Defining the role of ADP-ribosyltransferases in DNA repair and genome stability

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

Preserving the integrity of genetic material through repair of damaged DNA is critical for the health of an organism. My laboratory is focussed on understanding these processes, with specific reference to how a set of enzymes called ADP-ribosyltransferases (ARTs) regulate a variety of DNA repair processes.

The best defined role of ARTs is in promoting repair of breaks in the DNA double strand helix. Inhibition of this pathway using small molecule ART inhibitors (ARTi) is being exploited in the clinic to specifically kill tumours with defects in a DNA repair process known as homologous recombination. However, there are several different types of ART in a cell and an emerging theme is that these enzymes can regulate different DNA repair processes. The proposed work will build on our recent exciting new findings addressing how different ARTs function together to repair DNA damage and how inhibiting one ART over others more effectively kills cells following exposure to agents that induce DNA damage. Given currently available ARTi target multiple ARTs, these studies will provide information that underpins the development of more specific ARTi with increased efficacy in the clinic. We will also define novel factors that when inhibited along with ARTs either exaggerate or rescue the sensitivity of cells to DNA damage, identifying pathways that make ART inhibited cells more sensitive or tolerant to radio- or chemotherapy. This work will lead to efforts in improving the efficacy of these agents in the clinic and identify new targets whose inhibition will overcome drug resistance.

Technical Summary

This work is concerned with increasing our understanding of how different ADP-ribosyltransferases (ARTs) maintain genome integrity through DNA repair. We have exploited genome editing technology to generate cell lines defective in the principle DNA damage responsive ARTs alone, or in combination. Using this unique set of reagents we have identified exciting and novel redundancy between ARTs in resolving DNA damage during S-phase. We will build on this work using genome editing technology, in combination with cutting edge biochemical and cell based assays, to define how the ARTs PARP1 and PARP2 regulate these processes. These approaches will also be extended to characterising a novel ART we have uncovered that allows cells deficient in PARP1 and PARP2 to tolerate DNA damage. Using state of the art screening facilities in Oxford, we will perform a genome-wide CRISPR/Cas9 based screen to identify novel genes and pathways that when disrupted suppress or enhance the sensitivity of PARP-deficient cells to DNA damage. Given inhibition of ARTs is being exploited to treat homologous recombination-defective tumours, we will assess how disruption of these genes impacts on these events. These experiments will lead to efforts in improving the efficacy of these agents in the clinic and identify new targets whose inhibition will overcome drug resistance.

Planned Impact

This work fits within the MRC discovery research priority of 'Resilience, Repair and Replacement', specifically to the 'Natural Protection' objective, and has the potential to impact on human health and well-being in the short and longer terms.

This work constitutes Discovery Research. As such, it will have some immediate and many potential long-term impacts. Primary immediate beneficiaries will be the scientific community, as outlined in the 'Academic Beneficiaries' section. Longer term impacts are rooted in our increased understanding of fundamental concepts in DNA metabolism and how this impacts on human health and clinical practice. Synthetic lethality is emerging as an effective treatment for subsets of tumours defective in components of the DNA damage response. Therefore, in the longer term, this work will increase our understanding of this concept and provide important information that can be exploited to further refine these treatments. Additionally, they will identify potential biomarkers for malignancy, or resistance of tumours to chemo and/or radiotherapy. Longer term beneficiaries will include:

i) Commercial exploitation
- This work will provide commercial companies with information to develop therapies that specifically target malignant cells either alone or in combination with chemo/radiotherapy.
- It will identify genes that when deregulated render cells refractory to treatment with ARTi and/or DNA damaging agents. This will provide companies with potential therapeutic targets that when inhibited will overcome this resistance.
- It will provide information for companies wishing to screen for gene mutations in DNA repair genes that contribute towards malignancy, or resistance of tumours to clinical intervention. This may be extended to a variety of other disease states associated with defects in the DNA damage response including premature ageing, immune deficiencies and neurological degeneration.

ii) Public sector exploitation
- This work will provide information to increase efficacy of cancer treatment
- It will influence policy decisions regarding targeting the DNA damage response in the clinic both in terms of application to drug resistance mechanisms and how this may be overcome, in addition to applying this knowledge to disease states other than cancer.

iii) Wider public in general
- Developing new targeted cancer therapies will impact on the lives of future cancer patients and their families.
- An important impact is to inform the wider public of the importance of genome maintenance in human health and well-being. This will inform tailored, specific information regarding life-style, diet etc. to protect from the effects of DNA damage which accumulate during the aging process. This will allow lifestyles changes that will contribute to health and wellbeing.

Publications


10 25 50