Modelling ADP-ribosyltransferases as therapeutic targets in cancer therapy

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?

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.

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.