Harnessing TNF-mediated cell death in cancer

Lead Research Organisation: Institute of Cancer Research
Department Name: Division of Breast Cancer Research

Abstract

Inflammation and cell death are ancient processes of fundamental biological importance that enable survival and adaptation during infection and injury. Tumour necrosis factor (TNF) is the prototypical proinflammatory cytokine that signals, through its type 1 receptor (TNF-R1), either cell survival, cell proliferation or cell death1. TNF stimulates an inflammatory response whose 'purpose' is to remove the source of the disturbance, allowing the host to adapt to an abnormal condition, and, ultimately, to restore functionality and homeostasis to the tissue. However, when deregulated, inflammation can drive chronic remodelling and tissue repair, which contributes to chronic inflammatory diseases, cancer and treatment failure.

The principle idea addressed by this application is that cell survival and adaptation mechanisms of tumours are supported, at least in part, by cancer-related inflammation, and that this can be successfully targeted by switching the TNF response from pro-inflammation to cell death. Despite clear evidence that TNF can signal cell survival and death, the mechanisms that can switch between the distinct biological outcomes remain elusive. This is important as its resolution, and putative therapeutic intervention, would allow the diversion of cancer-related inflammation into activation of cell death. Our starting point for this proposal is substantial new data on the regulation of TNF-signalling and cell death.

We identified that the Ub-receptor function of cIAP1 critically controls TNF signalling, selectively regulating the pro-death effects of TNF, without inhibiting the NFkB pathway. Our preliminary data indicate that a point mutation in the Ub-binding domain of cIAP1 switches the TNF response to cell death, and completely blocks tumorigenesis. Here, we propose to use mouse models, imaging, and biochemical approaches to decipher how the Ub-receptor function of cIAP1 regulates TNF-induced cell death, and whether switching the TNF response from pro-inflammation to cell death causes tumours to permanently regress.

Cytokines of the TNF-superfamily are classic inducers of programmed necrosis (necroptosis). Although anticancer therapies that trigger the necrotic death of tumour cells may be particularly attractive to overcome apoptosis resistance, because TNF-induced necroptosis also facilitates inflammation, and deregulated inflammation can also support tumorigenesis, it will be important to carefully examine the physiological and pathological consequence of stimulating TNF-induced necrosis. At present, there is no clear evidence to indicate whether necrosis is beneficial or harmful in cancers. Here we will determine the therapeutic opportunities and pathological consequences of manipulating TNF signalling in cancer.

Given that TNF plays an eminent role in diverse pathological processes at the core of human diseases, our findings will provide new avenues for therapeutic intervention strategies.

Technical Summary

Deregulated cell death and inflammation can drive chronic remodelling and tissue repair, which can contribute to cancer and treatment failure. The principle idea addressed by this application is that cell survival and adaptation mechanisms of tumours are supported, at least in part, by cancer-related inflammation, and that this can be successfully targeted by switching the TNF response from pro-inflammation to activation of cell death, causing tumours to permanently regress.

Despite clear evidence that TNF can signal cell survival and death, the mechanisms that can switch between the distinct biological outcomes remain elusive. This is important as its resolution, and putative therapeutic intervention, would allow the diversion of cancer-related inflammation into activation of cell death. Our starting point for this proposal is substantial new data on the regulation of TNF-signalling and cell death.

We identified that the Ub-receptor function of cIAP1 critically controls TNF signalling, selectively regulating the pro-death effects of TNF, without inhibiting the NFkB pathway. Our preliminary data indicate that a point mutation in the Ub-binding domain of cIAP1, which abrogates its ability to bind to M1- and K63-linked Ub chains, switches the TNF response to cell death, and completely blocks tumorigenesis. Mechanistically, we find that the Ub-binding domain of cIAP1 regulates intracellular trafficking of TNF-R1, and the consequences of RIPK1 ubiquitylation. Our data are consistent with a scenario whereby Ub-binding of cIAP1 participates with ESCRT-0 in capturing and sorting ubiquitylated TNF-R1 and RIPK1 for recycling.

Here, we propose to use mouse models, imaging, and biochemical approaches to decipher how the Ub-receptor function of cIAP1 regulates TNF-induced cell death, and whether switching the TNF response from pro-inflammation to cell death is beneficial or harmful in cancer.

Planned Impact

Impact and potential beneficiaries of this research:

-Scientific community:
The successful conclusion of the proposed project is expected to provide a better understanding of the molecular mechanisms that control Ubiquitin (Ub)-dependent regulation of signal transduction pathways that influence the ability of multi-cellular organisms to adapt to perturbations. As such our research addresses fundamental issues relevant to normal animal physiology and following exposure to stress. Further, as Ub-mediated regulation of cell death and tissue repair is involved in almost every aspect of life, the proposed project will also impact the health-care community. Of particular interest is the notion that cell death regulatory proteins also fulfill nonlethal functions in differentiation and tissue remodeling. It is now clear that the cell death machinery is involved in releasing signals to communicate with their cellular environment, to promote cell division, tissue regeneration, and wound healing. Unraveling the molecular details governing this process could lead to a better understanding of aging, tissue regeneration and cancer.

- Cancer patients:
One of the key problems in cancer is the adaptive nature of tumours. It is now recognised that the Ub system modulates key signal transduction pathways that have direct implications in the 'evolvability' of tumours. Hence, cancer treatments would be significantly more successful if it was possible to target Ub-dependent signalling events. Our research addresses a fundamental aspect of Ub-dependent tissue repair. Ultimately, this work will provide new insights into the mechanisms through which Ub-dependent signalling contributes to cancer, and how we can translate this information for patient benefit.

- Public sector, Academia/Pharma based drug development teams:
Given TNF's importance under physiological and pathological conditions, and its central role in chronic inflammatory diseases, inflammation-associated cancer, and cancer-related inflammation, the results of this project will be of significant interest not only for academic but also private drug development groups. The identification of the molecular mechanisms through which TNF signalling can be switched from pro-inflammation to cell death will open up new avenues for therapeutic intervention strategies. It is anticipated that a clinical trial could follow from this study within five years. This will have obvious benefits in health and well being affecting the public sector, and can be exploited by the industry for the development of novel technologies for prevention and therapy of liver cancer in the early phases of the disease.

- General public, schools:
The project is a clear-cut example of the development of suitable experimental models for understanding the mechanism of disease and to develop new methods for prevention and cure. Thus results arising from the project can be used to illustrate the process in education, leading to obvious cultural benefits by emphasizing the importance of scientific approaches in health care.

Publications

10 25 50
 
Description Harnessing cell death mechanisms to overcome treatment resistance and improve tumour immunity
Amount £1,261,388 (GBP)
Funding ID 24399 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 01/2023
 
Title CRISPR knockout cells 
Description We have generated various cell lines that lack individual components of the TNF receptor signalling complex 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact The generation of specific knockout cell lines will help the dissection of inflammation and cell death in cancer. 
 
Title CRISPR mediated deletion of specific DNA regions 
Description we developed improved techniques to apply the CRISPR/CAS9 technique to delete specific genes from the genome 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? No  
Impact This will improve the way through which we analyse the function of specific genes. 
 
Title Detection of Ripoptosome assembly 
Description Here we have developed a proximity ligation assay to detect the assembly of the ripoptosome, a large (2kDa) protein complex consisting of RIPK1, FADD, Caspase-8, FLIP, RIPK3 and Myo7a. We use a combination of antibodies that generate a localized, discrete signal only when two proteins are in close proximity, that is, in a complex. Using specific primary antibodies against the respective members of the complex, which in turn are recognized by oligonucleotide-coupled secondary antibodies, we can obtaine discrete proximity labelling of protein:protein interactions, confirming their close proximity. To control for nonspecific binding between the various secondary antibodies, we generally add only one or no primary antibody to the reaction. This results in no signal. We find that MYO7A/CASP8 proximity labelling is entirely dependent on the presence of RIPK1. Accordingly, depletion of Ripk1 by RNAi completely abolishes CASP8/MYO7A proximity signals. Likewise, the interaction between CASP8 and RIPK3 is strictly RIPK1 dependent. This result also demonstrates that the observed proximity signal is not due to nonspecific binding of the primary antibodies. The requirement of RIPK1 for the generation of CASP8/MYO7A or CASP8/RIPK3 proximity signals strongly suggests that CASP8 and MYO7A or CASP8 and RIPK3 interact with one another within RIPK1- based protein complexes such as the ripoptosome. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact This method allows single cell analysis of protein complexes in situ, that is without breaking up cells. 
 
Title IAP research reagents 
Description we have generated various cell lines that harbour specific mutations in the cIAP gene 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact This allows the dissection of inflammation and cell death, and provide important insights for future anti-cancer therapies. 
 
Title Phosphorylation-specific antibody for RIPK1 S320/321 
Description This phosphorus-specific antibody detects phosphorylation of RIPK1 at position S321 of mouse and S320 of human RIPK1 
Type Of Material Antibody 
Year Produced 2017 
Provided To Others? Yes  
Impact This antibody is being used by many labs to study MK2-mediated phosphorylation of RIPK1 
 
Title Prescission mediated characterisation of Ubiquitin linkages on specific lysine residues 
Description We developed a new method that allows the characterisation of specific Ubiquitin linkage types on specific lysine residues. This is important because specific Ubiquitin linkages emanate specific signalling events. However, so far it has been impossible to characterise this. This is the first time that it will be possible to map the linkage types on specific lysine residues. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This represents a paradigm shift in the way Ub signalling will be analysed. This method will help to characterise specific signalling events in vivo. 
 
Title Ubiquitin restriction analysis 
Description We have improve the UbiCrest technique that allows the characterisation of Ubiquitin-dependent signalling events 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact This will allow to dissect the signalling events that control inflammation and cancer development 
 
Title cIAP1 UBA knockin mouse 
Description To study the function of the UBA domain of cIAP1 in vivo, we generated a conditional knock-in mouse bearing the MF>AA mutation in the absence of cIAP2. Previous work indicated that cIAP1 and cIAP2 function redundantly to each other. Therefore, we generated the conditional cIAP1UBAmut mouse from an ES cell clone that previously had been targeted at the cIAP2 locus. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? Yes  
Impact While it is beyond doubt that cIAPs suppress TNF-induced cell death, how this is achieved remains unclear. The main problem in dissecting cIAP-mediated regulation of TNF-induced cell death has been the fact that the signaling aspect of Ub (recruitment/ activation of TAK1, IKK, MK2, and NF-kB-mediated gene induction) and the direct Ub-dependent anti-apoptotic function of cIAPs cannot be separated. We now identified a point mutation in cIAP1 that selectively sensitizes cells to TNFinduced cell death, without interfering with TNF-mediated activation of NF-kB, and IKK- and MK2-mediated phosphorylation of RIPK1. This mutation affects the evolutionarily conserved ubiquitin-associated (UBA) domain of cIAP1. Mice with a knockin mutation in the UBA domain develop normally but are acutely sensitive to TNF-induced systemic inflammatory response syndrome (SIRS), which is caused by enhanced sensitivity to TNF-mediated cell death. Our data are consistent with the notion that the UBA domain is required for Ub-mediated regulation of RIPK1 kinase activity. 
 
Title primary and immortalised murine cell lines 
Description Using wild-type and mutant mouse strains, we generated different primary and immortalised mouse lines. These included mouse embryonic fibroblasts, mouse dermal fibroblasts, bone marrow derived macrophages and keratinocytes. Such cells were generated from the following mouse strains: WT, cIAP1-/-, cIAP2-/-, cIAP1cIAP2-DKO (double knockout), Ripk1-/-, Ripk3-/-, Mlkl-/-, cascade-8-/-, Ripk1casp8-DKO, cIAP1-UBAcIAP2-/-, cIAP1-UBA. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? Yes  
Impact Many of these reagents were requested by other research labs, and form the basis of future investigations. 
 
Description Harnessing cell death mechanisms to improve therapeutic responses in lung cancer 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We conducted several experiments to test what therapeutic combinations would be best suited to kill a particular lung cancer type
Collaborator Contribution Our collaborators provided an in vivo mouse model for lung cancer in which we could test the therapeutic benefit of a particular drug combination.
Impact Our experiment contributed to the finding that targeting LUBAC sensitises lung cancer cells to chemotherapy. This work was published in J. Exp Med doi: 10.1084/jem.20180742
Start Year 2016
 
Description IAP-mediated regulation of SIRS 
Organisation University of Ghent
Department Department of Biomedical Molecular Biology
Country Belgium 
Sector Academic/University 
PI Contribution We have generated double targets KO/KI animals of cIAP2/cIAP1 to test the role of the ubiquitin-binding domain of cIAP1. We find that cells from such animals are sensitive cytokine induced cell death.
Collaborator Contribution Our collaborator have tested the in vivo sensitivity of such animals using a model for Systemic Inflammatory Response Syndrome (SIRS).
Impact not available yet
Start Year 2016
 
Description Identification of specific Ubiquitin chains on components of the TNF receptor signalling complex 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution we have provide novel findings to address the question what types of signalling chains are conjugated to specific components of the TNF receptor signalling complex.
Collaborator Contribution The collaborator has provided us with recombinant enzymes that allowed us to investigate the Ubiquitin linkage types that are conjugated to components of the TNF receptor signalling complex.
Impact the study that reports the findings from this collaboration has been accepted for publication at Molecular Cell. This study has led to a better understanding of TNF signalling
Start Year 2015
 
Description Immunogenic Cell Death 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Different types of cell deaths have different immunological consequences. Here we are collaborate with scientists at the CRICK to learn the latest methodologies in measuring cross priming of CD8+ T cells.
Collaborator Contribution The scientists at the CRICK are teaching us methods of measuring cross priming of CD8+ T cells.
Impact no outputs have yet occurred
Start Year 2018
 
Description Immunogenic Cell Death-2 
Organisation Genentech, Inc
Country United States 
Sector Private 
PI Contribution We are collaborating with Genentech in identifying cell death mechanics that are most immunogenic, meaning that they activate a patient's own immune system.
Collaborator Contribution Our collaborators are sharing with us reagents and methodologies that help us to characterise immunogenic cell death.
Impact no outputs have yet arisen.
Start Year 2019
 
Description MIB-mediated regulation of CYLD 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology (LMCB)
Country United Kingdom 
Sector Academic/University 
PI Contribution We identify that the E3 ligase MIB2 targets the deubiquitylase CYLD for ubiquitylation and inactivation, providing a new concept through which DUBs can be regulated.
Collaborator Contribution Our collaborators structurally investigated how ubiquitylation of CYLD might influence its activity.
Impact This collaboration resulted in the identification of a novel concept of DUB regulation. The collaboration ended with MRC because the collaborator left for the Walter and Eliza Hall in Melbourne, Australia.
Start Year 2016
 
Description MIB-mediated regulation of inflammation 
Organisation University College Dublin
Department School of Medicine and Medical Science
Country Ireland 
Sector Academic/University 
PI Contribution We provided reagents and cell lines to investigate the role of Mind bomb E3 ligases in cytokine signalling. This provided the Martin lab with important reagents, and will provide them with co-authorship on a publication, which is currently submitted to Molecular Cell
Collaborator Contribution This collaboration allowed us to investigate the role of Mind bomb E3 ligases in the regulation of TNF-mediated induction of cytokines. Seamus Martin from Trinity College conducted cytokine profiles and TNF dose response curves for us.
Impact the publication that reports this collaboration is currently under review in Molecular Cell. The work established that Mind Bomb E3 ligases play no role in TNF-mediated activation of cytokines in a panel of cell lines. The observation contributes to our current understanding of inflammatory signalling and how cross-priming can be achieved, thereby improving anti-cancer strategies.
Start Year 2015
 
Description MIB2-mediated regulation of RIPK1 and CYLD 
Organisation Harvard University
Department Department of Biological Chemistry & Molecular Pharmacology (BCMP)
Country United States 
Sector Academic/University 
PI Contribution We identified a novel mechanism through which MIB E3 ligases regulate RIPK1-mediated cell death. This identified the Dead Domain of RIPK1 as an acceptor domain for Ubiquitin.
Collaborator Contribution Hao Wu modelled the Death Domain of RIPK1 to investigate whether the ubiquitylation of this domain might interfere with its function.
Impact This collaboration resulted in an improved understanding of RIPK1 signalling..
Start Year 2016
 
Description MK2-mediated regulation of RIPK1 
Organisation The Walter and Eliza Hall Institute of Medical Research (WEHI)
Department Cell Signalling and Cell Death Division
Country Australia 
Sector Charity/Non Profit 
PI Contribution We provided our collaborators with vital information with regards to how the MK2 kinase regulates RIPK1. We further provided them with experiment, which demonstrated that MK2 directly regulates cell death by phosphorylating RIPK1 in health and disease.
Collaborator Contribution Our collaborators provided us with AML tumour models to test whether inhibition of MK2 might provide a therapeutic benefit in combination with IAP inhibitors.
Impact this collaboration resulted in improved understanding of how MK2 contributes to tumour formation and treatment failure. The collaboration also resulted in a submission of a manuscript to Mol Cell.
Start Year 2016
 
Description MK2-mediated regulation of cell death 
Organisation Klinikum der Universität München
Country Germany 
Sector Academic/University 
PI Contribution this collaboration focuses on the complex relationship between cell death and inflammation. In particular we have been investigating how MK2 regulates RIP kinase activity. We identified that MK2 directly phosphorylates RIPK1 at residue S321, a site previously been identified by Manolis Pasparakis (University of Cologne) and John Silke (WEHI). We established this collaboration to establish how MK2 regulates RIPK1 in health and disease. Our collaborators provided us with biological materials from the knock-in animals, which we analysed for their sensitivity to death triggers
Collaborator Contribution Our collaborators generated a knock-in mouse that bears phospho-mimicking and non-phosphorylatable mutants of RIPK1. Moreover, our collaborator provided patient samples that we analysed.
Impact Generation of genetically engineered animals, and cells thereof.
Start Year 2016
 
Description MK2-mediated regulation of cell death 
Organisation The Walter and Eliza Hall Institute of Medical Research (WEHI)
Country Australia 
Sector Academic/University 
PI Contribution this collaboration focuses on the complex relationship between cell death and inflammation. In particular we have been investigating how MK2 regulates RIP kinase activity. We identified that MK2 directly phosphorylates RIPK1 at residue S321, a site previously been identified by Manolis Pasparakis (University of Cologne) and John Silke (WEHI). We established this collaboration to establish how MK2 regulates RIPK1 in health and disease. Our collaborators provided us with biological materials from the knock-in animals, which we analysed for their sensitivity to death triggers
Collaborator Contribution Our collaborators generated a knock-in mouse that bears phospho-mimicking and non-phosphorylatable mutants of RIPK1. Moreover, our collaborator provided patient samples that we analysed.
Impact Generation of genetically engineered animals, and cells thereof.
Start Year 2016
 
Description MK2-mediated regulation of cell death 
Organisation University of Cologne
Department The Institute for Genetics
Country Germany 
Sector Academic/University 
PI Contribution this collaboration focuses on the complex relationship between cell death and inflammation. In particular we have been investigating how MK2 regulates RIP kinase activity. We identified that MK2 directly phosphorylates RIPK1 at residue S321, a site previously been identified by Manolis Pasparakis (University of Cologne) and John Silke (WEHI). We established this collaboration to establish how MK2 regulates RIPK1 in health and disease. Our collaborators provided us with biological materials from the knock-in animals, which we analysed for their sensitivity to death triggers
Collaborator Contribution Our collaborators generated a knock-in mouse that bears phospho-mimicking and non-phosphorylatable mutants of RIPK1. Moreover, our collaborator provided patient samples that we analysed.
Impact Generation of genetically engineered animals, and cells thereof.
Start Year 2016
 
Description MLKL-mediated Necroptosis 
Organisation Auburn University
Country United States 
Sector Academic/University 
PI Contribution This collaboration focusses on the regulation of necroptosis, a controlled form of lytic cell death. We have identified a regulatory mechanism through which the key effector MLKL triggers necroptosis. In particular we have generated a MLKL knock-in mouse model in which we changed a particular residue that is important for MLKL regulation. We have done all the preliminary characterisation of this model with respect to its sensitivity to cel death induction.
Collaborator Contribution Jason Upton, a world leader in virus induced necroptosis, at Auburn University tested the in vivo relevance of the MLKL mutation. He assessed whether the mutant animal, and cells thereof, is more susceptible to viral infection.
Impact This collaboration is multi-disciplinary. Discipline involved are: cell death immunity inflammation virology
Start Year 2019
 
Description Regulation of cell competition 
Organisation Columbia University
Department Department of Genetics and Development
Country United States 
Sector Academic/University 
PI Contribution We have provide a novel molecular link in the regulation of cell competition and inflammatory signalling. In addition, we have provided reagents and research tools to our collaborator.
Collaborator Contribution Our collaborator investigated the involvement of our key protein in their assay system to gain a better understand of the regulatory mechanisms of cell competition.
Impact currently there are no outputs or outcomes of this collaboration
Start Year 2015
 
Description Regulation of necroptosis 
Organisation The Walter and Eliza Hall Institute of Medical Research (WEHI)
Country Australia 
Sector Academic/University 
PI Contribution Here we are studying the molecular mechanism through which the necroptosis effector MLKL is activated and how its cytotoxic potential is regulated. The key aspect is to understand how we can induce necroptosis in cancer cells. Necroptosis is an immunogenic form of cell death that will greatly help to activate a patient's own immune system against cancer.
Collaborator Contribution James Murphy is providing structural support and advise in studying MLKL, a key effector of necroptosis. James previously crystallised MLKL (Immunity. 2013 Sep 19;39(3):443-53; Biochem J. 2013 Nov 13), which has greatly helped us in resolving how MLKL is regulated.
Impact not applicable yet.
Start Year 2019
 
Description Saint Analysis of putative binding partners of components of the TNF receptor signalling complex 
Organisation University of Michigan
Country United States 
Sector Academic/University 
PI Contribution We provided extensive mass spec data sets for analysis.
Collaborator Contribution The collaborators used the provided mass spec data set to run Saint Algorithms. This identified putative binding partners of components of the TNF receptor signalling complex.
Impact the data reporting this analysis has been accepted at Cell Reports.
Start Year 2014
 
Description The role of IAPs and cytokine signalling in pancreatic cancer 
Organisation University of Lausanne
Country Switzerland 
Sector Academic/University 
PI Contribution We have generated double targeted KO (lacking cIAP1 and cIAP2) animals that we are currently characterising for their phenotype with regards to cell death, inflammation and cancer. We have identified that these animals are sensitised to cytokine induced cell death.
Collaborator Contribution Our collaborator will investigate whether conditional animals with targeted alleles in cIAP1 and cIAP2 are resistant to inflammation driven pancreatitis.
Impact not yet available
Start Year 2016
 
Description UBA mediated regulation of Th17 helper cells 
Organisation Technical University of Denmark
Department Department of Micro and Nanotechnology
Country Denmark 
Sector Academic/University 
PI Contribution Our collaborators investigated the role of the UBA domain in cIAP1 for the development and functionality of Th17 T helper cells. They examined bones from animals harbouring a point mutation in the Ubiquitin-associated domain of cIAP1 to test whether this domain affects the population of T-helper cells of the Th17 subtype
Collaborator Contribution We generated Knockin animals harbouring a point mutation in the UBA domain of cIAP1, and provided such animals to our collaborator
Impact Detailed analysis revealed that the UBA domain of cIAP1 does not affect Th17 cell function.
Start Year 2017
 
Description UbiCrest analysis of cytokine signalling 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Using the UbiCrest methodology, we are studying the ubiquitin linkage types that are mediated by IAPs. This provides us with a conceptional framework how inflammatory signalling is achieved and regulated.
Collaborator Contribution David Komander has provided us with the necessary reagents to conduct the UbiCrest analysis.
Impact Using the UbiCrest analysis tool set we determined the linkage repertoire on components of the TNF signalling complex. This has resulted in a better understanding of TNF signalling in health and disease.
Start Year 2015
 
Description Ubiquitin-mediated regulation of RIPK1 kinase activity 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We are investigating the mechanism of RIPK1 kinase regulation and have developed a unique model system to study how ubiquitylation of RIPK1 controls its kinase activity
Collaborator Contribution GSK has provided us with RIPK1 kinase inhibitors.
Impact This collaboration has led to a better understanding of how RIPK1 kinase is regulated in health and disease.
Start Year 2016
 
Description mass spectrometric identification of components of the TNF-receptor signalling complex 
Organisation Canadian Institutes of Health Research
Department Institute of Cancer Research
Country Canada 
Sector Public 
PI Contribution We have provided purified samples for mass spectrometric analysis.
Collaborator Contribution Our collaborators have analysed our samples using state-of-the art mass spectrometry. Moreover, using Saint Analysis, this has helped us to identify putative binding partners of components of the TNF signalling complex.
Impact The manuscript that the reports these mass spectrometric findings has been accepted for publication Cell Reports.
Start Year 2014
 
Description mass spectrometric identification of components of the TNF-receptor signalling complex 
Organisation Max Planck Society
Department Max Planck Institute for Terrestrial Microbiology
Country Germany 
Sector Academic/University 
PI Contribution We have provided purified samples for mass spectrometric analysis.
Collaborator Contribution Our collaborators have analysed our samples using state-of-the art mass spectrometry. Moreover, using Saint Analysis, this has helped us to identify putative binding partners of components of the TNF signalling complex.
Impact The manuscript that the reports these mass spectrometric findings has been accepted for publication Cell Reports.
Start Year 2014
 
Description mass spectrometric identification of components of the TNF-receptor signalling complex 
Organisation University of Basel
Department Biozentrum Basel
Country Switzerland 
Sector Academic/University 
PI Contribution We have provided purified samples for mass spectrometric analysis.
Collaborator Contribution Our collaborators have analysed our samples using state-of-the art mass spectrometry. Moreover, using Saint Analysis, this has helped us to identify putative binding partners of components of the TNF signalling complex.
Impact The manuscript that the reports these mass spectrometric findings has been accepted for publication Cell Reports.
Start Year 2014
 
Description Awareness event at the House of Lords 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Talk and discussion groups brought together clinicians, fundamental biologists, researchers from pharmaceutical companies, policymakers and end users to discuss how breast cancer research, and novel treatment regiments, could be improved via a change in clinical practice for patient benefit.

Apart from a positive feedback, there were no recordable impacts.
Year(s) Of Engagement Activity 2015,2016,2017,2018,2019
 
Description Cell Death Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact The activity was to educate medical student and Vets about the different forms of cell deaths.
Year(s) Of Engagement Activity 2020
 
Description Fundraising event at the Breast Cancer Now Research Centre 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A series of fund raising events took place throughout these years, each event saw 20 -30 people taking part. Ongoing research was communicated orally, which led to lively exchanges of questions and answers

Fundraisers liked the event, which prompted many repeat venues in which the current project was explained in great details
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015