and autophagic cell death

Autophagosome may function as a platform facilitating pro-caspase-8 activation and thereby influencing cell fate. ESR3 will use a proteomics approach to investigate the full components of the caspase-8 activating complex, including the inhibitors and regulators of the complex.

Genetic approaches (overexpression, siRNA, Crispr/CAS9) will be used to test the effect of different components of caspase-8 activating complex on autophagy and cell fate.

ESR4: Deciphering and exploiting aggresome-associated cell death initiation to predict anti-cancer treatment efficacy.

ESR4 will investigate the kinetics and quantities of apoptosis vs. necroptosis activation using semi-HTS flow cytometry and biochemistry in melanoma models.

Highly sensitive FRET-based time-lapse imaging of caspase activation in combination with biophotonic indicators for necroptotic membrane permeabilisation will provide insight into whether both cell death modes can be co-activated at the single cell level.

Components of systems biological network analysis will be employed for data interpretation and additional mechanistic insight, leading towards the development of predictive treatment response models.

ESR5: Functional role of the newly discovered PERK-interacting partners

Understanding the functional impact of the PERK-interactome and its dynamic changes during ER stress will be crucial to understand the global role of PERK in disease conditions (e.g., cancer) and to develop strategies aimed at blocking specific interactions, while sparing others.

The host laboratory has identified new molecular partners of PERK, which modulate the dynamic of interorganellar contact sites and response to Ca2+ fluxes, independent of the UPR.

ESR5 will investigate how specific perturbations/disruptions of the newly identified PERK-interactome (shRNA, CRrisp-CAS9, pharmacological inhibition) affect key cancer cell features under basal or ER stress conditions.

Work Package 5

ESR6: UPR as a novel therapeutic target in paediatric cancers

This project will focus on exploring the potential of targeting the UPR in paediatric cancers.

ESR6 will screen a large number paediatric cancer cell lines and primary patient material for sensitivity to ER stress inducers to identify childhood cancers that are particularly prone to ER stress.

Combinations of ER stress inducers and other cytotoxic drugs will be tested to identify synergistic drug interactions and a synthetic lethality screening assay using a genome-wide shRNA library will be used to discover novel candidate genes that regulate ER stress-triggered cell death in childhood cancers.

Candidate genes will be characterized and validated in cell culture and in vivo using the chorioallantois membrane model.

ESR7: Assessing the potential of IRE1/XBP1s for therapeutics and diagnostics in breast cancer (BC)

ESR7 will determine whether the IRE1α/XBP1s signalling axis can serve as a viable diagnostic or prognostic marker or therapeutic target in BC, by analysing XBP1s expression in approximately 100 BC and normal matched patient tissue samples.

ESR7 will also examine the therapeutic potential of blocking IRE1α/XBP1s signalling in a panel of human BC cell lines (T47D, SKBR3, MDA-MB231, SKBR3, BT474) and the non-tumorigenic breast cell line MCF10A.

ESR8: ER stress in neurodegenerative disease

Protein misfolding is a common feature of several neurodegenerative diseases which leads to a profound upregulation of ER stress markers.

ESR8 will determine whether the IRE1α/XBP1 signalling component of UPR is a viable target for treatment of amyotrophic lateral sclerosis/ motor neuron disease (ALS), in view of the involvement of this pathway in ALS and the finding that a mutation in the familial form of ALS attenuates this pathway.

This hypothesis will be tested by analysing the therapeutic potential of IRE1 activating peptides and peptide mimetics in a range of ALS models including  cell lines harbouring mutations associated with familial ALS and  a motor neuron cell line, NSC-34, to assess their effects on formation of ubiquitinated protein aggregates, autophagy and motor neuron survival.

ESR9: ER stress and UPR in inflammatory liver disease and shock

ESR9 will identify specific inflammatory mediator patterns inducing ER stress and will clarify the impact of ER stress/UPR on the development of liver dysfunction upon shock and inflammation using in vitro models and  in vivo models of shock (hemorrhagic shock and local ischemia) and inflammation (bacterial endotoxin induced systemic inflammation).

The main objective of this study will be to understand the impact of ER-stress/UPR on organ/cellular function and inflammatory response in these experimental models. These data will provide a rationale for the development of novel diagnostic and therapeutic strategies focused on modulation of UPR.

Work Package 6

ESR10: Development of inhibitors to block essential protein-protein interactions

ESR10 will use a range of in silico software for homology modelling, protein-protein interaction studies, and virtual high-throughput screening in order to develop small molecule and peptidomimetic inhibitors targeting RtcB-XBP1-mRNA interaction, IRE1 activity and interactions, and ATF6.

Part of the work is done in close collaboration with ESR11 and 12, and also involves experimental work (secondments) at participating laboratories to verify mechanisms of action of said compounds.

ESR11: Developing IRE1-activating agents for the treatment of degenerative diseases

ESR11 will combine and analyse all the data available on the peptide activators of IRE1 and the binding site interactions of IRE1 activators (Hsp72, Bim, PUMA) to model the interactions and to screen virtual compound libraries for hits that mimic the effects of the activator peptides or proteins.

These IRE1-activating compounds will be synthesised and tested in in vitro and in cell based assays for effects on cellular processes, on signalling networks, on survival under ER stress conditions and on the secretome.

ESR12: Targeting ATF6 as an anti-cancer strategy

ESR12 will develop strategies to block ATF6 activation by targeting its processing at the Golgi.

ESR12 will carry out a virtual screen for compounds that inhibit ATF6 processing and develop a GFP-tagged ATF6 cell-based assay in order to test and prioritize hit compounds from the virtual screen.

ATF6 inhibitors will be tested for efficacy in breast cancer models to determine if targeting this branch of the UPR is a viable therapeutic strategy.

ESR13: Development of a multiplexed serum test based on ER stress biomarkers

ESR13 will develop a biochip that can be used to detect pathological levels of ER stress markers in serum.

ESR13 will assess novel candidate markers of ER-related stress identified in hepatic, motor neuron and leukaemia cells with/without ER stress-inducing agents and identify common ER stress-related secreted factors and cell type specific ER stress related secreted factors.

Based on this data ESR13 will develop biochips that can detect ER stress-related secreted factors in well-defined clinical sample cohorts to investigate their potential for disease state identification.

ESR14: Assessing the UPR for predictive value in melanoma patients

ESR14 will deep sequence and quantitate ER stress-related factors in tumour tissue samples taken from a cohort of 100 melanoma patients who were treated with Pembrolizumab (Keytruda) or Ipilimumab (Yervoy).

Subsequently, ESR14 will use the OPTI high level multiscale modelling platform to determine the value of these markers for predicting response to immunotherapy prior to treatment.