Defining the role of ERK5 kinase and ERK5 transcriptional activities in cell migration and EMT using novel ERK5 inhibitors

Lead Research Organisation: Babraham Institute
Department Name: Signalling

Abstract

The cells in our body are constantly subjected to changes in their environment and they contain an extensive network of signalling pathways that coordinate appropriate responses. In the developing embryo, cells may receive stimuli or cues telling them to divide (so called growth factors) or they may receive cues telling them to cease dividing and undergo 'differentiation', a process in which cells acquire the characteristics of specialized cell types that make up the discrete tissues in our adult bodies such as nerves, blood cells in the immune system or our skin. This process of cell division and differentiation continues in adults in certain tissues; which constantly renew themselves such as our skin.
For cells to respond to growth or differentiation cues they must activate key growth or differentiation proteins; this often involves increasing the abundance of these proteins. The genetic information for these proteins is stored in discrete pieces of DNA (genes), which reside on chromosomes in the nucleus. When a cell receives a growth signal these genes are 'read' by 'transcription factors', discrete proteins that bind to DNA and transcribe the DNA information the into messenger RNA (mRNA) molecules, which are in turn 'translated' into the relevant proteins. This coupled process of transcription and translation is called 'gene expression'.

This whole complex process is orchestrated by signalling pathways, which control every step. Control is the key word here. For example, if the cells divide too much or fail to differentiate correctly they may become cancerous. The signalling pathways controlling cell division and differentiation typically involve cascades of enzymes called protein kinases. These enzymes 'tag' other proteins with a phosphate group (a process called phosphorylation) and this changes the activity, abundance or localisation of the protein. The tagged protein is referred to as the 'substrate' of the protein kinase enzyme. This project concerns a protein kinase called ERK5.

1. There is much interest in finding drugs that block ERK5 activity (ERK5 inhibitors or ERK5i) as they may help to treat inflammation, cardiovascular disease or cancer. Indeed, we have been working with a team of scientists to identify new ERK5i that inhibit ERK5 kinase activity. However, to our surprise they actually promote gene reading or transcription. The ERK5 protein is unusual in that it has two quite different functional regions or domains. The first is the kinase domain, which phosphorylates substrates; the second is a transcription factor domain, which binds DNA to read genes. Our results suggest that when an ERK5i inhibits the kinase domain it causes structural changes that activate the transcription factor domain. So one aim is to understand at the molecular level how this happens and whether this is a good thing or a bad thing for designing ERK5 inhibitors.
2. Second, we want to identify the genes that ERK5 binds to so we can better understand the role of ERK5 - and specifically the two functional domains of ERK5 - in gene expression.
3. Our recent experiments have suggested that ERK5 activity is important in regulating a differentiation process called epithelial-to-mesenchymal transition (or EMT). EMT is important during development of the embryo, during wound repair and for cancer cells to spread around the body and invade new sites - a process called metastases. Indeed, we have found that blocking ERK5 activity reverses EMT and prevents the movement of cells. So a final aim of this project is to understand how ERK5 controls this EMT process and whether it is controlled by the kinase domain or the gene reading domain of ERK5.

This study should tell us more about the normal role and regulation of ERK5. ERK5 may also be important in clinical conditions (inflammation, cardiovascular disease, cancer) so our results may have wider impacts and we will work with scientists in these areas to progress this.

Technical Summary

ERK5 is unusual in consisting of a kinase domain (KD), similar to ERK1/2, and a C-terminal domain with an NLS and transcriptional transactivation domain (TAD) that can autonomously bind chromatin and promote gene expression. The relationship between the KD and TAD is poorly understood. ERK5 controls angiogenesis & neural differentiation in the developing embryo. It also promotes B cell survival, inflammation, cardiovascular disease and fibrosis so there is widespread interest in finding ERK5 inhibitors (ERK5i). We have been involved in an ERK5 drug discovery programme that has identified nanomolar ERK5i. In the course of this work we have found that:
1. Whilst ATP-competitive ERK5is completely inhibit the isolated ERK5 KD, they cause the paradoxical, kinase-independent activation of the ERK5 TAD in full length ERK5.
2. ERK5 inhibition reduces cell migration and reverses the epithelial-to-mesenchymal transition suggesting that ERK5 may be important in cell movement, wound repair and conceivably tumour cell invasion.

We suggest that binding of ERK5is inhibits kinase activity but also elicits a conformational change that exposes the ERK5 TAD allowing ERK5 to enter the nucleus to drive gene expression. Understanding these dual effects is critical to understanding how ERK5 functions and exerts its biological effects. It is also critical if we are to understand how best to employ ERK5 kinase inhibitors that are being developed for a variety of indications. Is it most important to inhibit the KD or the TAD? To inform these issues we will define how ERK5i-dependent inhibition of the ERK5 KD elicits activation of the ERK5 TAD, identify genomic targets of the ERK5 KD and ERK5 TAD signalling functions and investigate the role of ERK5 (KD and TAD domains) in EMT.
Our basic biology study may have far reaching implications for colleagues studying diseases where ERK5 is implicated and where the significance of paradoxical ERK5 TAD activation has not even been considered.

Planned Impact

The primary impact will come from new knowledge of mechanisms of signal transduction, related to the role of ERK5 in various biological contexts (see Academic beneficiaries).

Impacts on industry and other stakeholders:
1. Industry: All major pharmaceutical companies remain interested in protein kinases as drug targets for a variety of diseases. Several of these companies have active ERK5 inhibitor programmes (AstraZeneca, Boehringer Ingelheim, etc) and ERK5 is being positioned for a variety of indications including cancer, inflammation, fibrosis and cardiovascular disease. ERK5 is clearly druggable and this proposal developed out of a collaboration with the University of Newcastle including Astex Pharmaceuticals and CRT as commercial partners. Our research will therefore be relevant to a range of BioPharma companies contributing to UK economic competitiveness and we have clear pathway to progress this.

2. BBSRC: Within the BBSRC 2010 Strategic Plan this work maps to BBSRC Strategic Priority 3: Basic Bioscience Underpinning Health. In particular 'basic molecular science underpinning the translation of knowledge about drug targets into chemical and biological tools and drugs'. Relevant areas include: new tools in chemical biology, lipidomics and genomics; molecular cell biology, chemical biology and biochemistry to drive the discovery and validation of new drug targets or selective pharmaceuticals. The project exemplifies the use of Partnerships, with contributions across sectors (Institutes, Universities, Industry).
Within the BBSRC Strategic Plan 2013/14 refresh this research maps to Strategic Research Priority 3 - Bioscience For Health - and is relevant to the Societal Grand Challenge of 'maintaining health across the whole lifecourse' and the Key Priority 'Generate new knowledge of the biological mechanisms of development and the maintenance of health across the lifecourse'. In particular, our work on 'signalling mechanisms will provide new insights to potential strategies for health monitoring and intervention, including drug targets and pharmaceuticals', consistent with the aspiration that 'basic bioscience funded by BBSRC underpins the pharmaceutical and healthcare industries'.

3. Healthcare and 3rd sector charities: ERK5 is implicated in a variety of processes that promote disease and infirmity in old age and is therefore of interest to the health sector. ERK5 is implicated in myogenesis and adipogenesis. Age-related loss of muscle mass significantly impairs quality of life in the elderly. Similarly, adipocytes are critical regulators of metabolism and are involved in a variety of metabolic diseases including obesity. In addition, ERK5 is directly implicated in inflammation, fibrosis and cardiovascular disease - all of which have ageing as a key risk factor. Thus understanding how ERK5 functions may contribute to future intervention strategies aimed at these problems. In addition, ERK5 is amplified in some cancers and our new results studying EMT suggest a role in metastases. Thus, our basic biology will be of interest to a variety of disease charities as well as the healthcare professions.

Training: This project will provide further training for key researchers (Lochhead & Cook) in new scientific skills in growth areas (chemical genomics/chemical biology, genomics, bioinformatics). It will build on Lochheads's excellent organisational skills, honed in industry, providing training for her future contribution to UK science & economic output.

Science & Society: We will continue to contribute to public STEM (science, technology, engineering and maths) understanding through our public engagement activities. Indeed, Lochhead has been closely involved in Cook lab public engagement activities, communicating her knowledge and enthusiasm to the next generation of scientists and informing interested adults through activities such as science exhibitions and science visits to schools and local community groups.

Publications

10 25 50
 
Description The ERK5 protein includes an N-terminal kinase domain (50% identical to ERK1/2). However, unlike other MAPKs, it contains a large, unique C-terminal extension that includes a nuclear localization signal (NLS) and a transcriptional activation domain (TAD). In seeking to assess the cell-based efficacy of ERK5 inhibitors we employed a transcription-based assay in which full length activated ERK5 (activated by co-expression with its activating kinase MEK5) phosphorylates the transcription factor MEF2D which in turn drives Luciferase expression; this was motivated by the lack of well-validated ERK5 substrates that would report ERK5 inhibition. Using this assay we found that two different ERK5 inhibitors caused a saturable inhibition of ERK5 but were only able to inhibit 60-70% of ERK5 activity. A residual 30% was always unaffected. When we repeated the assay with an ERK5 construct that contained only the kinase domain we observed 100% inhibition. Finally, we were able to show that ERK inhibitors could actually stimulate the activation of MEF2D driven by inactive full length ERK5 and this required the C-terminus of ERK5. So ERK5 inhibitors could cause a paradoxical activation of the ERK5 TAD.

Our high-level Aims were:
1. Investigate how ERK5 inhibitors induce MEF2D-mediated gene expression independently of ERK5 kinase activity.
2. Identify genes that are direct targets of ERK5.
3. Define the role of ERK5 in promoting EMT.

1. Investigate how ERK5 inhibitors induce MEF2D-mediated gene expression independently of ERK5 kinase activity
This Aim is complete. We have shown that ERK5 kinase inhibitors bind to the kinase domain of ERK5 and cause intramolecular conformational changes that expose the C-terminus, thereby de-repressing/activating the TAD. This work has now been written up and has been accepted for publication in Nature Communications. It completes all the key elements of Aim 1 and has defined a completely new mode of paradoxical kinase activation. This work will be of great relevance to ERK5 researchers, kinase researchers in general, those interested in kinase-independent effects of protein kinases and the Biotech/Pharma sector who are searching for ERK5 kinase inhibitors.

2. Identify genes that are direct targets of ERK5 Ongoing
Important groundwork for this Aim has now been completed. We have established HCT116 cell liens which exhibit Tet-regulated expression of mutationally activated MEK5D (also relevant to Aim 3) and are preparing for planned ChIP-seq experiments

3. Define the role of ERK5 in promoting EMT Ongoing
Important groundwork for this Aim has now been completed. We have optimized qPCR probes and antibodies for all key EMT regulators and markers, designed constructs for CRISPR-mediated ERK5 deletion and started gene targeting in HCT116 cells.

Summary. Very good progress towards objectives. Aim 1 complete; groundwork for Aims 2 & 3 completed. 3 papers published to date.

Papers and other outputs Status and/or Google Scholar citations
1. Lochhead PA, Clark J, Wang L-Z, Gilmour L, Squires M, Gilley R, Foxton C, Newell, DR, Wedge SR & Cook SJ (2016) Tumour cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation. Cell Cycle. 15:506-518 GS 15

2. Myers S, Molyneux L, Miller D, Arasta M, Blackburn T, Cook SJ, Edwards N, Endicott JA, Hammonds T, Hardcastle IR, Harnor S, Heptinstall A, Golding BT, Griffin RJ, Lochhead PA, Martin MP, Martin N, Newell DR, Reuillon T, Rigoreau LJ, Thomas HD, Tucker JA, Wang L-Z, Wong A-C, Noble MEM, Wedge SR & Cano C (2019) Identification of a Novel Orally Bioavailable Tool Compound for Studying the Role of ERK5 in Cancer. Eur J Med Chem. 178:530-543 GS 2

3. Lochhead PA, Tucker JA, Tatum NJ, Wang J, David Oxley D, Johnson VP*, Gray NS, Martin E.M. Noble MEM & Cook SJ (2020) Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors Nature Commun. In press

4. A mini-review on ERK5 signalling is also in preparation arising from this work.

5. We hosted a Summer Intern, Victoria Johnson, an undergraduate from the University of Cambridge. The PDRA, Pamela Lochhead successfully wrote a grant to secure funding for this internship. It was very successful and Victoria will receive co-authorship on one of the papers arising from this grant (see Paper 3 above *)
Exploitation Route Our findings will have a strong influence on ERK5 kinase inhibitor drug discovery projects in the Biotech/Pharma sector as well as with our academic collaborators
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our findings are already being used by academic and pharma collaborators undertaking ERK5 drug discovery projects. We have an active collaboration with a small company called Oppilotech Ltd and have onbgougn discussions with a second company
First Year Of Impact 2019
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Member of Scientific Advisory Board of Cancer Research Technology Discovery Lab
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Membership of Scientific Advisory Board for Cancer Research Technology
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Scientific Advisory Board for CRUK Programme Grant awarded to Northern Institute for Cancer Research, Univeristy of Newcastle
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a advisory committee
 
Title ERK5 KO cells lines 
Description Generated by CRISPR/Cas9; charcterisation ongoing 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? No  
Impact Too early to say 
 
Title ERK5 cell based assay 
Description A method for assessing the cellular activity of the ERK5 protein kinase 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact It has facilitated a joint academic/pharma ERK5 drug discovery project It has also facilitated academic research in this area leading to one research publication and a second in preparation 
 
Title ERK5 mutants 
Description A panel of mutants of ERK5 have been generated that are defective for ERK5 kinase inhibitor binding. These mutants no longer exhibit paradoxical activation of ERK5 transcriptional transactivation domain (TAD) by ERK5 kinase inhibitors, confirming that the TAD activating function is due to 'on target' activity of the inhibitors. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The wider impact of this is too early to say. These tools have important implications for understanding the role of ERK5 in any and all biological responses in which ERK is implicated as they allow separation of ERK5 kinase and TAD activities. 
 
Title ERK5 WT and KO gene data set 
Description We have generated an Illumina mRNA array gene expression data set in which we have compared WT and ERK5 KO immortalised MEFs 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact The results of this work have afforded new lines of enquiry and new directions for our research and have led to further research funding applications including BB/N015886/1 It will also lead to primary research publications and the results will be of interest to collaborators in the Pharma sector 
 
Description Astex Pharmaceuticals - Chem Biol 
Organisation Astex Pharmaceuticals
Department Astex Therapeutics Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have established assays that allow us separate the kinase and TAD functions of ERK5. We will now investigate the role of these activities in ERK5-driven cell motility and EMT. This willin part use unique ERK5 chmical biology probes provided by Astex
Collaborator Contribution This will in part use unique ERK5 chemical biology probes provided by Astex, which allow us to visualise ERK5 in cells and to capture ERK5 and associated proteins from cell extracts
Impact Too soon for tangible outcomes and outputs.
Start Year 2016
 
Description Developing novel cell based assays to find new inhibitors the RAS-RAF-MEK-ERK pathway 
Organisation PhoreMost
Country United Kingdom 
Sector Private 
PI Contribution We have developed cell-based transcriptional reporter assays that allow screening in cells for novel inhibitors of the RAS-RAF-MEK-ERK signalling pathways
Collaborator Contribution Our partners have sued these cell based assays to screen for novel peptide-based inhibitors using their proprietary technology
Impact This collaboration led to a successful Innovate UK funding award between PhoreMost and the Cook lab at the Babraham Institute It has also led to a separate 3-way research collaboration between PhoreMost, the Cook lab at the Babraham Institute and Plexxikon, a structure-based drug discovery SME in California
Start Year 2017
 
Description Machine learning to inform drug discovery 
Organisation Oppilotech Ltd
Sector Private 
PI Contribution Oppilotech are a systesm biology/modelling company who employ machine learning to identify new drug targets. We have provided them with wet lab data to parameterise their models and have tested predictions arising from them
Collaborator Contribution They have performed modelling of the ERK5 pathway based on our data and information in the literature. This collaboration supported by Institute KEC funding to promote Campus Company:Institute collaborations at the Babraham Institute
Impact Too early to say but results are encouraging
Start Year 2019
 
Description Newcastle/CRT/MRC ERK5 
Organisation Newcastle University
Department Northern Institute for Cancer Research Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution We have established a cell based assay that can be used to monitor ERK5 activity and screen for ERK5 inhibitors
Collaborator Contribution High throughput screening and follow up testing to identify ERK5 inhibitors
Impact One manuscript published; a second in preparation; further research funding application submitted; identification of ERK5 inhibitors
Start Year 2012
 
Description Escape Room Installation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A 'Signalling' Escape Room was designed by students within the Signallign Laboratory, working with the Public Engagement team. This was then presented by studnets and post-docs, including members of the Cook lab at both the Cambridge Science Festival and the Latitude Music Festival.
Year(s) Of Engagement Activity 2019,2020
 
Description In conversation with the Babraham Institute - part of Cambridge Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 1:1 dialogue with general public (open invitation but registration required for numbers).
Evening reception in which we explain our science and answer questions
Part of a programme of events for the annual Cambridge Science Festival
Year(s) Of Engagement Activity 2017
 
Description Participated in Babraham Institute exhibit at Royal Society Summer Science Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Babraham Institute prepared an exhibit - The Ageing Clock - which exemplified aspects of our ageing research portfolio for a public audience. Tjis was selcted tp be part of the prestigious Royal Society Summer Science Exhibition and I was involved in presentign this exhbit to the Public togehter with colleagues.
Year(s) Of Engagement Activity 2018
 
Description School visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The students were enthused about the topic of my presentation and this led to dialogue and discussion about several issues including new cancer therapies, evolution of drug resistance in cancer, the use of animals in research.


Anecdotally, the institute received requests for summer placement students following this visit.
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018,2019
 
Description Science Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Students visited the lab and undertook small lab-based proejcts supervised by students/post-docs and myself. I explained the research that we do and discussed ethical issues such as the use of animals in research.
This precipitated excellent discussion and dialogue.

We received excellent feedback from the schools involved and requests for further outreach activities
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017,2018,2019,2020
 
Description Visits by Teachers 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact My lab has hosted 6th form Biology teachers who were visiting my Institution during Half Term to update their knowledge as part of their CPD
Year(s) Of Engagement Activity 2016,2017,2018,2019,2020