Loss of POLE3-POLE4 unleashes replicative gap accumulation upon treatment with PARP inhibitors.

The advent of PARP inhibitors (PARPis) has profoundly changed the treatment landscape of BRCA1/BRCA2-mutated cancers. Despite this, the development of resistance to these compounds has become a major challenge. Hence, a detailed understanding of the mechanisms underlying PARPi sensitivity is crucially needed. Here, we show that loss of the POLE3-POLE4 subunits of DNA polymerase epsilon (Polε) strongly sensitizes cancer cells to PARPis in a Polε level-independent manner. Loss of POLE3-POLE4 is not associated with defective RAD51 foci formation, excluding a major defect in homologous recombination. On the contrary, treatment with PARPis triggers replicative gap accumulation in POLE3-POLE4 knockout (KO) cells in a PRIMPOL-dependent manner. In addition to this, the loss of POLE3-POLE4 further sensitizes BRCA1-silenced cells to PARPis. Importantly, the knockdown of 53BP1 does not rescue PARPi sensitivity in POLE3-POLE4 KO cells, bypassing a common PARPi resistance mechanism and outlining a potential strategy to sensitize cancer cells to PARPis.

Disrupted control of origin activation compromises genome integrity upon destabilization of Polε and dysfunction of the TRP53-CDKN1A/P21 axis.

The maintenance of genome stability relies on coordinated control of origin activation and replication fork progression. How the interplay between these processes influences human genetic disease and cancer remains incompletely characterized. Here we show that mouse cells featuring Polε instability exhibit impaired genome-wide activation of DNA replication origins, in an origin-location-independent manner. Strikingly, Trp53 ablation in primary Polε hypomorphic cells increased Polε levels and origin activation and reduced DNA damage in a transcription-dependent manner. Transcriptome analysis of primary Trp53 knockout cells revealed that the TRP53-CDKN1A/P21 axis maintains appropriate levels of replication factors and CDK activity during unchallenged S phase. Loss of this control mechanism deregulates origin activation and perturbs genome-wide replication fork progression. Thus, while our data support an impaired origin activation model for genetic diseases affecting CMG formation, we propose that loss of the TRP53-CDKN1A/P21 tumor suppressor axis induces inappropriate origin activation and deregulates genome-wide fork progression.