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

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.

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.

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.