EXO1 inhibition selectively kills replication repair-deficient cancer cells

Deficiencies in DNA repair pathways create context-specific vulnerabilities that may be exploited therapeutically. Recent studies have established EXO1 as a druggable nuclease, but the genetic contexts that confer sensitivity to EXO1 inhibition remain incompletely defined. Here, we assessed whether pharmacological inhibition of EXO1 selectively targets repair-deficient cancer cells across multiple DNA repair backgrounds. Using potent and selective EXO1 inhibitors, we demonstrate impaired viability in cells deficient for Fanconi Anaemia pathway components (FANCG and APITD1/MHF1), BRCA1-A complex factor BRCC36, and the spliceosome factor ZRSR2. EXO1 inhibition in FANCG or ZRSR2 KO cells induced accumulation of micronuclei, γH2AX, and RPA32 phosphorylation at serine 33, consistent with enhanced replication stress leading to cell death, and supporting a role for EXO1 in processing replication-associated DNA structures and maintaining genome stability under conditions of replication stress. Combining EXO1 inhibitor with PARP inhibitors exacerbated cytotoxicity, suggesting partially nonoverlapping repair functions and therapeutic potential for EXO1/PARP inhibitor combination strategies. Collectively, these findings establish that pharmacological inhibition of EXO1 recapitulates the sensitivity to genetic EXO1 loss in certain DNA repair deficient backgrounds, supporting its development as a targeted therapeutic strategy in molecularly stratified cancers.