Targeting epigenetic regulators induces transcription-replication conflicts to overcome ATR inhibitor resistance.

Many cancer cells exhibit elevated replication stress and are highly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity to maintain genomic stability and survival. While ATR inhibitors (ATRi) have great promise as therapeutic agents, intrinsic or acquired resistance will likely be a significant challenge. We previously showed that loss of the RNA polymerase II Mediator subunits CDK8 and Cyclin C (CCNC) confers resistance to ATRi by suppressing transcription-dependent replication stress. To identify vulnerabilities that could be exploited to restore ATRi sensitivity in these resistant settings, we performed genome-wide CRISPR screens in wild-type and CDK8-deficient cells. These screens revealed impairment of epigenetic components HDAC3 or the PRC2 complex as synthetic vulnerabilities that enhance ATRi sensitivity, particularly in contexts of loss of CDK8 or CCNC. This ATRi sensitivity is driven by the induction of transcriptional dysregulation, leading to increased transcription-replication collisions, replication stress, and apoptosis upon ATR inhibition. Importantly, we show that HDAC3 loss limits growth of ATRi-resistant tumours in vivo. Moreover, pharmacological inhibition of HDAC3 or PRC2 phenocopies their genetic loss. Collectively, our findings highlight the therapeutic potential of targeting epigenetic regulators to induce transcriptional dysregulation and ensuing replication stress to overcome ATRi resistance.