Defining the mechanism and specificity of the 53BP1-Rev7 non-homologous end joining pathway in immunity and oncogenesis

The major aim of this research project is to refine our understanding of the 53BP1 pathway: a poorly understood sub-pathway of the vertebrate non-homologous end joining (NHEJ) DNA repair system that plays a vital role in the human adaptive immune response, yet also drives the initiation and progression of breast, ovarian and other tumours in patients harbouring a faulty copy of the BRCA1 tumour suppressor gene. A key objective of the project is to challenge a widely accepted paradigm for the molecular function of this pathway. We will set out to test a hypothesis that proposes an alternative explanation for this pathway's activity. Namely, that the single-stranded DNA enrichments detected at DNA double-strand break (DSB) sites in cells deficient for the 53BP1 pathway, do not represent product of a failure to protect DNA ends from resection as is widely perceived, but instead represents a structured DNA repair intermediate/substrate that cannot be overcome when the pathway fails. If correct, the genetically modified mice we plan to engineer will be immune-deficicient as a result of an inability to efficiently generate antibody diversity via 53BP1-dependent NHEJ. Nevertheless, cells from these animals will be able to survive and divide relatively healthily in the absence of the activity of BRCA1, a protein normally essential for sustaining normal cell proliferation and the maintenance of genome stability. Both scenarios will provide us with valuable biological tools with which we will attribute mechanism to the 53BP1 DNA repair system, in biological contexts directly related to human health and disease. Other distinct, yet complementary experiments will examine secondary predictions of our model, including one that may explain the context specificity of the 53BP1-dependent DNA repair system, thereby linking its immune system functions to its potent oncogenic activities in cancer.

This project aims to determine the mechanism and specificity of the 53BP1 pathway in regulating DNA double-strand break (DSB) repair in immunity and cancer. I hypothesise that the three known situations in which 53BP1 mediates NHEJ (class switch recombination (CSR), fusion of uncapped telomeres, and genomic instability in BRCA1-deficient cancers) are connected to a similar DNA intermediate: staggered DSBs comprising secondary-structure forming single-stranded DNA termini.

Supported by a body of compelling new experimental evidence, we make the prediction that the 53BP1-dependent repair system integrates a previously unexpected activity to convert staggered DNA ends into DSBs amenable to NHEJ, and that this activity is toxic when such structures form inappropriately, such as at upcapped telomeres or in HR-defective cancers. We will test these predictions using a combination of pre-existing and newly generated transgenic mouse models, with which we will dissect DSB repair mechanisms in the murine immune system and in cellular models of BRCA1-deficiency. In doing so, we will challenge the currently accepted paradigm for the 53BP1 pathway (resection inhibition), and propose an alternative explanation for the molecular events that occur when it fails. If our predictions are correct, this work will underpin a new paradigm to explain the context specificity of 53BP1-dependent NHEJ, with important consequences for understanding vital molecular mechanisms in normal cell physiology and oncogenic transformation.

The likely beneficiaries from this research include:

- The scientific community (as described in academic beneficiaries).
- 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, 53BP1 pathway-loss renders breast cancer cells resistant to what normally represent highly effective therapies. These findings may therefore highlight mutations that may be 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. We will be receptive to commercially viable findings as the project progresses, any of which will be discussed with Oxford University innovations, an internationally renowned technology transfer company.