RESUMO
Defects in DNA double-strand break repair are thought to render BRCA1 or BRCA2 (BRCA) mutant tumors selectively sensitive to PARP inhibitors (PARPis). Challenging this framework, BRCA and PARP1 share functions in DNA synthesis on the lagging strand. Thus, BRCA deficiency or "BRCAness" could reflect an inherent lagging strand problem that is vulnerable to drugs such as PARPi that also target the lagging strand, a combination that generates a toxic accumulation of replication gaps.
Assuntos
Proteína BRCA1 , Proteína BRCA2 , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Neoplasias , Proteína BRCA1/genética , Proteína BRCA2/genética , DNA , Reparo do DNA/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.
Assuntos
Proteína BRCA1/genética , Replicação do DNA/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Linhagem Celular , Cisplatino/farmacologia , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Endogâmicos NOD , RNA Helicases/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
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
Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.
Assuntos
Nucleotidiltransferases , Bibliotecas de Moléculas Pequenas , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Relação Estrutura-Atividade , Ligação Proteica , Estrutura Molecular , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , DNA Polimerase Dirigida por DNA/metabolismo , Camundongos , Síntese de DNA TranslesãoRESUMO
Homologous recombination (HR) is an important conserved process for DNA repair and ensures maintenance of genome integrity. Inappropriate HR causes gross chromosomal rearrangements and tumorigenesis in mammals. In yeast, the Srs2 helicase eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has been elusive. Here, we identify C. elegans RTEL-1 as a functional analog of Srs2 and describe its vertebrate counterpart, RTEL1, which is required for genome stability and tumor avoidance. We find that rtel-1 mutant worms and RTEL1-depleted human cells share characteristic phenotypes with yeast srs2 mutants: lethality upon deletion of the sgs1/BLM homolog, hyperrecombination, and DNA damage sensitivity. In vitro, purified human RTEL1 antagonizes HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control after deregulation of RTEL1 may be a critical event that drives genome instability and cancer.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , DNA Helicases/metabolismo , Instabilidade Genômica , Recombinação Genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , DNA/metabolismo , DNA Helicases/genética , Reparo do DNA , Humanos , Mutação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Hereditary cancers derive from gene defects that often compromise DNA repair. Thus, BRCA-associated cancers are sensitive to DNA-damaging agents such as cisplatin. The efficacy of cisplatin is limited, however, by the development of resistance. One cisplatin resistance mechanism is restoration of homologous recombination (HR), which can result from BRCA reversion mutations. However, in BRCA2 mutant cancers, cisplatin resistance can occur independently of restored HR by a mechanism that remains unknown. Here we performed a genome-wide shRNA screen and found that loss of the nucleosome remodeling factor CHD4 confers cisplatin resistance. Restoration of cisplatin resistance is independent of HR but correlates with restored cell cycle progression, reduced chromosomal aberrations, and enhanced DNA damage tolerance. Suggesting clinical relevance, cisplatin-resistant clones lacking genetic reversion of BRCA2 show de novo loss of CHD4 expression in vitro. Moreover, BRCA2 mutant ovarian cancers with reduced CHD4 expression significantly correlate with shorter progression-free survival and shorter overall survival. Collectively, our findings indicate that CHD4 modulates therapeutic response in BRCA2 mutant cancer cells.
Assuntos
Autoantígenos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Genes BRCA2/fisiologia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Feminino , Humanos , Mutação/genética , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
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
FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.
Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Instabilidade Genômica/genética , Chaperonas de Histonas/genética , RNA Helicases/genética , Rad51 Recombinase/genética , Instabilidade Cromossômica/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Técnicas de Inativação de Genes , Células HeLa , Chaperonas de Histonas/deficiência , Humanos , Mitomicina/farmacologia , Reparo de DNA por Recombinação/genéticaRESUMO
Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.
Assuntos
Proteína BRCA1/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Dano ao DNA , Reparo de Erro de Pareamento de DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína BRCA1/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular/efeitos dos fármacos , Aberrações Cromossômicas , Dano ao DNA/efeitos dos fármacos , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , 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 D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Humanos , Camundongos , Camundongos Mutantes , Mitomicina/farmacologia , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ-BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an 'undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Síndrome de Bloom/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , RecQ Helicases/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Núcleo Celular/metabolismo , Células Cultivadas , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Helicases/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Instabilidade Genômica/genética , Células HeLa , Humanos , Insetos , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas , RecQ Helicases/genética , Distribuição TecidualRESUMO
BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance.
Assuntos
Acetilação , Fatores de Transcrição de Zíper de Leucina Básica , Dano ao DNA/genética , DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi , Lisina/metabolismo , Proteína BRCA1/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Recombinação Homóloga , Humanos , MutaçãoRESUMO
The effectiveness of poly (ADP-ribose) polymerase inhibitors (PARPi) in creating single-stranded DNA gaps and inducing sensitivity requires the FANCJ DNA helicase. Yet, how FANCJ relates to PARP1 inhibition or trapping, which contribute to PARPi toxicity, remains unclear. Here, we find PARPi effectiveness hinges on S-phase PARP1 activity, which is reduced in FANCJ deficient cells as G-quadruplexes sequester PARP1 and MSH2. Additionally, loss of the FANCJ-MLH1 interaction diminishes PARP1 activity; however, depleting MSH2 reinstates PARPi sensitivity and gaps. Indicating sequestered and trapped PARP1 are distinct, FANCJ loss increases PARPi resistance in cells susceptible to PARP1 trapping. However, with BRCA1 deficiency, the loss of FANCJ mirrors PARP1 loss or inhibition, with the detrimental commonality being loss of S-phase PARP1 activity. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA1 deficient cells and emphasize the importance of understanding drug mechanisms for enhancing therapeutic response.
Assuntos
DNA Helicases , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi , Poli(ADP-Ribose) Polimerase-1 , Linhagem Celular Tumoral , DNA Helicases/genética , Reparo do DNA , Proteína 2 Homóloga a MutS/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fase S , Humanos , Proteínas de Grupos de Complementação da Anemia de Fanconi/genéticaRESUMO
Single-stranded DNA gaps are postulated to be fundamental to the mechanism of anti-cancer drugs. Gaining insights into their induction could therefore be pivotal for advancing therapeutic strategies. For poly (ADP-ribose) polymerase inhibitors (PARPi) to be effective, the presence of FANCJ helicase is required. However, the relationship between FANCJ dependent gaps and PARP1 catalytic inhibition or trapping-both linked to PARPi toxicity in BRCA deficient cells-is yet to be elucidated. Here, we find that the efficacy of PARPi is contingent on S-phase PARP1 activity, which is compromised in FANCJ deficient cells because PARP1, along with MSH2, is "sequestered" by G-quadruplexes. PARP1's replication activity is also diminished in cells missing a FANCJ-MLH1 interaction, but in such cells, depleting MSH2 can release sequestered PARP1, restoring PARPi-induced gaps and sensitivity. Our observations indicate that sequestered and trapped PARP1 are different chromatin-bound forms, with FANCJ loss increasing PARPi resistance in cells susceptible to canonical PARP1 trapping. However, in BRCA1 null cells, the loss of FANCJ mirrors the effects of PARP1 loss or inhibition, with the common detrimental factor being the loss of PARP1 activity during DNA replication, not trapping. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA deficient cells and emphasize the importance of understanding drug mechanisms for enhancing precision medicine.
RESUMO
We showed in this study that cells deficient of the BRCA1-associated BACH1 helicase, also known as BRIP1, failed to elicit homologous recombination (HR) after DNA double-stranded breaks (DSBs). BACH1-deficient cells were also sensitive to mitomycin C (MMC) and underwent MMC-induced chromosome instability. Moreover, we identified a homozygous nonsense mutation in BACH1 in a FA-J patient-derived cell line and could not detect BACH1 protein in this cell line. Expression of wild-type BACH1 in this cell line reduced the accumulation of cells at G2/M phases following exposure to DNA crosslinkers, a characteristic of Fanconi anemia (FA) cells. These results support the conclusion that BACH1 is FANCJ.
Assuntos
Anemia de Fanconi/genética , Recombinação Genética , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina Básica , Neoplasias da Mama , Divisão Celular , Linhagem Celular Tumoral , Mapeamento Cromossômico , Primers do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi , Feminino , Fase G2 , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Zíper de Leucina , Fatores de Transcrição/deficiência , TransfecçãoRESUMO
It is clear that FANCJ, also known as BACH1 or BRIP1, is an essential tumor suppressor gene based on the identification of clinically relevant mutations not only in breast cancer, but also the childhood cancer syndrome, Fanconi anemia. This conclusion is further supported by the direct and functional interaction between FANCJ and the hereditary breast cancer-associated gene product BRCA1. In the absence of the FANCJ DNA helicase or its interaction with BRCA1, cells have defects in several aspects of the DNA damage response. In particular, the BRCA1-FANCJ interaction is essential for promoting error-free repair, checkpoint control and for limiting DNA damage tolerance. As the number of FANCJ clinical mutations and affected patients accumulate, it will be critical to understand whether the associated tumors resemble BRCA-associated tumors. If so, FANCJ patients could also benefit from new therapies that selectively sensitize DNA repair-defective tumors and spare healthy cells. In this article, we summarize the breast cancer-associated FANCJ mutations and discuss functional outcomes for DNA repair and tumor suppression.
Assuntos
Proteína BRCA1/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Mutação , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína BRCA1/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Reparo do DNA , Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
The toxic lesion emanating from chemotherapy that targets the DNA was initially debated, but eventually the DNA double strand break (DSB) ultimately prevailed. The reasoning was in part based on the perception that repairing a fractured chromosome necessitated intricate processing or condemned the cell to death. Genetic evidence for the DSB model was also provided by the extreme sensitivity of cells that were deficient in DSB repair. In particular, sensitivity characterized cells harboring mutations in the hereditary breast/ovarian cancer genes, BRCA1 or BRCA2, that function in the repair of DSBs by homologous recombination (HR). Along with functions in HR, BRCA proteins were found to prevent DSBs by protecting stalled replication forks from nuclease degradation. Coming full-circle, BRCA mutant cancer cells that gained resistance to genotoxic chemotherapy often displayed restored DNA repair by HR and/or restored fork protection (FP) implicating that the therapy was tolerated when DSB repair was intact or DSBs were prevented. Despite this well-supported paradigm that has been the impetus for targeted cancer therapy, here we argue that the toxic DNA lesion conferring response is instead single stranded DNA (ssDNA) gaps. We discuss the evidence that persistent ssDNA gaps formed in the wake of DNA replication rather than DSBs are responsible for cell killing following treatment with genotoxic chemotherapeutic agents. We also highlight that proteins, such as BRCA1, BRCA2, and RAD51 known for canonical DSB repair also have critical roles in normal replication as well as replication gap suppression (RGS) and repair. We review the literature that supports the idea that widespread gap induction proximal to treatment triggers apoptosis in a process that does not need or stem from DSB induction. Lastly, we discuss the clinical evidence for gaps and how to exploit them to enhance genotoxic chemotherapy response.
Assuntos
Proteína BRCA2RESUMO
Introduction: Translesion synthesis (TLS) is a DNA damage tolerance (DDT) mechanism that employs error-prone polymerases to bypass replication blocking DNA lesions, contributing to a gain in mutagenesis and chemo-resistance. However, recent findings illustrate an emerging role for TLS in replication gap suppression (RGS), distinct from its role in post-replication gap filling. Here, TLS protects cells from replication stress (RS)-induced toxic single-stranded DNA (ssDNA) gaps that accumulate in the wake of active replication. Intriguingly, TLS-mediated RGS is specifically observed in several cancer cell lines and contributes to their survival. Thus, targeting TLS has the potential to uniquely eradicate tumors without harming non-cancer tissues. Areas Covered: This review provides an innovative perspective on the role of TLS beyond its canonical function of lesion bypass or post-replicative gap filling. We provide a comprehensive analysis that underscores the emerging role of TLS as a cancer adaptation necessary to overcome the replication stress response (RSR), an anti-cancer barrier. Expert Opinion: TLS RGS is critical for tumorigenesis and is a new hallmark of cancer. Although the exact mechanism and extent of TLS dependency in cancer is still emerging, TLS inhibitors have shown promise as an anti-cancer therapy in selectively targeting this unique cancer vulnerability.
Assuntos
Dano ao DNA/genética , Terapia de Alvo Molecular , Neoplasias/terapia , Animais , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Humanos , Neoplasias/genética , Neoplasias/patologiaRESUMO
FANCJ (BRIP1/BACH1) is a hereditary breast and ovarian cancer (HBOC) gene encoding a DNA helicase. Similar to HBOC genes, BRCA1 and BRCA2, FANCJ is critical for processing DNA inter-strand crosslinks (ICL) induced by chemotherapeutics, such as cisplatin. Consequently, cells deficient in FANCJ or its catalytic activity are sensitive to ICL-inducing agents. Unfortunately, the majority of FANCJ clinical mutations remain uncharacterized, limiting therapeutic opportunities to effectively use cisplatin to treat tumors with mutated FANCJ. Here, we sought to perform a comprehensive screen to identify FANCJ loss-of-function (LOF) mutations. We developed a FANCJ lentivirus mutation library representing approximately 450 patient-derived FANCJ nonsense and missense mutations to introduce FANCJ mutants into FANCJ knockout (K/O) HeLa cells. We performed a high-throughput screen to identify FANCJ LOF mutants that, as compared with wild-type FANCJ, fail to robustly restore resistance to ICL-inducing agents, cisplatin or mitomycin C (MMC). On the basis of the failure to confer resistance to either cisplatin or MMC, we identified 26 missense and 25 nonsense LOF mutations. Nonsense mutations elucidated a relationship between location of truncation and ICL sensitivity, as the majority of nonsense mutations before amino acid 860 confer ICL sensitivity. Further validation of a subset of LOF mutations confirmed the ability of the screen to identify FANCJ mutations unable to confer ICL resistance. Finally, mapping the location of LOF mutations to a new homology model provides additional functional information. IMPLICATIONS: We identify 51 FANCJ LOF mutations, providing important classification of FANCJ mutations that will afford additional therapeutic strategies for affected patients.