Probing mechanisms of synergy between PARP inhibitors and inhibitors of ATR, CHK1 and WEE1 in gynaecological cancers

Abstract

PARP inhibitors (PARPi) inhibit the repair of DNA single-strand breaks, resulting in collapsed replication forks, which are repaired by homologous recombination repair (HRR). PARPi are synthetically lethal to cancers which are HRR defective (HRD), although resistance often develops. Replication-associated DNA lesions signal to S and G2/M cell cycle checkpoints via ATR, CHK1 and WEE1. Loss of G1 DNA damage checkpoint control is common in cancer cells, so they are predicted to be more dependent on S and G2/M checkpoints. ATR, CHK1 and WEE1 inhibitors are synergistic with PARPi, although the mechanisms underpinning this synergy are not fully understood. The aim of this thesis is to determine the mechanisms of synergy between inhibitors of PARP (rucaparib), and ATR (VE-821), CHK1 (PF-477736) and WEE1 (MK-1775) in gynaecological cancer cells. PARPi induced replication stress (RS) (γH2AX foci formation) 3 to 10-fold across the HRR competent (HRC) cells, with modest induction of RS in HRD cells, which had significantly higher basal RS. Rucaparib (10 µM) increased ATR and CHK1 activity as measured by phosphorylation of targets CHK1Ser345 (1.8 to 3.1-fold) and CHK1Ser296 (1.4 to 4.5-fold), respectively. Rucaparib also increased levels of pCDK1Tyr15 (1.3 to 1.7-fold) targeted directly by WEE1 and indirectly by CHK1 and ATR. VE-821 (1 µM) inhibited rucaparib-induced phosphorylation of all 3 targets (40 – 100%). PF-477736 (50 nM) inhibited CHK1Ser296 and CDK1Tyr15 phosphorylation (50 – 100 %) but caused a 5 to 31-fold feedback activation of ATR. MK-1775 (0.1 µM) inhibited CDK1Tyr15 phosphorylation (80- 100%) but activated CHK1 and ATR (up to 20-fold). VE-821, PF-477736 and MK-1775 each significantly potentiated rucaparib in HRC cells (2 to 5-fold). However, no synergy was observed in matched BRCA defective cells suggesting the checkpoint inhibitors impaired HRR. Rucaparib increased RAD51 foci (indicative of HRR) 3 to 5-fold across all HRC cells and this increase was completely ablated by VE-821, PF-477736 and MK-1775, suggesting that they induced an HRD phenotype that was synthetically lethal with PARPi. Similar effects were observed in patient-derived malignant ascites cells. Rucaparib-induced S-phase and G2-M arrest was also abrogated by VE-821, PF-477736 and MK-1775. The data described in this thesis provide compelling evidence that impairment of HRR is a major mechanism underpinning the synergy of PARPi with ATR, CHK1 or WEE1 inhibition.