G-quadruplex homeostasis is a determinant of PARP inhibitor toxicity in BRCA2-deficient cells.

Inhibition of PARPs is a key strategy to treat tumours with defects in homologous recombination (HR), including those with mutations in the tumour suppressor gene BRCA2. PARP inhibitors generate replication stress, creating a dependence on HR to repair the resulting DNA damage. However, the DNA lesions generated upon PARP inhibition that impede replication fork progression and trigger a requirement for BRCA2 in cell survival are poorly defined. Here, we demonstrate that elevated levels of G-quadruplex (G4) DNA structures is a determinant of genome instability and PARP inhibitor toxicity, while suppressing these structures results in PARP inhibitor resistance. The HUWE1-associated stress response protein HAPSTR1 and BRCA2 function in parallel pathways to PARP1/PARP2 to suppress G4 levels during S-phase. Mechanistically, PARP1/PARP2 disruption in HAPSTR1 or BRCA2-deficient cells leads to G4-replication conflicts, ssDNA gaps, replication-associated DNA damage and genome instability. HAPSTR1 turnover is regulated through HUWE1-dependent proteasome degradation. As such, HUWE1 disruption results in elevated HAPSTR1 and suppression of elevated G4 levels in BRCA2-deficient cells, resulting in PARP inhibitor resistance. Together, these data identify G4 structures as a determinant of PARP inhibitor toxicity, while the HAPSTR1/HUWE1 axis is essential to suppress these structures and confer PARP inhibitor resistance.