Uncovering function and mode of activation of the central Fanconi Anemia FANCD2/FANCI DNA repair protein complex, a potential cancer drug target.

The human genome contains thousands of genes, activities of which are important for a functioning healthy human body. Our organism is made up of billions of cells, all originating from one single cell. This is possible, since that original cell divided into two cells, which subsequently produced four cells, and so on. It is of crucial importance that the entire genome is duplicated accurately during each division. The genome is constantly subjected to damage from a variety of sources from inside or outside the cell. If cells fail to maintain an intact genome,, mutations can arise, which can cause a number of severe diseases, such as cancer. To counter-act the effects of damage, our cells have evolved sophisticated mechanisms to protect DNA, so called DNA repair pathways. These pathways safeguard the genome to prevent harmful mutations from arising. When one of these pathways fails to function properly, it allows for mutations to arise unhindered. One particularly serious example of this is when the Fanconi Anemia DNA repair pathway is not operational. When that happens, it causes the disease Fanconi Anemia (FA). FA is a genetic disease characterized by various developmental defects and predisposition to cancer in patients. Many patients do not survive beyond their teenage years, underscoring the seriousness of the disease. So far, 18 FA proteins have been identified, which includes the BRCA1 and BRCA2 genes, which are mutated in breast cancer. Together, these proteins compose the FA pathway, which is responsible for repairing DNA crosslinks, a dangerous type of DNA damage. Mutation in any of the corresponding 18 genes can cause FA. At the same time, since these DNA crosslinks are so dangerous, drugs causing such crosslinks are used in the clinic to treat cancer in non-FA cancer patients. However, patients often develop resistance to these otherwise so successful drugs. It is believed that resistance is caused by an increased ability of the cancer cells to repair DNA crosslinks. If we could prevent the cancer cells from repairing the crosslinks, we could treat these deadly cancers and cure the patients.

The aim of this proposal is to learn more about how cancer cells repair DNA crosslinks, so that we can use this information to design drugs that can treat patients with currently untreatable cancers. More specifically, we will study a protein complex called the FANCD2/FANCI complex. This complex is central and absolutely critical for cells to repair DNA crosslinks. We have recently discovered a novel domain, or part, of this complex, which is fundamental to its function. We are going to make use of this unique discovery to further our understanding of how the FANCD2/FANCI complex controls DNA crosslink repair. The increased understanding will in turn pave the way for the development of novel cancer drugs.

Fanconi Anemia (FA) is a genetic disease predisposing to cancer. 18 FA proteins, including BRCA1 and BRCA2, often mutated in breast cancer, compose the FA pathway, which repairs dangerous DNA interstrand crosslinks (ICLs). Mutation in any of these genes can cause FA. Due to the severe cellular toxicity of ICLs, ICL-forming drugs (e.g. cisplatin) are used successfully to treat cancers in non-FA patients. However, patients often develop resistance, which can be caused by an increased ability of the cancer cells to repair ICLs. An inhibitor of the FA pathway would prevent cancer cells from repairing ICLs, thereby sensitizing them to ICL-forming drugs.

Two of the central FA proteins, FANCD2 and FANCI, form a complex that is activated by an essential monoubiquitination step. Absence of either of the subunits, or their modification, completely shuts down the pathway, and sensitizes cells to ICL-forming drugs. Despite the importance of this complex in DNA repair, we still do not know what regulates its activation and what its functional activity is in ICL repair. We have recently obtained the structure of full-length human FANCD2/FANCI complex and discovered a novel domain, which we have termed the Tower domain. This domain is essential for function of the complex. We aim to uncover the mechanisms underlying the activation of DNA repair by the FA complex and understand how the Tower domain controls this process.

We will undertake a multidisciplinary approach including a) study the structure of the FANCD2/FANCI complex bound to DNA by cryo EM, b) apply various biochemical techniques such as in vitro reconstitution of monoubiquitination c) live-cell imaging to monitor DNA repair in live cells.

The information resulting from this research will be valuable for development of drugs targeting the pathway, thereby sensitizing drug-resistant cancers to existing chemotherapeutic drugs, in turn enabling clinicians to treat these currently untreatable patients.

A detailed and thorough description of exactly how our research will benefit industry, healthcare professionals, cancer charities as well as the wider public and population, is present in the "Pathways to Impact" document as part of this application.

In brief:

This project will investigate the role of the critical FANCD2/FANCI protein complex in the Fanconi Anemia (FA) DNA repair pathway and DNA interstrand crosslink (ICL) repair. FA patients suffer from bone marrow failure, anemia and cancer predisposition. There is an interest in the pharmaceutical industry to develop new diagnostic tools and in particular improved therapeutic measures for these patients. Results from our research might benefit such commercial entities.

Also, the availability of drugs inactivating FANCD2/FANCI, could potentially become a powerful tool used clinically to sensitize cancer cells in non-FA cancer patients, benefiting a large number of cancer patients both in the UK and elsewhere. Several pharmaceutical companies are very interested in developing specific inhibitors against proteins involved in ubiquitin signaling for these purposes. Such improved clinical tools will naturally tremendously benefit healthcare professionals, such as medical doctors and nurses, in their care for cancer patients. Such benefits will enhance quality of life and health for many people, in turn contribute to the nation's health generally, also considering the societal effects of a patient in a family. Also important, the ability to treat the thousands of currently non-treatable cancer patients (patients presenting with ICL-resistant cancers), will have a profound positive economical effect both on the NHS medical system, as well as employers, whether government or companies.

An important part of our work is to train the next generation of scientists. The present project will entail careful training and mentoring of a postdoctoral researcher. This person will in turn acquire extremely valuable skills, critical for him/her to move to the next stage in his/her career.