Antoine Simoneau

The cyclic GMP-AMP synthase (cGAS), a sensor of cytosolic DNA, is critical for the innate immune response. Here, we show that loss of cGAS in untransformed and cancer cells results in uncontrolled DNA replication, hyperproliferation, and genomic instability. While the majority of cGAS is cytoplasmic, a fraction of cGAS associates with chromatin. cGAS interacts with replication fork proteins in a DNA binding-dependent manner, suggesting that cGAS encounters replication forks in DNA. Independent of cGAMP and STING, cGAS slows replication forks by binding to DNA in the nucleus. In the absence of cGAS, replication forks are accelerated, but fork stability is compromised. Consequently, cGAS-deficient cells are exposed to replication stress and become increasingly sensitive to radiation and chemotherapy. Thus, by acting as a decelerator of DNA replication forks, cGAS controls replication dynamics and suppresses replication-associated DNA damage, suggesting that cGAS is an attractive target for exploiting the genomic instability of cancer cells.

PARP inhibitor (PARPi) is widely used to treat BRCA1/2-deficient tumors, but why PARPi is more effective than other DNA-damaging drugs is unclear. Here, we show that PARPi generates DNA double-strand breaks (DSBs) predominantly in a trans cell cycle manner. During the first S phase after PARPi exposure, PARPi induces single-stranded DNA (ssDNA) gaps behind DNA replication forks. By trapping PARP on DNA, PARPi prevents the completion of gap repair until the next S phase, leading to collisions of replication forks with ssDNA gaps and a surge of DSBs. In the second S phase, BRCA1/2-deficient cells are unable to suppress origin firing through ATR, resulting in continuous DNA synthesis and more DSBs. Furthermore, BRCA1/2-deficient cells cannot recruit RAD51 to repair collapsed forks. Thus, PARPi induces DSBs progressively through trans cell cycle ssDNA gaps, and BRCA1/2-deficient cells fail to slow down and repair DSBs over multiple cell cycles, explaining the unique efficacy of PARPi in BRCA1/2-deficient cells.

Prostate cancer harboring BRCA1/2 mutations are often exceptionally sensitive to PARP inhibitors. However, genomic alterations in other DNA damage response genes have not been consistently predictive of clinical response to PARP inhibition. Here, we perform genome-wide CRISPR-Cas9 knockout screens in BRCA1/2-proficient prostate cancer cells and identify previously unknown genes whose loss has a profound impact on PARP inhibitor response. Specifically, MMS22L deletion, frequently observed (up to 14%) in prostate cancer, renders cells hypersensitive to PARP inhibitors by disrupting RAD51 loading required for homologous recombination repair, although this response is TP53-dependent. Unexpectedly, loss of CHEK2 confers resistance rather than sensitivity to PARP inhibition through increased expression of BRCA2, a target of CHEK2-TP53-E2F7-mediated transcriptional repression. Combined PARP and ATR inhibition overcomes PARP inhibitor resistance caused by CHEK2 loss. Our findings may inform the use of PARP inhibitors beyond BRCA1/2-deficient tumors and support reevaluation of current biomarkers for PARP inhibition in prostate cancer.