A novel regulator of DNA double strand break repair fate with roles in immunity and oncogenesis

Lead Research Organisation: University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics

Abstract

DNA double-strand breaks (DSBs) are a highly toxic form of DNA damage that can kill cells and cause the types of mutations to the DNA in our cells that can trigger cancer. This cell-killing property of DSBs explains why they are intentionally generated in radiotherapy and chemotherapy treatments to eliminate cancer cells. However, DSBs also arise spontaneously in normal dividing cells, when the cell's DNA copying machinery encounters problems. These DSBs are typically recognised and processed by error-free DNA repair pathways, preventing them from causing the mutations that trigger cancer development. Perhaps paradoxically, in certain specialised tissues mutagenic DSB repair is actually favoured, providing a molecular mechanism by which genetic material can be transferred between different regions of the genomes in our cells to create genetic diversity. These mutagenic events are fundamental for the plasticity of our adaptive immune systems, reshuffling antibody gene segments in white blood cells so that they can generate the many different types of antibody that are needed to fight the many different pathogens that we are exposed to.

To counteract the potentially hazardous effect of unscheduled DSBs and respond appropriately when they are programmed, cells have evolved two dedicated DNA repair pathways: Homologous Recombination (HR) uses a copy and paste mechanism to repair DSBs accurately. It is therefore the most important pathway in dividing cells, as this is when most DSBs occur and the threat of new cells inheriting new mutations means accurate DNA repair is mandatory; On the other hand, Non-homologous end-joining (NHEJ) is a more simple pathway that can glue nearly any two DNA ends together, not caring about their sequence or whether they are the right ones. NHEJ normally repairs DSBs in non-dividing cells where they occur as isolated events, yet when numerous DNA ends are available it can be mutagenic. NHEJ is also crucial in our immune systems, where the fusing of DNA ends originating from two DSBs generated at different locations on our chromosomes is the required outcome. Because of this intrinsic discrepancy in desired DNA repair outcome between different cellular contexts, achieving the right equilibrium between these repair pathways is vital to ensure DSBs are appropriately resolved.

Recently, we have found imbalances in DNA repair pathway equilibrium to link normal immune-function to human breast cancer development. Indeed a specialised subset of proteins that normally facilitate antibody gene rearrangements, are also responsible for the mutations that accompany loss of the human breast cancer tumour suppressor 'BRCA1', driving malignant transformation. In new work, we have identified a new protein whose loss in BRCA1-deficient cells can rescue the DNA repair defect that normally afflicts them. However, this then renders them resistant to important anti-cancer drugs that are normally extremely effective at killing them. Importantly, we have found that this protein is likely to be crucial for normal immune function, promoting DNA repair in antibody genes. Our proposal extends on these exciting findings, using molecular, biochemical, and cell biology approaches to question the molecular basis of this protein's unanticipated function in DSB repair. We will also inactivate this gene in the mouse, to study its actual role in the immune system, and generate important reagents for in vitro studies. The benefit of this work will be two-fold: Firstly, it will provide new insight into normal immune function, aiding the understanding of human immunodeficiency disorders; Secondly, it will reveal the molecular basis of common human cancers, also yielding insight into potential mechanisms of drug resistance that face personalised medicine approaches. These findings may pave way to improved approaches to diagnose, treat and better manage cancer.

Technical Summary

DNA double-strand breaks (DSBs) are highly toxic and must be accurately repaired to counteract the threat of oncogenic mutations. However, in some tissues mutagenic DSB repair is actually favoured by the cell, providing a molecular mechanism by which genetic material can be transferred between genetic loci to create genetic diversity. To cope with this intrinsic discrepancy in desired DNA repair outcome between different cellular contexts, cells have evolved a complex regulatory system to maintain the right equilibrium between repair pathways, ensuring DSBs are appropriately resolved. Recently, we have found misregulation of this system to link normal immune-function to the genomic instability that characterises cancer. Specifically, a chromatin component of the non-homologous end joining pathway that normally mediates antibody gene rearrangements in lymphocytes, is also responsible for the genomic instability that accompanies loss of the tumour suppressor BRCA1.

We now identify a new protein whose depletion in BRCA1-deficient cells can rescue the HR defect that normally afflicts them, in turn rendering them resistant to clinically important anti-cancer drugs. Notably, we report that this protein's DSB repair activities are also crucial for normal immune function, protecting the integrity of DSB ends in antibody genes to promote their efficient repair. We propose several integrated approaches to elucidate the molecular basis of this protein's unanticipated function in DSB repair. The benefit of this work will be two-fold: Firstly, it will provide new insight into normal immune function, aiding the understanding of human primary immunodeficiency disorders; Secondly, it will reveal the molecular basis of genomic instability in common human cancers, also yielding insight into the potential mechanisms of drug resistance that face modern personalised medicine approaches. These findings may pave way to improved approaches to diagnose, treat and better manage cancer.

Planned Impact

The likely beneficiaries from this research include:

- The scientific community (as described in academic beneficiaries). If our hypothesis is correct we will identify a novel DSB repair player whose function in cooperation with or in parallel to 53BP1 and RIF1, modulate chromatin to regulate DNA repair pathway choice in immune function and cancer.

- Patients and the health service. Breast cancer is by far the most common cancer among women in the UK (2010), accounting for 31% of all new cases of cancer in females. It affects over 50,000 people/year in the UK and is responsible for more than 11,500 deaths/year. Our original route into this research came from a anti-cancer drug resistance perspective relevant for understanding patient responses to anti-breast cancer therapies. In tumour models, Mad2l2-loss renders breast cancer cells resistant to what normally represent highly effective therapies. These findings may therefore highlight mutations that are selected for during cancer evolution and therapy regimes. Although one would predict that the identification of such mutations in human cancer would lead to poor patient prognosis, they would at least help better predict responses to secondary treatment regimes, and help avoid the administration of futile treatments that result in patient suffering without giving therapeutic gain.

- Commercial beneficiaries such as the pharmaceutical industry. We also hope that our aim to understand the basic molecular mechanisms underlying tumourigenesis and drug resistance may facilitate the identification of compensatory pathways in cancer. These might then be targeted to selectively sensitise sub-classes of cancer in modern personalised medicine approaches. Any commercially viable finding will be discussed with Isis innovations, a nationally renowned technology transfer company.
 
Description Defining the mechanism and specificity of the 53BP1-Rev7 non-homologous end joining pathway in immunity and oncogenesis
Amount £877,449 (GBP)
Funding ID MR/R017549/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2018 
End 07/2021
 
Description Genome Editing Mice for Medicine (GEMM); at MRC Harwell; FLJ26957/ENSMUST00000133280.1 Floxed allele
Amount £0 (GBP)
Organisation MRC Harwell 
Sector Academic/University
Country United Kingdom
Start 03/2018 
End 03/2018
 
Title A knockout model for Rev7 function in Class-Switch Recombination using the CH12F3 murine B cell lymphoma cell-line 
Description - We implemented an efficient CRISPR-Cas9 genome-editing pipeline in the CH12F3 cell-line to engineer loss-of-function alleles in the Rev7 and other genes. - Rev7 knockout cell-lines faithfully recapitulate the class-switch recombination immune system defects presented in knockout mice. 
Type Of Material Cell line 
Provided To Others? No  
Impact - Replacement & Reduction: Substituting in vivo experiments with experiments using an in vitro system has substantially reduced our reliance on the use of transgenic mice to undertake significant portions of our research programme. As a result we have been able to substantially reduce animal numbers used in our experiments. 
 
Title Genetically engineered model of Rev7 function in the adaptive immune response 
Description - Generated a conditional knockout allele for Rev7 (Mad2l2) in B cells by generating and intercrossing mice bearing the Mad2l2-tm1c(WTSI) allele with the B cell specific Cre-recombinase driver line Mb1-cre 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Research in progress 
 
Description DNA repair pathway choice in therapeutic resistance in BRCA1-deficient cancer 
Organisation Netherlands Cancer Institute (NKI)
Country Netherlands 
Sector Academic/University 
PI Contribution Collaborative research outcomes from my team: - Molecular determination of the contribution of anti-cancer drug resistance factors to DNA double-strand break repair regulation. - We are continuing to identify novel mechanisms of drug resistance (PARP inhibitors) in BRCA1-mutation associated breast cancer. - Annual joint meetings between members of our groups
Collaborator Contribution Collaborative research outcomes from Sven Rottenberg's team: - Identification and molecular characterisation of therapeutic resistance factors in pre-clinical breast cancer models of BRCA1-deficiency - Annual joint meetings between members of our groups
Impact Xu G, Chapman JR, Brandsma I, Yuan J, Mistrik M, Bouwman P, et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature. 2015 Mar 23. Two new manuscripts in preparation.
Start Year 2013
 
Description DNA repair pathway choice in therapeutic resistance in BRCA1-deficient cancer 
Organisation University of Bern
Country Switzerland 
Sector Academic/University 
PI Contribution Collaborative research outcomes from my team: - Molecular determination of the contribution of anti-cancer drug resistance factors to DNA double-strand break repair regulation. - We are continuing to identify novel mechanisms of drug resistance (PARP inhibitors) in BRCA1-mutation associated breast cancer. - Annual joint meetings between members of our groups
Collaborator Contribution Collaborative research outcomes from Sven Rottenberg's team: - Identification and molecular characterisation of therapeutic resistance factors in pre-clinical breast cancer models of BRCA1-deficiency - Annual joint meetings between members of our groups
Impact Xu G, Chapman JR, Brandsma I, Yuan J, Mistrik M, Bouwman P, et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature. 2015 Mar 23. Two new manuscripts in preparation.
Start Year 2013
 
Description Bone Marrow Drive 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Media (as a channel to the public)
Results and Impact Catarina Oliveira, a Postdoctoral Scientist employed on these awards organised two bone marrow registration drives through the charity DKMS. The intended purpose was to raise awareness of blood cancers for World Blood Cancer Day and to raise awareness for the need to recruit people of different nationalities to the registry, primarily Southeast Asian nationalities. These were day-long events in two different University buildings which recruited 230 people in total. Two of laboratory colleagues helped with running the event (Dr Jordan Becker, Dr Kirstin Bilham). The second of these drives was covered by local media: http://www.banburycake.co.uk/news/headlines/15324061.Donor_appeal_for_poorly_toddler___39_may_have_saved_other_lives__39_/
http://www.oxfordmail.co.uk/news/15324061.Donor_appeal_for_poorly_toddler___39_may_have_saved_other_lives__39_/
http://www.oxfordtimes.co.uk/news/15324061.Donor_appeal_for_poorly_toddler___39_may_have_saved_other_lives__39_/. Further to these drives, Dr Oliveira was been named a finalist in the DKMS Let's Make a Spark awards in the Company Donor Recruitment Award category (awards ceremony 21st March 2018). Dr Oliveira was also been alerted to the fact that one of the people who was recruited during the drives she organised was 'matched' to a person needing a stem cell transplant, representing a life saved through her efforts. Many people who were not able to attend the registration events contacted Dr Oliveira indecentlly to learn more information regarding the bone marrow registry and went on to sign up directly with the charity which has stimulated interest and also educated people to the processes involved in stem cell collection.
Year(s) Of Engagement Activity 2016,2017
 
Description Oxford Science Fair 'Health day' (2nd of July 2016) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Suzanne Snellenberg, a Postdoctoral Scientist in my lan participated in this event:

A stall was set up at the Templars Square shopping centre. Children could make their own DNA strain with lego or pipe cleaners. While they were doing that the children and parents could ask about DNA/genetics.
Year(s) Of Engagement Activity 2016
 
Description Science Oxford's 'DNA After Dark' 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Peter Yeow, a PhD student from the Chapman lab participated on this engagement event:

The Wellcome Trust Centre for Human Genetics (WTCHG) took part in Science Oxford's 'DNA After Dark' event on Thursday 10th of November. It was an adults-only ecent where participants get a chance to have a go at selected hands-on science experiments. I volunteered to be apart of the team that helped to organize and run the event for that night.

It was a good opportunity for me have conversations in a reasonable amount of depth about my scientific research in a relaxed environment. The event was impactful, as many of the participants showed keen interest in the work we do at the Wellcome Trust Centre. Finally, a large number of the participants gave positive feedback at the end of the activity.
Year(s) Of Engagement Activity 2016
 
Description WTCHG Art and Science Collaboration 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The primary aim of this public engagement project was to create an art exhibition focusing on the research undertaken in the Wellcome Trust Centre for Human Genetics. For that purpose, we work in collaboration with a local art group -the Oxford Printmakers Co-op- to produce art pieces based on the work of different DPhil students within the centre.
Artist and DPhil student were paired up and, through several meetings, the students went through the research they were undertaking with the artist to help them understand the science. At the same time, while the artist was developing the artwork based on the research, the student itself should generate a small poster-summary to be exhibited alongside the final piece. The pieces were exhibited in the research Centre and at several centres within Oxford (Fusion Arts Centre, John Radcliffe Hospital).
Along that, a Family Day was also organised in the exhibition venue, where artist and DPhil students performed different activities, such as making DNA bracelets, playing with the genetic code
The exhibition was covered by local media.
Year(s) Of Engagement Activity 2016,2017
 
Description Windmill Primary School DNA Day 
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 • Cathy Oliveira, a postdoctoral researcher in my laboratory put together a detailed application to apply for seed funds from The Wellcome Trust to start a new programme describing DNA and genetics to 5-6 year old primary school children.
• The application was successful and provided funds to purchase items for a 2-hour long workshop for 90 children and their teachers/teaching assistants
• The activities which took place during the workshop were:
o Assembling a life-sized human body made of foam, which encompassed bones, muscles, organs and skin
o Putting together a 'cell' which was made of felt, and putting together the basic parts including the cell membrane, cytoplasm, nucleus and mitochondria
o Observing cells down a microscope
o Making a model of DNA out of pipe cleaners and beads, which the children could take home- parents fed back to me that this sparked discussion at home about DNA and the human body in general
o All of the hand-crafted items were produced by me utilising the funds made available, and are now held in the institute's Public Engagement cupboard for anyone else to use for future workshops.
• Two postdocs, a lab manager and a D.Phil student from the Chapman laboratory helped put together the workshop and attended all three workshop days at the school
Year(s) Of Engagement Activity 2016