The protein kinase ATR is an apical regulator of the DNA damage response and coordinates DNA repair, cell cycle checkpoint engagement, and DNA replication to ensure genome stability. To achieve this, ATR signalling often cooperates with, or antagonises, other DNA repair pathways, including many known and predicted interplays with the related kinase ATM. Accordingly, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring ATM mutations. As with all therapeutics, there is therefore a need to understand which functions and pathways drive ATRi efficacy, and to identify biomarkers of response to facilitate patient stratification and pre-empt drug resistance. This thesis describes the systematic characterisation of interplays between ATR and ATM by using CRISPR-Cas9 genome-wide screens to identify factors which promote hypersensitivity or resistance to the ATRi ceralasertib in ATM-proficient and/or ATM-deficient cells. We identify and validate factors which modulate ATRi efficacy, with a subset of these requiring functional ATM expression. These data provide a rich resource to inform future studies into interplays between ATR and ATM, strategies for treating ATM-deficient tumours, and the clinical use of ATRi.

Strikingly, two of the strongest resistance hits in both ATM-proficient and ATM-deficient cells encoded Cyclin C and CDK8, which function as a complex to regulate RNA polymerase II-mediated transcription. We show that Cyclin C/CDK8 loss promotes resistance to inhibitors of the replication stress response (RSR), and reduces S-phase DNA:RNA hybrid formation and transcription-associated replication stress. Our results highlight that replication stress arisen from transcription-replication encounters is a predominant driver of cell death when the RSR is inactivated. By modulating such conflicts, we show that it is possible to influence sensitivities to ATRi or CHK1i in ways that might in due course be exploitable in the clinical arena. Finally, we identify conserved ATM/ATR consensus SQ motifs in the C-terminus of Cyclin C, and show that these are phosphorylated upon DNA damage. We discover that, when phosphorylated, the C-terminus of Cyclin C interacts with the phosphoribosyl pyrophosphate synthetase (PRPS) complex involved in nucleotide synthesis. These preliminary findings suggest the existence of a novel regulatory mechanism at the intersection of DNA damage, transcription, and nucleotide metabolism, which may function to promote genome stability.