Role of BRCA2 in the response to replication inhibitors

Some cancer patients have specific genetic defects in their cancer cells that influence their response to anti-cancer treatment. Patients with cancers such as breast and pancreatic cancer can have mutations in the BRCA2 gene, which makes their cancer cells less able to repair damage in their chromosomal DNA. This is important for therapy because many anti-cancer treatments work by causing deadly damage to the DNA. Patients with and without mutations in BRCA2 do therefore respond differently to some of these treatments. DNA-damaging drugs include agents called replication inhibitors such as Gemcitabine, which are used for chemotherapy of breast and pancreatic cancer.
How cancer cells with BRCA2 mutations respond to replication inhibitors is not well understood. This project aims to characterise how BRCA2 prevents and repairs DNA damage in cells treated with replication inhibitors to generate new insights into the treatment response in genetic backgrounds frequently found in cancer patients.

I will use my expertise in the field of replication fork progression and the DNA damage response to replication inhibitors to further our understanding of the role of BRCA2 and HR in the response to replication inhibitors. We will use Gemcitabine (20,20-difluorodeoxycytidine) as a model replication inhibitor and BRCA2-deficient and BRCA2-complemented normal and cancer cell lines of human and hamster origin. We will use DNA fibre analyses to measure replication fork progression in presence of Gemcitabine and replication fork restart after removal of Gemcitabine. Cells will be labelled with chlorodeoxyuridine (CldU) for 20 minutes followed by incubation with Gemcitabine for periods from 30 min to several hours. Fork stalling and restart will be monitored by adding iododeoxyuridine (IdU) in presence or after washing out of Gemcitabine. Chromosomal DNA will be spread out on microscope slides and labelled DNA detected by immunofluorescence using specific antibodies.
We will further investigate the role of BRCA2 in the repair of Gemcitabine-induced DNA damage using immunostaining for DNA damage markers phospho-Serine S4/S8 RPA, phospho-Serine 139 H2AX (gamma-H2AX) and 53BP1 to detect excessive single-stranded DNA and double-strand breaks. We will also measure DNA breaks by comet assay and pulse-field gel electrophoresis. To investigate the role of BRCA2 in cellular sensitivity and resistance to Gemcitabine we will use clonogenic survival and apoptosis assays.
To better understand the mechanism of BRCA2 function at stalled forks, we will use a combination of mutant cell lines, siRNA depletion and small molecule inhibitors to investigate the roles of DNA repair proteins (RAD51, BRIP1, PALB2 and MRE11) that functionally interact with BRCA2 during the response to Gemcitabine. The known molecular functions of these proteins will allow us to work towards a model of the molecular transaction promoted by BRCA2 at stalled replication forks.

The proposed research is a basic research that will contribute to improved health and treatment outcomes in the mid- to long term. Findings from basic research can be translated into clinical trials within a few years, as happened for the use of PARP inhibitors in BRCA1 and BRCA2-defective cancers. Public beneficiaries of this research will include cancer patients, patients with Fanconi Anaemia, the NHS and cancer charities.

1. Cancer patients.
There is a lot of interest among the public in general and cancer patients in particular in better treatments for cancer. To achieve this, we need to understand how cancer patients carrying different genetic mutations respond to treatments, and why some patients fail to respond to treatments that work well in others. The results from this study will help to generate hypotheses about the interaction of replication inhibitors such as Gemcitabine with genetic defects in homologous recombination that can then be tested in the pre-clinical or clinical setting. This could ultimately lead to a more personalised approach to cancer therapy, making sure that patients receive the treatments that they are most likely to respond to. This would help improve treatment efficacy and reduce unwanted side effects of treatments, increasing quality of life and well being for the patients.
Cancer patients that are treated with replication inhibitors will benefit from a better understanding of the action of these drugs, and cancer patients carrying mutations in BRCA2, FANCN or FANCJ will benefit form a better understanding of the functions of these genes in the response to replication inhibitor treatments.

2. Fanconi Anaemia patients.
Homozygous mutations in BRCA2, FANCN and FANCJ cause the genetic disorder Fanconi Anaemia in children, characterised by bone marrow failure and cancer predisposition (http://www.fanconi.org.uk/). Fanconi Anaemia patients carrying homozygous mutations in BRCA2, FANCN or FANCJ will benefit form a better understanding of the functions of these genes in DNA replication and DNA repair.

3. The NHS and Cancer Charities.
These are also major stakeholders in the search for better and more personalised cancer treatments. Using the drugs that are most likely to work will help to reduce the cost of cancer treatment, which is also a major issue for the public, the NHS and cancer charities.