RESUMO
Kinases are signaling enzymes that regulate diverse cellular processes. As such, they are frequently mutated in cancer and therefore represent important targets for drug discovery. However, until recently, systematic approaches to identify vulnerabilities and resistances of kinases to DNA-damaging chemotherapeutics have not been possible, partially due to the lack of appropriate technologies. With the advent of CRISPR-Cas9, a comprehensive study has investigated the cellular survival of more than 300 kinase-deficient isogenic cell lines to a diverse panel of DNA-damaging agents, enriched for chemotherapeutics. Here, we discuss how this approach has allowed for the rational development of combination therapies that are aimed at using synthetic lethal interactions between kinase deficiencies and DNA-damaging agents that are used as chemotherapeutics.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fosfotransferases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/efeitos dos fármacos , Descoberta de Drogas/métodos , Humanos , Neoplasias/metabolismoRESUMO
We provide a catalog for the effects of the human kinome on cell survival in response to DNA-damaging agents, covering all major DNA repair pathways. By treating 313 kinase-deficient cell lines with ten diverse DNA-damaging agents, including seven commonly used chemotherapeutics, we identified examples of vulnerability and resistance that are kinase specific. To investigate synthetic lethal interactions, we tested the response to carmustine for 25 cell lines by establishing a phenotypic fluorescence-activated cell sorting (FACS) assay designed to validate gene-drug interactions. We show apoptosis, cell cycle changes, and DNA damage and proliferation after alkylation- or crosslink-induced damage. In addition, we reconstitute the cellular sensitivity of DYRK4, EPHB6, MARK3, and PNCK as a proof of principle for our study. Furthermore, using global phosphoproteomics on cells lacking MARK3, we provide evidence for its role in the DNA damage response. Our data suggest that cancers with inactivating mutations in kinases, including MARK3, are particularly vulnerable to alkylating chemotherapeutic agents.
Assuntos
Dano ao DNA/fisiologia , Humanos , Transdução de SinaisRESUMO
Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.
Assuntos
Reparo do DNA/genética , Reparo do DNA/fisiologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Instabilidade Cromossômica , Dano ao DNA , Anemia de Fanconi/terapia , Proteína do Grupo de Complementação A da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/deficiência , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Terapia Genética , Histonas/metabolismo , Humanos , Mutação , Rad51 Recombinase/metabolismo , Proteases Específicas de Ubiquitina/deficiência , UbiquitinaçãoRESUMO
DNA lesions caused by UV damage are thought to be repaired solely by the nucleotide excision repair (NER) pathway in human cells. Patients carrying mutations within genes functioning in this pathway display a range of pathologies, including an increased susceptibility to cancer, premature aging, and neurological defects. There are currently no curative therapies available. Here we performed a high-throughput chemical screen for agents that could alleviate the cellular sensitivity of NER-deficient cells to UV-induced DNA damage. This led to the identification of the clinically approved anti-diabetic drug acetohexamide, which promoted clearance of UV-induced DNA damage without the accumulation of chromosomal aberrations, hence promoting cellular survival. Acetohexamide exerted this protective function by antagonizing expression of the DNA glycosylase, MUTYH. Together, our data reveal the existence of an NER-independent mechanism to remove UV-induced DNA damage and prevent cell death.