Abstract
Exonuclease EXO1 performs multiple roles in DNA replication and DNA damage repair (DDR). However, EXO1 loss is well-tolerated, suggesting the existence of compensatory mechanisms that could be exploited in DDR-deficient cancers. Using CRISPR screening, we find EXO1 loss as synthetic lethal with many DDR genes somatically inactivated in cancers, including Fanconi Anaemia (FA) pathway and BRCA1-A complex genes. We also identify the spliceosome factor and tumour suppressor ZRSR2 as synthetic lethal with loss of EXO1 and show that ZRSR2-deficient cells are attenuated for FA pathway activation, exhibiting cisplatin sensitivity and radial chromosome formation. Furthermore, FA or ZRSR2 deficiencies depend on EXO1 nuclease activity and can be potentiated in combination with PARP inhibitors or ionizing radiation. Finally, we uncover dysregulated replication-coupled repair as the driver of synthetic lethality between EXO1 and FA pathway attributable to defective fork reversal, elevated replication fork speeds, post-replicative single stranded DNA exposure and DNA damage. These findings implicate EXO1 as a synthetic lethal vulnerability and promising drug target in a broad spectrum of DDR-deficient cancers unaddressed by current therapies.