Mechanistic insights into ATR dependency during replication resulting from BRCA or ATM deficiency

The DNA damage response (DDR) is a fundamental process that impacts on many aspects of biology. In mammalian cells, the DDR affects the regulation of the cell cycle and DNA replication processes, DNA repair and genomic stability, transcriptional regulation, senescence and cell death, and interplays with immune responses. Furthermore, defects in various DDR proteins can result in pathologies including growth defects, accelerated aging, cancer and neurodegenerative disease.

To cope with the diverse forms of DNA lesions that occur, various DDR pathways have evolved that collectively comprise several hundred distinct proteins. It is known that that different DDR pathways and components functionally cooperate and we have so far only glimpsed a small proportion of these, with many more remaining to be discovered. Consequently, a better understanding of DDR signalling and the interplay between DDR pathways will provide important new insights into human biology, disease and health.

One key role of DDR proteins is in DNA replication, where various cellular stresses can result in replication fork stalling and, if not effectively dealt with, generate cytotoxic DNA double strand breaks (DSBs) through replication fork 'collapse'. One of the key DDR proteins that plays pivotal functions during S-phase is the ATR (Ataxia Telangiectasia and Rad3 related) protein. ATR plays multiple roles in the replication stress response including the stabilization of stalled replication forks, regulation of late replication origin firing, and repair of DSBs by homologous recombination (HR). Recently, DSB repair-independent mechanisms for BRCA1 and 2 have also been identified, showing that they too play distinct roles at the site of stalled replication forks.

We plan to gain mechanistic insight into the roles of BRCA1, BRCA2 and ATM in the replication stress response as well as understanding the dependency on ATR in cells that lack functionality in any of these three DDR factors. Enhancing our mechanistic understanding of BRCA1/2's role at stalled replication forks would not only contribute to our understanding of core replication biology, but would also provide insight into how HR proteins are able to influence other DDR pathways. Importantly, BRCA's role in replication fork protection has already been implicated in drug resistance, which can be overcome by a loss of ATM. Emerging data also suggest that ATR is not epistatic with BRCA1, BRCA2 and ATM, but rather deficiencies in these factors confer a significant increase in sensitivity to ATR inhibition. Understanding the mechanism(s) behind this interplay could therefore differentiate BRCA's link with ATR from its interaction with ATM, alongside providing insights into the differences and parallels between BRCA's role in DSB repair and in replication fork stabilisation.

Recent work by members of the Steve Jackson lab has shown that CRISPR-Cas9 based resistance screens are able to detect novel genetic and functional interactions in the DDR. We therefore propose using resistance screens with the ATR inhibitor AZD6738 as a tool to allow us to identify mechanisms of overcoming ATR dependency, and in doing so enhance our understanding of the roles of BRCA1, BRCA2 and ATM in the replication stress response pathway. Following the identification of new functional-interaction partners, we will then undertake detailed probing of the mechanisms behind these interactions as well as those that have arisen from past and ongoing screens in the Jackson group.

In addition to building upon our fundamental understanding of core DDR and replication biology, further understanding the role of BRCA and ATM in other DDR pathways could also help AstraZeneca's expanding DDR portfolio by i) suggesting novel synthetically lethal partners for therapeutic intervention; ii) predicting resistance mechanisms to DDR therapeutics and iii) supporting patient stratification.