RESUMO
Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Idoso , Animais , Proteína BRCA1/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Análise Mutacional de DNA/métodos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Embrionárias Murinas , Mutagênese , Neoplasias/genética , Neoplasias/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Mutação Puntual , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Medicina de Precisão/métodos , Sequenciamento Completo do Genoma/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Dedos de Zinco/genéticaRESUMO
Although PARP inhibitors target BRCA1- or BRCA2-mutant tumor cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or "revertant" mutations in BRCA1 or BRCA2 Whether secondary mutant tumor cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR mutagenesis, we generated isogenic tumor cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments in which the clonal composition of tumor cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pretreatment frequency of secondary mutant tumor cells influencing the eventual clonal composition of the tumor cell population. In xenograft studies, the presence of secondary mutant cells in tumors impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor-sensitive and PARP inhibitor-resistant BRCA2 mutant tumor cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumors, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in "revertant" tumor cells, vulnerabilities still exist that could be therapeutically exploited. Mol Cancer Ther; 16(9); 2022-34. ©2017 AACR.
Assuntos
Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA2/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Animais , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas , Seleção Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In 2014, olaparib (Lynparza) became the first PARP (Poly(ADP-ribose) polymerase) inhibitor to be approved for the treatment of cancer. When used as single agents, PARP inhibitors can selectively target tumour cells with BRCA1 or BRCA2 tumour suppressor gene mutations through synthetic lethality. However, PARP inhibition also shows considerable promise when used together with other therapeutic agents. Here, we summarise both the pre-clinical and clinical evidence for the utility of such combinations and discuss the future prospects and challenges for PARP inhibitor combinatorial therapies.