Telomere maintenance pathways and their role in genome stability.

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

Most cancer cells have the capacity to replicate indefinitely. This is partly achieved by activation of a telomere maintenance mechanism (TMM) and we are trying to understand the molecular basis of some of these pathways. Telomeres are repetitive DNA sequences that cap chromosomes and so prevent damage to the genome. The majority of tumours activate telomerase or the Alternative Lengthening of Telomeres (ALT) pathway to maintain telomeres but the TMM is unknown in a subset of tumours. In addition the molecular mechanism that underlies ALT is poorly understood. Recently we have shown that activation of the ALT pathway can destabilize other repeated sequences in the genome but the significance of this observation is unclear. Therefore using single telomere and single molecule approaches we plan to investigate telomeric DNA and other sequences in order to elucidate the underlying TMM in tumours. In addition in cell lines we plan to disrupt the expression of genes that are thought to play a role in ALT and study the effect on telomeres and other DNA sequences in the genome. In the long term we hope this will lead to identification of targets for novel anti-cancer therapies.

Technical Summary

We have shown that human telomere repeat arrays are prone to mutations, with a germline mutation rate of 0.6% per kb. We proposed that the germline mutation process is dominated by intra-allelic events and that inter-allelic recombination is uncommon. Our recent analysis of sequences adjacent to the Xp/Yp telomere, encompassing a hyper variable minisatellite (DXYS14), strongly supports this view. We have also shown the telomere mutation frequency is significantly higher in sporadic colon cancers, increasing to 35% per allele in tumours with defects in mismatch repair (MMR). Again the mutation process appears to be dominated by intra-allelic events. In contrast complex telomere mutations, discovered in cells that use the Alternative Lengthening of Telomeres (ALT) pathway to maintain telomere length, are highly likely to arise by recombination-like processes that underlie ALT. Control of the ALT pathway and genes required for telomere lengthening are poorly understood. Recently we have shown that the MS32 minisatellite, which normally mutates by recombination in the human germline, becomes highly unstable upon activation of the ALT pathway but four other minisatellites remain stable. To investigate the link between the minisatellite and telomere instability in ALT+ cell lines we will measure mutation frequencies at a variety of minisatellites, selected by their location in the genome and the nature of the repeat array (sequence composition, repeat length). To determine whether the destabilization of MS32 is a localized perhaps chromatin mediate effect, we will study other repeated sequences in the vicinity of the MS32 locus and we will examine gene expression in related cell lines with and without ALT activity. We will investigate the frequency of extraordinary MS32 instability in soft tissue and other sarcomas to determine whether it has value as diagnostic marker. We also plan to utilize the MS32 minisatellite as a tool to dissect the ALT pathway. Initially we will disrupt the expression of helicase genes thought to play a role in ALT and study the effect on the mutation rate and profile at the MS32 locus and at single telomeres. We plan to take a similar approach to study the effect of disruption of the MMR pathway on telomere stability and length. Finally we plan to develop PCR based tools for the analysis of a subset of telomeres in the mouse. Such tools will be valuable in the investigation of the ALT pathway and of telomere length dynamics in mutant strains that model some human diseases.

Publications

10 25 50
 
Description CRUK Leicester Centre Development Fund
Amount £9,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2014 
End 08/2015
 
Description CRUK Project Grant
Amount £180,000 (GBP)
Funding ID C17992/A8641 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2007 
End 09/2010
 
Description Eastley-MRC Clinical training fellowship
Amount £140,000 (GBP)
Funding ID MR/N02107X/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2018
 
Description Hope Against Cancer, Leicestershire and Rutland
Amount £15,000 (GBP)
Organisation Hope Against Cancer (Rutland and Leicestershire) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 05/2018
 
Description MRC Project Grant
Amount £421,204 (GBP)
Funding ID G0901657 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2010 
End 08/2013
 
Description Rosetrees Trust project
Amount £30,000 (GBP)
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2016 
End 04/2018
 
Description UHL Charitable Funds - Foxtrot
Amount £5,000 (GBP)
Organisation University Hospitals of Leicester NHS Trust 
Department UHL Charitable Funds
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 04/2018
 
Description UHL Charitable Funds - Foxtrot
Amount £6,000 (GBP)
Organisation University Hospitals of Leicester NHS Trust 
Department UHL Charitable Funds
Sector Charity/Non Profit
Country United Kingdom
Start 02/2015 
End 01/2017
 
Title single molecule telomere analysis 
Description The method combines single molecule telomere amplification (smSTELA) with characterisaton of sequence-variant telomere repeats (TVR-PCR). This facilitates detailed telomere mutation analysis on DNA samples from human tissues samples. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact Using this approach we have been able to make a significant contribution to understanding the turnover of human telomeric DNA in normal human tissues and in tumours. 
 
Title STS sample collection 
Description We have collected fresh frozen tissue samples from patients with soft tissue sarcomas ( tumour, normal adjacent tissue, blood). The samples are held in the Nottingham Health Sciences Biobank for our project but will be available to other projects at a later date. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact Currently there is one published research paper from this sample collection. Other publications will follow. 
 
Description ALT in liposarcomas 
Organisation Fondazione IRCCS Istituto Nazionale dei Tumori
Country Italy 
Sector Charity/Non Profit 
PI Contribution The work for this project was initiated and conducted in my lab and the results analysed and written by me. Our collaborator kindly provided us with a panel of DNA samples from liposarcomas in which telomere length, telomerase activity and the presence / absence of APBs had been determined.
Collaborator Contribution Our collaborator provided DNA samples and some clinical information from a panel of liposarcomas
Impact PMID: 18311780
Start Year 2006
 
Description Collaboration re CTAGGG repeats 
Organisation National Museum of Natural History
Country United States 
Sector Academic/University 
PI Contribution My group initiated the invesitgation. We identified the CTAGGG repeats within human telomeres and showed that they cause localized instability in the telomeric DNA in somatic cells and in the germ-line. I proposed the model by which the CTAGGG repeat might cause instability and initiated the collaboration to explore it.
Collaborator Contribution Our collaborators conducted some DNA binding and DNA damage assays and showed that the CTAGGG repeats bind efficiently to hPot1 and to other Shelterin components in vitro. in addition they investigated the unusual structure formed by the CTAGGG repeats.
Impact PMID: 19656953 PMID: 19692585
Start Year 2008
 
Description Collaboration re TRF2 in ALT+ cells 
Organisation Medical Research Council (MRC)
Department MRC Toxicology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution We did the telomere length analysis and cytogenetics to look for chromosome fusions, indicative of uncapped telomeres repaired by non homologous end joining. We contributed to the model that was developed to explain the observations.
Collaborator Contribution As a result of our collaboration we expanded our expertise, to include the use of shRNA technology.
Impact PMID: 18056407
Start Year 2006
 
Description Jacqui Shaw 
Organisation Cancer Research UK
Department Leicester Cancer Research UK Centre
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Royle is the academic lead with expertise in telomere biology; makes significant intellectual contribution to the research project involving the molecular characterisation of tumours and other samples that are collected. Royle is also responsible for training clinical and no-clinical staff involved in the project.
Collaborator Contribution Shaw contributes expertise in the analysis of circulating cell free tumour DNA and provides access to a GCLP standard laboratory and dedicated equipment for ddPCR. Shaw and team provide training for staff involved in the project.
Impact doi.org/10.1136/jclinpath-2016-204151
Start Year 2015
 
Description Nottingham Biobank 
Organisation Nottingham Health Science Biobank
Country United Kingdom 
Sector Hospitals 
PI Contribution Patient recruitment; analysis of samples collected.
Collaborator Contribution Collection of samples from operating theatres and clinics; processing of samples adn long term storage
Impact Two papers: Eastley, N et al. Collaboration is multi-disciplinary. Biobank staff suuport translational research and rovide longterm storage of samples. University group with NHS collegaues lead the study and provide expertise in molecular genetics; cell biology, bioinformatic analysis of samples collected and stored by the Biobank.
Start Year 2015
 
Description Robert Ashford 
Organisation University Hospitals of Leicester NHS Trust
Department UHL Charitable Funds
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Royle is a co-applicant an ethically approved study with Ashford (Genetic profile and telomere characteristics and of high-grade soft tissue sarcomas; IRAS 141820). Royle is the academic lead on the laboratory research project involving the molecular characterisation of tumours and other samples that are collected. Royle makes significant intellectual contribution to the research project and is responsible for training clinical and no-clinical staff involved in the project.
Collaborator Contribution Mr Ashford (Consultant Orthopaedic and Musculoskeletal Tumour Surgeon in UHL Trust) is the lead applicant on an ethically approved research project: Genetic profile and telomere characteristics and of high-grade soft tissue sarcomas (IRAS 141820). He is responsible for all the clinical aspects of the project. He facilitates the recruitment of patients to the study and is responsible for compliance with the ethical considerations surrounding informed consent; for training and supervision of staff involved in sample collection.
Impact Ethically approved research project: Genetic profile and telomere characteristics and of high-grade soft tissue sarcomas REC reference:14/NE/1192; IRAS ID: 141820. doi:10.1136/jclinpath-2016-204151
Start Year 2014
 
Description Tumour testing -IT 
Organisation Imagen Therapeutics
Country United Kingdom 
Sector Private 
PI Contribution Send tumour samples; generate whole exome data and conduct bioinformatic analyses
Collaborator Contribution Personalised drug sensitive testing of individual tumours
Impact Presentation to NHS staff at research away day. A publication is planned. The collaboration is multi-disciplinary the company has expertise in tumour drug testing and University group conducts molecular genetic analysis on samples.
Start Year 2017
 
Description Barwell- Developing a shared vision for Genomic Medicine 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Approximately 80 people attend this meeting with the aim of identifying how genomic medicine could be take forward and be integrated in to the NHS now and in the future. Several ideas emerged including working closely with a neighbouring country to form an international Genomic Medicine Centre that could us mutually beneficial in understanding how best to store and used genomic data now (arising from the 100,000 genome project) and in the future. In addition ideas about provision of short modular training courses for NHS staff were proposed.
Year(s) Of Engagement Activity 2014,2017
 
Description CRUK- fundraising events 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Informed members of public about role of telomeres in maintenance of genome stability an role in cancer formation / progression.

Helped to raise the profile of cancer research conducted at the University of Leicester
Year(s) Of Engagement Activity 2008,2009,2010
 
Description Cancer charity - outreach event 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact Approximately 70 people attended and evening of talks and poster presentations about cancer research. This sparked discussions and greater awareness of our ongoing research.
Year(s) Of Engagement Activity 2017
 
Description Flagship lecture 1st year undergraduates 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Approximately 280 first year undergraduates in Biological Sciences attended a flagship lecture entitled: Genetics, Ageing & Telomeres: the ends insight
Year(s) Of Engagement Activity 2016
 
Description Hope - outreach event 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact Estimated 70 adults attend this event and they were able to engage various genetics outreach events and view posters that explain various projects funded by the charity including our work of soft tissue sarcomas.
Year(s) Of Engagement Activity 2016
 
Description Outreach to primary school in Leicester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I organized a half day visit to the Department of Genetics for 60 children (year 6) and teachers. This was done in conjunction with a CETL 'Genie' at University of Leicester.

Feed back was very positive. Hope this event served to enthuse young pupils about science and about studying at University.
Year(s) Of Engagement Activity 2007
 
Description Outreach to secondary school in Leicester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I organised and conducted a practical class in a science club at a local secondary school (duration 2hrs; 35 students + teachers).

Feed back was very positive. Hope this event served to enthuse pupils about science and about studying at University.
Year(s) Of Engagement Activity 2006
 
Description Pint of Science 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Royle gave an 'outreach' talk about telomeres and her research in the first year a 'Pint of Science' event to be held in Leicester
Year(s) Of Engagement Activity 2019
URL https://pintofscience.co.uk/
 
Description Poster at BSG 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presented, as a poster, our pilot study that aims to determine whether testing patients soft tissue sarcomas, removed at surgery, for sensitivity to over 50 chemotherapeutic drugs has the potential to be used to improve selection of adjuvant treatment regiome in a highly to personalised approach. We have demonstrated variability in response between tumours and identified some drugs that have the portenial to be more effetive than others currently used to treat recurrence or metastatic disease.
Year(s) Of Engagement Activity 2019
URL http://www.britishsarcomagroup.org.uk/events/event/bsg-conference/
 
Description Short talk at Genetics 50th Celebrations: Open Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Talk stimulated interest, evidenced by comments received following the event

Not measured
Year(s) Of Engagement Activity 2014
 
Description TEN UK 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Royle from the University of Leicester and two other colleagues, from Univeristy of Sussex and University of Newcastle, organised the second Telomere network UK (TEN UK) 2 day workshop, in Leicester Sept 2019. The purpose was to bring together UK research group leaders; potsdoctoral research associates and PhD students who investigate telomeres in a wide variety of species. The workshop promoted interacting between groups and gave early career researchers the opportunity to presnt their work and develop their own networks.
Year(s) Of Engagement Activity 2019
URL https://www2.le.ac.uk/departments/genetics/news/ten-uk-meeting-2019
 
Description TEN-UK 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Royle from the University of Leicester and two other colleagues, from Univeristy of Sussex and University of Newcastle, organised the first Telomere-network-UK (TEN-UK) 2 day workshop, in Newcastle, Sept 2018. The purpose was to bring together UK research group leaders; potsdoctoral research associates and PhD students who investigate telomeres in a wide variety of species. The workshop promoted interacting between groups and gave early career researchers the opportunity to presnt their work and develop their own networks.
Year(s) Of Engagement Activity 2018
 
Description TalK at BSG 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Selected presentation delivered by Clinical Fellow; sparked questions;
Year(s) Of Engagement Activity 2018