Modelling ADP-ribosyltransferases as therapeutic targets in cancer therapy

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

The genome contains a blueprint to produce all the machinery required for cells to function. As such, it is important to maintain the integrity of its constituent DNA. Unfortunately cells are exposed to agents that damage DNA. For example, ionizing radiation (IR) coverts water into reactive oxygen species (ROS) that introduces breaks into the DNA, or alters the bases that encode for proteins. Therefore, cells have evolved multiple mechanisms to repair different types of DNA damage, and these pathways are increasingly well defined. However, a remaining challenge is to understand how these pathways integrate to allow cells to survive IR and other mutagenic agents when one repair pathways fails. Understanding this redundancy has important implications for health and wellbeing. For example, DNA repair pathways are often inactivated in cancer cells and the relative sensitivity of these cells to IR and other DNA damaging agents is exploited in radio/chemotherapy. Defining compensatory pathways that allow cancer cells to tolerate these agents will facilitate the development of drugs that inhibit these pathways and further sensitize cancers to radio/chemotherapy. Importantly, ROS are produced naturally in cells. Therefore, this knowledge will also provide insights into how cancer cells can be sensitized to this endogenous DNA damage, circumventing the requirement for radio/chemotherapy and thus eliminating the inherent toxicity and unwanted side effects of these treatments.

An example of such a strategy is inhibition of proteins known as ADP-ribosyltransferases (ARTs) that sense DNA breaks and chemically modify proteins at the DNA damage sites to promote repair. ART inhibitors (ARTi) are currently being developed in the clinic to treat breast and ovarian cancers with defects in the ability to repair DNA breaks by homologous recombination (HR). Importantly, treatment with ARTi is most promising in combination with IR or other mutagens. However, many questions remain regarding how and why ARTi kill HR-defective cancers that, if resolved, will improve the efficacy of these agents. For example, cells contain multiple ARTs that respond to different types of DNA damage and we have identified a significant degree of redundancy between ARTs. Understanding these relationships will facilitate the development of more specific ARTi that refine treatment strategies. Further, whilst ARTi kill certain tumour cells, it is important to establish how they impact on DNA integrity in non-cancer cells, especially in combination with IR. Finally, whilst certain cancers are highly sensitive to ARTi, they rapidly adapt to treatment with these agents. Identifying genes that, when de-regulated, render cancer cells refractory to ARTi treatment will provide potential targets that when inhibited will overcome this resistance.

DNA repair pathways function in a similar manner in a wide variety of organisms. Therefore, a powerful approach to address these questions is to exploit the ease of experimentation in relatively simple model organisms and extend the findings to humans. Unfortunately, this approach is hampered by the lack of certain DNA repair proteins in the most commonly used model organisms to study DNA repair. Recently, however, we initiated a study of DNA repair in the microorganism Dictyostelium and have established that it contains a number of DNA repair proteins, including ARTs, absent in other model organisms. Therefore, Dictyostelium will prove an important model to investigate selected DNA repair pathways and redundancy. The overall objectives of this proposal are to exploit the advantages of Dictyostelium and human cells to address the following:

i) How do multiple ARTs regulate compensatory repair mechanisms following DNA damage?
ii) What is the impact of long term ART inhibition on genome stability in the presence or absence of agents used in radiotherapy?
iii) How do HR-defective cells become refractory to ARTi?

Technical Summary

This application describes complementary experimental approaches in Dictyostelium, mouse and human cells to investigate three outstanding questions with regards to the mechanisms by which ADP-ribosyltransferases (ARTs) maintain genome stability following exposure to DNA damaging agents: a) How do alternate DNA repair pathways compensate for defective single strand break repair induced by disruption of ARTs? b) What is the long-term impact of disrupting ARTs, alone or in combination with DNA damage induced by IR, on genome stability? c) How do HR-defective cells become refractory to treatment with ARTi.

Redundancy between different ARTs will be assessed in MEFs disrupted for multiple ARTs, or by depletion of ARTs in human cells using siRNA. The role of specific repair pathways in this redundancy (e.g. BER, NHEJ, HR etc.) will be tested by depleting selected repair pathway components in the appropriate genetic background by siRNA, or chemical inhibition. The ability to tolerate and resolve DNA damage will be assessed using standard assays including sensitivity of cells to DNA damage, assessing DNA repair kinetics by comet assays, and recruitment of repair factors to DNA lesions by cell imaging and chromatin fractionation technologies. The impact of disrupting ARTs on genome stability will be assessed in Dictyostelium using cell-based assays to determine mutation frequencies at specific loci, or whole genome sequencing to assess the types and frequency of global mutations. Genetic screens to identify genes that when de-regulated render HR-defective cells resistant to ARTi will be performed by generating mutation libraries in HR-defective backgrounds and selecting for cells that tolerate ARTi.

Planned Impact

The immediate beneficiaries will primarily be the scientific community. This work will provide data, reagents and methodologies of interest to the DNA repair and Dictyostelium communities. These experiments will consolidate Dictyostelium as a model to study DNA repair and, importantly, this information will be exploited to increase our understanding of how these pathways function in humans.

In the longer term, this work will impact on human health and wellbeing. Given that DNA repair pathways are targeted in traditional chemo and/or radiotherapy regimens, this work will provide conceptual advances that can be exploited to improve these treatments. Further, synthetic lethality is emerging as an effective treatment for subsets of tumours defective in components of the DNA damage response. 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:

Academic Impacts:
i) Worldwide academic advancement
This work will improve our understanding of the molecular basis of DNA repair.
ii) Innovative methodologies
Innovative methodologies developed in Dictyostelium will be transferable to other organisms. This work will also improve existing technologies in Dictyostelium (e.g. disrupting genes by targeted HR and generating mutants by REMI)
iii) Training highly skilled researchers and improving teaching
This work will provide a training ground for the postdoc and technician in a variety of scientific and transferable skills. It will also provide future research projects to train/teach undergraduate and postgraduate researchers.

Economic and Social Impacts:
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 effects of DNA damage during stem cell propagation and embryonic development.
iii) Wider public in general
- Developing new targeted cancer therapies will impact on the lives of future cancer patients and their families.
- This work will facilitate 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.
 
Title Homologous recombination deficient Dictyostelium strain 
Description This strain is defective in homologus recombination and as such sensitive to inhibitors of Poly-ADP-ribose polymerases (PARP) 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2015 
Provided To Others? Yes  
Impact It is being used to screen for pathways that drive resistance of HR-defective cells to PARP inhibitors. 
 
Description Analysis of ADP-ribose binding proteins in Dictyostelium 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Genetic and cell biology analysis of ADP-ribose binding proteins in Dictyostelium
Collaborator Contribution Structural and biochemical analysis of Dictyostelium ADP-ribose binding proteins
Impact Gunn A.R., Banos-Pinero B., Paschke P., Sanchez-Pulido P., Ariza A., Day J., Emrich M., Leys D., Ponting C.P., Ahel I. and Lakin N.D. (2016). The role of ADP-ribosylation in regulating DNA interstrand crosslink repair. J. Cell Sci. 129:3845-3858.
Start Year 2014
 
Description Identification of ADP-ribose binding proteins in Dictyostelium 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Genetci and cell biology anlysis novel ADP-ribose binding proteins in Dictyostelium
Collaborator Contribution Bioinfiormatics to identify novel ADP-ribose binding proteins in Dictyostelium
Impact Gunn A.R., Banos-Pinero B., Paschke P., Sanchez-Pulido P., Ariza A., Day J., Emrich M., Leys D., Ponting C.P., Ahel I. and Lakin N.D. (2016). The role of ADP-ribosylation in regulating DNA interstrand crosslink repair. J. Cell Sci. 129:3845-3858.
Start Year 2013
 
Description 7th EMBO Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk at the 7th EMBO Meeting, Mannheim, Germany. Between 50-100 participants in a special interest group symposium on ADP-ribosylation
Year(s) Of Engagement Activity 2016
 
Description DNA Repair Conference (California, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Poster presentation at a Gordon Conference on 'DNA Damage, Mutation and Cancer'
Year(s) Of Engagement Activity 2016
 
Description Department of Biochemistry Open Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 2 Open days attended to publisise the Biochemistry course at Oxford, encourage students to apply to study this subject, an addition to stimulating a more general interest in science
Year(s) Of Engagement Activity 2015
 
Description Department of Biochemistry, University of Oxford Open Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 2 Open days attended to publisise the Biochemistry course at Oxford, encourage students to apply to study this subject, an addition to stimulating a more general interest in science
Year(s) Of Engagement Activity 2014
 
Description Department of Biochemistry, University of Oxford Open Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 2 Open days attended to publisise the Biochemistry course at Oxford, encourage students to apply to study this subject, an addition to stimulating a more general interest in science
Year(s) Of Engagement Activity 2013,2014
 
Description Department of Biochemistry, University of Oxford Open Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 2 Open days attended to publisise the Biochemistry course at Oxford, encourage students to apply to study this subject, an addition to stimulating a more general interest in science
Year(s) Of Engagement Activity 2016
 
Description Keynote address, CR-UK Leicester Centre, MRC Toxicology Unit Genome Sciences Theme Workshop, University of Leicester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact An invited Keynote address at a workshop to open the CR-UK Leicester Centre, MRC Toxicology Unit & Genome Sciences Unit at the University of Leicester

To publicise our research funded by the RCUK and network with researchers.
Year(s) Of Engagement Activity 2014
 
Description National Science & Engineering Saturday Club 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Scientific activities with local school pupils aged 13-16 to encourage an interest and career in science and technology
Year(s) Of Engagement Activity 2016
 
Description PARP Conference, Cold Spring Harbor, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk at conference to disseminate latest research findings.
Year(s) Of Engagement Activity 2016
URL https://meetings.cshl.edu/meetings.aspx?meet=PARP&year=16
 
Description PARP Conference, Cold Spring Harbor, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Data was presented to an audience of researchers interested in PARP biology.

An international collaboration was intiated during this activity.
Year(s) Of Engagement Activity 2014
 
Description Poster Presentation - International Dictyostelium Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact The poster presentation stimulated discussion of how this model organism can be used to study DNA repair

Resulted in the initiation of collaborations with laboratories for whole genome sequencing
Year(s) Of Engagement Activity 2015
 
Description Research talk - International Dictyostelium Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact The talk stimulated discussion of using Dictyostelium to assess histone post-translational modifications in DNA repair and other rpocesses

Stimulated discussion of future research directions and potential collaborations.
Year(s) Of Engagement Activity 2015
 
Description SGM Annual Meeting 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation at Annual Conference for Society of General Microbiology 2014, Liverpool
Year(s) Of Engagement Activity 2014
 
Description School Visit (Oxford) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Approximately 20 pupils attended a talk describing an overview of my research interests. This resulted in discussions regarding biomedical reserach, in addition to stimulating interest from pupils to embark on a university education/ research career.

This is difficult to assess, although the talk stimulated significant discussion and and invitation to talk again at the school.
Year(s) Of Engagement Activity 2014
 
Description Seminar, UK/EU Dictyostelium Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact A research senminar was presented to UK/EU researchers interested in various aspects of Dictyostelium biology.

This event allowed me to publicise work funded by BBSRC and to network with other researchers.
Year(s) Of Engagement Activity 2011
 
Description UNIQ summer school 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Presentation at UNIQ summer school for improving access
Year(s) Of Engagement Activity 2011,2015