CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

Abstract

SummaryThe observation that BRCA1- and BRCA2-deficient cells are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors spurred their development into cancer therapies that target homologous recombination (HR) deficiency1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1–4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here were present a high-confidence set of 73 genes whose mutation causes increased PARP inhibitor sensitivity. In addition to an expected enrichment for HR-related genes, we discovered that mutation in all three genes encoding RNase H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP inhibitor hypersensitivity of RNase H2-deficient cells is impaired ribonucleotide excision repair (RER)5. Embedded ribonucleotides, abundant in the genome of RER-deficient cells, are substrates for topoisomerase 1 cleavage, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukemia could provide an opportunity to exploit these findings therapeutically.