RESUMO
Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.
Assuntos
Neoplasias da Mama/patologia , Heterogeneidade Genética , Organoides/patologia , Bancos de Tecidos , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Camundongos , Camundongos Nus , Organoides/efeitos dos fármacos , Medicina de Precisão/métodosRESUMO
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
Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Reparo do DNA , Dano ao DNA , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Endonucleases Flap/uso terapêutico , Exodesoxirribonucleases/genética , Enzimas Reparadoras do DNA/genéticaRESUMO
Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1-9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.
Assuntos
Éxons , Deleção de Genes , Terapia de Alvo Molecular , Neoplasias , Oncogenes , Inibidores de Proteínas Quinases , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Animais , Éxons/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Oncogenes/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.
Assuntos
Replicação do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Deleção de Genes , Genes BRCA1 , Genes BRCA2 , Neoplasias/patologia , Proteínas Nucleares/deficiência , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA/biossíntese , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Recombinação Homóloga , Proteína Homóloga a MRE11 , Camundongos , Neoplasias/genética , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Neoplasias Mamárias Animais/patologia , Organoides/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Animais , Proteína BRCA1 , Proteína BRCA2/deficiência , Feminino , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteínas Supressoras de Tumor/deficiênciaRESUMO
Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas Mad2/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Reparo de DNA por Recombinação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/metabolismo , Humanos , Switching de Imunoglobulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mad2/deficiência , Proteínas Mad2/genética , Camundongos , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2017;35:147-157.
Assuntos
Reprogramação Celular , Epigênese Genética , Células-Tronco Pluripotentes/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Proliferação de Células , Reprogramação Celular/genética , Cromatina/metabolismo , Retrovirus Endógenos/metabolismo , Técnicas de Silenciamento de Genes , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Lisina/metabolismo , Metilação , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , RNA Interferente Pequeno/metabolismo , Regulação para Cima/genéticaRESUMO
Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.
Assuntos
Proteína BRCA1/deficiência , Neoplasias Mamárias Experimentais/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Proteína BRCA1/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinossarcoma/tratamento farmacológico , Carcinossarcoma/genética , Carcinossarcoma/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/metabolismo , Proto-Oncogene Mas , Análise de SobrevidaRESUMO
Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.
Assuntos
Proteína BRCA1 , Proteína BRCA2 , Replicação do DNA , Resistencia a Medicamentos Antineoplásicos , Histonas , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Feminino , Humanos , Camundongos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Camundongos NusRESUMO
Cells produce considerable genotoxic formaldehyde from an unknown source. We carry out a genome-wide CRISPR-Cas9 genetic screen in metabolically engineered HAP1 cells that are auxotrophic for formaldehyde to find this cellular source. We identify histone deacetylase 3 (HDAC3) as a regulator of cellular formaldehyde production. HDAC3 regulation requires deacetylase activity, and a secondary genetic screen identifies several components of mitochondrial complex I as mediators of this regulation. Metabolic profiling indicates that this unexpected mitochondrial requirement for formaldehyde detoxification is separate from energy generation. HDAC3 and complex I therefore control the abundance of a ubiquitous genotoxic metabolite.
Assuntos
Células , Histona Desacetilases , Humanos , Células/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Complexo I de Transporte de ElétronsRESUMO
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
BRCA1/2-mutated ovarian cancers (OCs) are defective in homologous recombination repair (HRR) of double-strand breaks (DSBs) and thereby sensitive to platinum and PARP inhibitors (PARPis). Multiple PARPis have recently received US Food and Drug Administration (FDA) approval for treatment of OCs, and resistance to PARPis is a major clinical problem. Utilizing primary and recurrent BRCA1/2-mutated carcinomas from OC patients, patient-derived lines, and an in vivo BRCA2-mutated mouse model, we identified a microRNA, miR-493-5p, that induced platinum/PARPi resistance exclusively in BRCA2-mutated carcinomas. However, in contrast to the most prevalent resistance mechanisms in BRCA mutant carcinomas, miR-493-5p did not restore HRR. Expression of miR-493-5p in BRCA2-mutated/depleted cells reduced levels of nucleases and other factors involved in maintaining genomic stability. This resulted in relatively stable replication forks, diminished single-strand annealing of DSBs, and increased R-loop formation. We conclude that impact of miR-493-5p on multiple pathways pertinent to genome stability cumulatively causes PARPi/platinum resistance in BRCA2 mutant carcinomas.
Assuntos
Carcinoma/genética , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Instabilidade Genômica , MicroRNAs/genética , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Proteína BRCA2/genética , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Recombinação Homóloga , Humanos , Camundongos , MicroRNAs/metabolismo , Mutação , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection.
Assuntos
Proteína BRCA1/deficiência , Quebras de DNA de Cadeia Dupla , Complexos Multiproteicos/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Telômero/metabolismoRESUMO
Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically.
Assuntos
Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Camundongos da Linhagem 129 , Camundongos Knockout , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/efeitos dos fármacosRESUMO
Mutations in homologous recombination (HR) genes BRCA1 and BRCA2 predispose to tumorigenesis. HR-deficient cancers are hypersensitive to Poly (ADP ribose)-polymerase (PARP) inhibitors, but can acquire resistance and relapse. Mechanistic understanding how PARP inhibition induces cytotoxicity in HR-deficient cancer cells is incomplete. Here we find PARP inhibition to compromise replication fork stability in HR-deficient cancer cells, leading to mitotic DNA damage and consequent chromatin bridges and lagging chromosomes in anaphase, frequently leading to cytokinesis failure, multinucleation and cell death. PARP-inhibitor-induced multinucleated cells fail clonogenic outgrowth, and high percentages of multinucleated cells are found in vivo in remnants of PARP inhibitor-treated Brca2-/-;p53-/- and Brca1-/-;p53-/- mammary mouse tumours, suggesting that mitotic progression promotes PARP-inhibitor-induced cell death. Indeed, enforced mitotic bypass through EMI1 depletion abrogates PARP-inhibitor-induced cytotoxicity. These findings provide insight into the cytotoxic effects of PARP inhibition, and point at combination therapies to potentiate PARP inhibitor treatment of HR-deficient tumours.
Assuntos
Anáfase/efeitos dos fármacos , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Citocinese/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Experimentais/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mitose/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo de DNA por Recombinação/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The emergence of resistance to poly-ADP-ribose polymerase inhibitors (PARPi) poses a threat to the treatment of BRCA1 and BRCA2 (BRCA1/2)-deficient tumours. Stabilization of stalled DNA replication forks is a recently identified PARPi-resistance mechanism that promotes genomic stability in BRCA1/2-deficient cancers. Dissecting the molecular pathways controlling genomic stability at stalled forks is critical. Here we show that EZH2 localizes at stalled forks where it methylates Lys27 on histone 3 (H3K27me3), mediating recruitment of the MUS81 nuclease. Low EZH2 levels reduce H3K27 methylation, prevent MUS81 recruitment at stalled forks and cause fork stabilization. As a consequence, loss of function of the EZH2/MUS81 axis promotes PARPi resistance in BRCA2-deficient cells. Accordingly, low EZH2 or MUS81 expression levels predict chemoresistance and poor outcome in patients with BRCA2-mutated tumours. Moreover, inhibition of Ezh2 in a murine Brca2-/- breast tumour model is associated with acquired PARPi resistance. Our findings identify EZH2 as a critical regulator of genomic stability at stalled forks that couples histone modifications to nuclease recruitment. Our data identify EZH2 expression as a biomarker of BRCA2-deficient tumour response to chemotherapy.