CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions
Michal Zimmermann1, Olga Murina2, Martin A M Reijns2
1The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Abstract:
The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. 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 (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, 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 leukaemia could provide an opportunity to exploit these findings therapeutically.
Insights
Poly(ADP-ribose) polymerase (PARP) inhibitors are effective cancer therapies. New research reveals that impaired ribonucleotide excision repair, caused by ribonuclease H2 deficiency, leads to PARP-trapping DNA lesions and increased sensitivity to PARP inhibitors.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Therapeutics
Background:
- BRCA1/2-deficient cells exhibit sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, driving their use in homologous recombination deficiency-targeted cancer therapies.
- PARP inhibitor cytotoxicity is linked to PARP trapping, a process involving non-covalent protein-DNA adducts at DNA lesions of uncertain origin.
- Understanding the nature of these lesions and the cellular consequences of PARP trapping is crucial for optimizing cancer treatments.
Purpose of the Study:
- To identify genes and pathways conferring cellular resistance to olaparib, a PARP inhibitor, using CRISPR screens.
- To elucidate the mechanisms underlying PARP inhibitor sensitivity in cells with specific genetic deficiencies.
Main Methods:
- Conducted three genome-wide CRISPR screens to identify genes impacting cellular sensitivity to olaparib.
- Analyzed genetic alterations associated with increased sensitivity to PARP inhibition.
- Investigated the role of ribonuclease H2 and ribonucleotide excision repair in mediating PARP inhibitor response.
Main Results:
- Identified 73 high-confidence genes, mutations in which confer hypersensitivity to PARP inhibitors.
- Discovered that mutations in all three ribonuclease H2 genes sensitize cells to PARP inhibition, beyond expected homologous recombination pathway genes.
- Established that impaired ribonucleotide excision repair in ribonuclease H2-deficient cells leads to hypersensitivity via PARP-trapping lesions.
Conclusions:
- Genomic embedded ribonucleotides are a significant, previously unappreciated source of PARP-trapping DNA lesions.
- Impaired ribonucleotide excision repair leads to an accumulation of genomic ribonucleotides, creating substrates for topoisomerase 1, resulting in PARP-trapping lesions.
- Frequent RNASEH2B deletions in metastatic prostate cancer and chronic lymphocytic leukemia present therapeutic opportunities by exploiting PARP inhibitor sensitivity.
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