ena-STUDY-UMCG-15-02-2019-12:50:14:102-490
single-cell sequencing of BRCA2 depleted HeLa cells treated with PARP and ATR inhibitors
Poly(ADP-ribose)-polymerase (PARP) inhibitors are selectively cytotoxic in homologous recombination (HR)-defective cancer cells, for instance due to BRCA1/2 or mutations. However, not all HR-deficient tumors efficiently respond to PARP inhibition and often acquire  resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR-deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2-depleted and Brca2 knock-out cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors to accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome-wide single cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib-treated BRCA2-depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING-associated inflammatory response in BRCA2-deficient cells. ATR inhibition further increased the numbers of cGAS-positive micronuclei and the extent of cytokine production in olaparib-treated BRCA2-deficient cancer cells. Combined, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR-deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling.
Poly(ADP-ribose)-polymerase (PARP) inhibitors are selectively cytotoxic in homologous recombination (HR)-defective cancer cells, for instance due to BRCA1/2 or mutations. However, not all HR-deficient tumors efficiently respond to PARP inhibition and often acquire  resistance. It is therefore important to uncover how PARP inhibitors induce cytotoxicity and develop combination strategies to potentiate PARP inhibitor efficacy in HR-deficient tumors. In this study, we found that forced mitotic entry upon ATR inhibition potentiates cytotoxic effects of PARP inhibition using olaparib in BRCA2-depleted and Brca2 knock-out cancer cell line models. Single DNA fiber analysis showed that ATR inhibition does not exacerbate replication fork degradation. Instead, we find ATR inhibitors to accelerate mitotic entry, resulting in the formation of chromatin bridges and lagging chromosomes. Furthermore, using genome-wide single cell sequencing, we show that ATR inhibition enhances genomic instability of olaparib-treated BRCA2-depleted cells. Inhibition of CDK1 to delay mitotic entry mitigated mitotic aberrancies and genomic instability upon ATR inhibition, underscoring the role of ATR in coordinating proper cell cycle timing in situations of DNA damage. Additionally, we show that olaparib treatment leads to increased numbers of micronuclei, which is accompanied by a cGAS/STING-associated inflammatory response in BRCA2-deficient cells. ATR inhibition further increased the numbers of cGAS-positive micronuclei and the extent of cytokine production in olaparib-treated BRCA2-deficient cancer cells. Combined, we show that ATR inhibition induces premature mitotic entry and mediates synergistic cytotoxicity with PARP inhibition in HR-deficient cancer cells, which involves enhanced genomic instability and inflammatory signaling.