Abstract
The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR–Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.
Highlights
Ataxia telangiectasia and Rad3-related (ATR) is a fundamental DNA damage response (DDR) protein kinase involved in DNA double-strand break (DSB) signalling and cell cycle checkpoint engagement, and is an apical regulator of the replication stress response (RSR) [1,2,3]
We found that loss of Cyclin C or CDK8 conferred resistance to a clinical ATR inhibitors (ATRi), VE-822, that is chemically distinct from AZD6738, as well as towards the CHK1i (LY2603618) or WEE1i (AZD1775), but not towards various DNA-damaging agents that we tested: ionising radiation (IR) which causes various forms of DNA damage, the strong apoptosis inducers etoposide and carboplatin, as well as hydroxyurea (HU) or aphidicolin which induce DNA replication stress by impairing DNA polymerase processivity (Figure 3C, Supplementary Figure S3F–J)
We identified substantially fewer gene hits in Atm KO compared to WT cells, which may reflect the numerous interplays between ATR and ataxia telangiectasia mutated (ATM) to regulate DNA repair, DNA replication and cell cycle checkpoint engagement
Summary
Ataxia telangiectasia and Rad3-related (ATR) is a fundamental DNA damage response (DDR) protein kinase involved in DNA double-strand break (DSB) signalling and cell cycle checkpoint engagement, and is an apical regulator of the replication stress response (RSR) [1,2,3]. Replication stress [5] has been identified as a hallmark of cancer [6] While this may be in-part due to faster proliferation rates and nucleotide shortages in S-phase, amplification of the oncogenes CCNE1 and MYC induce replication stress by shortening G1 and promoting firing of intragenic origins that would otherwise be repressed by near-completed transcription [7]. This increases conflicts between DNA replication and transcription [8], with such collisions causing genome instability through replication fork collapse [9,10]. ATR’s fundamental roles in the RSR, including limiting origin firing and promoting nucleotide synthesis, act to prevent RPA exhaustion and replication catastrophe, and likely present a key survival mechanism for cancer cells with high endogenous replication stress
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