RESUMO
The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.
Assuntos
Proteína Fosfatase 2C , Superóxido Dismutase-1 , Proteína Fosfatase 2C/metabolismo , Proteína Fosfatase 2C/genética , Humanos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Linhagem Celular Tumoral , Leucemia/genética , Sistemas CRISPR-Cas , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Mutações Sintéticas Letais , MutaçãoRESUMO
Using CRISPR-Cas9 nicking enzymes, we examined the interaction between the replication machinery and single-strand breaks, one of the most common forms of endogenous DNA damage. We show that replication fork collapse at leading-strand nicks generates resected single-ended double-strand breaks (seDSBs) that are repaired by homologous recombination (HR). If these seDSBs are not promptly repaired, arrival of adjacent forks creates double-ended DSBs (deDSBs), which could drive genomic scarring in HR-deficient cancers. deDSBs can also be generated directly when the replication fork bypasses lagging-strand nicks. Unlike deDSBs produced independently of replication, end resection at nick-induced seDSBs and deDSBs is BRCA1-independent. Nevertheless, BRCA1 antagonizes 53BP1 suppression of RAD51 filament formation. These results highlight distinctive mechanisms that maintain replication fork stability.
Assuntos
Proteína BRCA1 , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Replicação do DNA , Rad51 Recombinase , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Rad51 Recombinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Reparo de DNA por Recombinação , Recombinação Homóloga , Reparo do DNARESUMO
Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.