RESUMEN
Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.
Asunto(s)
Anomalías Múltiples/etiología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , G-Cuádruplex , Intercambio de Cromátides Hermanas , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Proliferación Celular , ARN Helicasas DEAD-box/química , ADN Helicasas/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Estabilidad Proteica , Seudogenes , ARN Helicasas/genética , ARN Helicasas/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Síndrome , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.
Asunto(s)
Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/genética , ARN Helicasas DEAD-box/genética , ADN Helicasas/genética , Células Epiteliales/enzimología , Fibroblastos/enzimología , Acetiltransferasas/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Transformada , Proliferación Celular , Cromátides/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Rotura Cromosómica , Segregación Cromosómica , Anomalías Craneofaciales/enzimología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patología , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Ectromelia/enzimología , Ectromelia/genética , Ectromelia/patología , Células Epiteliales/patología , Fibroblastos/patología , Expresión Génica , Humanos , Hipertelorismo/enzimología , Hipertelorismo/genética , Hipertelorismo/patología , Ratones , Mitosis , Modelos Biológicos , Mutación , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , CohesinasRESUMEN
Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31(comet). A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/fisiología , Intercambio de Cromátides Hermanas/fisiología , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Segregación Cromosómica , Humanos , Mitosis/fisiología , Morfolinas/farmacología , Nocodazol/farmacología , Paclitaxel/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Intercambio de Cromátides Hermanas/efectos de los fármacos , Moduladores de Tubulina/farmacologíaRESUMEN
The encouraging response rates of BRCA1- and BRCA2-mutated cancers toward PARP inhibitors make it worthwhile to identify other potential determinants of PARP inhibitor responsiveness. Since the Fanconi anemia (FA) pathway coordinates several DNA repair pathways, including homologous recombination in which BRCA1 and BRCA2 play important roles, we investigated whether this pathway harbors other predictors of PARP inhibitor sensitivity. Lymphoblastoid cell lines derived from individuals with FA or clinically related syndromes, such as Warsaw breakage syndrome, were tested for PARP inhibitor sensitivity. Remarkably, we found a strong variability in PARP inhibitor sensitivity among different FANCD1/BRCA2-deficient lymphoblasts, suggesting that PARP inhibitor response depends on the type of FANCD1/BRCA2 mutation. We identified the DNA helicases FANCM and DDX11 as determinants of PARP inhibitor response. These results may extend the utility of PARP inhibition as effective anticancer treatment.
