Stromal　antigen　1　and　2　(STAG1　and　STAG2)　are　two　mutually　exclusive　components　of　the　cohesin　complex　that　is　crucial　for　centromeric　and　telomeric　cohesion.　Beyond　its　structural　role,　STAG2　also　plays　a　pivotal　role　in　homologous　recombination　(HR)　repair　and　has　emerged　as　a　promising　therapeutic　target　in　cancer　treatment.　Here,　we　employed　a　fluorescence　polarization　(FP)-based　high-throughput　screening　and　identified　KPT-6566　as　a　dual　inhibitor　of　STAG1　and　STAG2.　Biochemical　and　biophysical　analyses　demonstrated　that　KPT-6566　directly　binds　to　STAG1　and　STAG2,　disrupting　their　interactions　with　SCC1　and　double-stranded　DNA.　A　metaphase　chromosome　spread　assay　showed　that　KPT-6566　causes　premature　chromosome　separation　and　induces　chromosome　damages　in　HeLa　cells.　Furthermore,　KPT-6566　also　impairs　DNA　damage　repair,　leading　to　the　accumulation　of　double-strand　breaks　and　cell　apoptosis.　Finally,　KPT-6566　can　sensitize　HeLa　and　HepG2　cells　to　PARP　inhibitor　Olaparib　and　the　NHEJ　inhibitor　UMI-77,　exhibiting　a　synergistic　effect　in　suppressing　cell　proliferation.　Our　findings　highlight　the　potential　of　STAG1/2　as　promising　therapeutic　targets　in　cancer　treatment,　particularly　when　they　are　targeted　in　combination　with　other　DNA　damage　response　inhibitors.　©　2024　Elsevier　B.V.