RESUMO
Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.
Assuntos
Proteína BRCA1/genética , DNA Ligase Dependente de ATP/genética , DNA de Cadeia Simples , Proteína Homóloga a MRE11/genética , Neoplasias Ovarianas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Biópsia , Sistemas CRISPR-Cas , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Aberrações Cromossômicas , Dano ao DNA , DNA Ligase Dependente de ATP/metabolismo , Feminino , Humanos , Lentivirus/genética , Neoplasias Mamárias Animais , Camundongos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Interferente Pequeno/metabolismo , TransgenesRESUMO
BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanisms that do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance in vivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.
Assuntos
Neoplasias , Neoplasias Ovarianas , Animais , Camundongos , Feminino , Humanos , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Multiômica , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias/genética , Neoplasias Ovarianas/genéticaRESUMO
We have previously developed 3D tumoroids derived from genetically engineered mouse models (GEMMs) of BRCA1/2-deficient breast cancer. Here, we describe how to genetically modify tumoroids, use them for functional genetic dropout screens, and carry out orthotopic transplantation of modified tumoroids. The purpose of this protocol is to screen for therapeutic targets and allow rapid and straightforward in vivo validation of the candidate targets. For complete details on the use and execution of this protocol, please refer to Paes Dias et al. (2021b).
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/genética , Feminino , Humanos , CamundongosRESUMO
Tumors with loss of breast cancer type 1 susceptibility protein (BRCA1) are homologous recombination (HR) deficient and hypersensitive to poly(ADP-ribose) polymerase inhibitors (PARPi). However, these tumors may restore HR and acquire PARPi resistance via loss of end-protection of DNA double-strand breaks. We found that loss of nuclear DNA ligase III resensitizes HR-restored BRCA1-deficient cells to PARPi by exposing post-replicative single-stranded DNA (ssDNA) gaps. Our work, and that of others, identifies ssDNA gaps as a key determinant of PARPi response.
RESUMO
The stalled fork protection pathway mediated by breast cancer 1/2 (BRCA1/2) proteins is critical for replication fork stability. However, it is unclear whether additional mechanisms are required to maintain replication fork stability. We describe a hitherto unknown mechanism, by which the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily-A containing DEAD/H box-1 (SMARCAD1) stabilizes active replication forks, that is essential to maintaining resistance towards replication poisons. We find that SMARCAD1 prevents accumulation of 53BP1-associated nucleosomes to preclude toxic enrichment of 53BP1 at the forks. In the absence of SMARCAD1, 53BP1 mediates untimely dissociation of PCNA via the PCNA-unloader ATAD5, causing frequent fork stalling, inefficient fork restart, and accumulation of single-stranded DNA. Although loss of 53BP1 in SMARCAD1 mutants rescues these defects and restores genome stability, this rescued stabilization also requires BRCA1-mediated fork protection. Notably, fork protection-challenged BRCA1-deficient naïve- or chemoresistant tumors require SMARCAD1-mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.