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Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
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The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.
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BACKGROUND: Several hereditary disorders, with highly variable and sometimes difficult to recognize manifestations, can present with a spontaneous pneumothorax. Options to perform DNA-testing have changed rapidly, as a result of which physicians of diverse disciplines are coming into contact with hereditary disorders. CASE DESCRIPTION: Two patients with a history of multiple spontaneous pneumothoraxes were seen at the outpatient clinic of the department of Clinical Genetics. Based on family history and physical examination, a suspicion of an underlying hereditary disorder arose. Birt-Hogg-Dubé syndrome and vascular Ehlers-Danlos syndrome were diagnosed through DNA-testing. Based on this, additional screening advices were given and DNA-testing became possible in the family. CONCLUSION: A spontaneous pneumothorax may be a manifestation of an underlying hereditary disorder. With attention to clinical symptoms and family history, physicians can contribute to timely diagnosis. In many cases this results in significant health benefits for both the patient and affected family members, such as screening for kidney cancer in the case of Birt-Hogg-Dubé syndrome.
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Síndrome de Birt-Hogg-Dubé , Neoplasias Renales , Neumotórax , Humanos , Neumotórax/etiología , Neumotórax/genética , Síndrome de Birt-Hogg-Dubé/complicaciones , Síndrome de Birt-Hogg-Dubé/diagnóstico , Síndrome de Birt-Hogg-Dubé/genética , AnamnesisRESUMEN
BACKGROUND: CRISPR screens provide large-scale assessment of cellular gene functions. Pooled libraries typically consist of several single guide RNAs (sgRNAs) per gene, for a large number of genes, which are transduced in such a way that every cell receives at most one sgRNA, resulting in the disruption of a single gene in that cell. This approach is often used to investigate effects on cellular fitness, by measuring sgRNA abundance at different time points. Comparing gene knockout effects between different cell populations is challenging due to variable cell-type specific parameters and between replicates variation. Failure to take those into account can lead to inflated or false discoveries. RESULTS: We propose a new, flexible approach called ShrinkCRISPR that can take into account multiple sources of variation. Impact on cellular fitness between conditions is inferred by using a mixed-effects model, which allows to test for gene-knockout effects while taking into account sgRNA-specific variation. Estimates are obtained using an empirical Bayesian approach. ShrinkCRISPR can be applied to a variety of experimental designs, including multiple factors. In simulation studies, we compared ShrinkCRISPR results with those of drugZ and MAGeCK, common methods used to detect differential effect on cell fitness. ShrinkCRISPR yielded as many true discoveries as drugZ using a paired screen design, and outperformed both drugZ and MAGeCK for an independent screen design. Although conservative, ShrinkCRISPR was the only approach that kept false discoveries under control at the desired level, for both designs. Using data from several publicly available screens, we showed that ShrinkCRISPR can take data for several time points into account simultaneously, helping to detect early and late differential effects. CONCLUSIONS: ShrinkCRISPR is a robust and flexible approach, able to incorporate different sources of variations and to test for differential effect on cell fitness at the gene level. These improve power to find effects on cell fitness, while keeping multiple testing under the correct control level and helping to improve reproducibility. ShrinkCrispr can be applied to different study designs and incorporate multiple time points, making it a complete and reliable tool to analyze CRISPR screen data.
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Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Reproducibilidad de los Resultados , Teorema de Bayes , Técnicas de Inactivación de GenesRESUMEN
Previously, we reported a series of families presenting with trichodiscomas, inherited in an autosomal dominant pattern. The phenotype was named familial multiple discoid fibromas (FMDF). The genetic cause of FMDF remained unknown so far. Trichodiscomas are skin lesions previously reported to be part of the same spectrum as the fibrofolliculoma observed in Birt-Hogg-Dubé syndrome (BHD), an inherited disease caused by pathogenic variants in the FLCN gene. Given the clinical and histological differences with BHD and the exclusion of linkage with the FLCN locus, the phenotype was concluded to be distinct from BHD. We performed extensive clinical evaluations and genetic testing in ten families with FMDF. We identified a FNIP1 frameshift variant in nine families and genealogical studies showed common ancestry for eight families. Using whole exome sequencing, we identified six additional rare variants in the haplotype surrounding FNIP1, including a missense variant in the PDGFRB gene that was found to be present in all tested patients with FMDF. Genome-wide linkage analysis showed that the locus on chromosome 5 including FNIP1 was the only region reaching the maximal possible LOD score. We concluded that FMDF is linked to a haplotype on chromosome 5. Additional evaluations in families with FMDF are required to unravel the exact genetic cause underlying the phenotype. When evaluating patients with multiple trichodisomas without a pathogenic variant in the FLCN gene, further genetic testing is warranted and can include analysis of the haplotype on chromosome 5.
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Síndrome de Birt-Hogg-Dubé , Fibroma , Neoplasias Renales , Humanos , Neoplasias Renales/genética , Cromosomas Humanos Par 5/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Proto-Oncogénicas/genética , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patología , Fibroma/genética , Proteínas Portadoras/genéticaRESUMEN
Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called "hotspots." In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.
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Proteínas Asociadas a CRISPR , Neoplasias , Humanos , Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas , Edición Génica , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , ADN/genética , Mutación , Endonucleasas/genética , Neoplasias/diagnóstico , Neoplasias/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Fanconi anaemia (FA) is a rare chromosomal-instability syndrome caused by mutations of any of the 22 known FA DNA-repair genes. FA individuals have an increased risk of head-and-neck squamous-cell-carcinomas (HNSCC), often fatal. Systemic intolerance to standard cisplatin-based protocols due to somatic-cell hypersensitivity underscores the urgent need to develop novel therapies. Here, we performed unbiased siRNA screens to unveil genetic interactions synthetic-lethal with FA-pathway deficiency in FA-patient HNSCC cell lines. We identified based on differential-lethality scores between FA-deficient and FA-proficient cells, next to common-essential genes such as PSMC1, PSMB2, and LAMTOR2, the otherwise non-essential RBBP9 gene. Accordingly, low dose of the FDA-approved RBBP9-targeting drug Emetine kills FA-HNSCC. Importantly both RBBP9-silencing as well as Emetine spared non-tumour FA cells. This study provides a minable genome-wide analyses of vulnerabilities to address treatment challenges in FA-HNSCC. Our investigation divulges a DNA-cross-link-repair independent lead, RBBP9, for targeted treatment of FA-HNSCCs without systemic toxicity.
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Anemia de Fanconi , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Proteínas de Ciclo Celular/genética , ADN , Emetina/uso terapéutico , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Estudio de Asociación del Genoma Completo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
BACKGROUND: We present a family consisting of a father and his two children with an exceptional phenotype of childhood renal cell carcinoma and brain tumors. Extensive genetic testing revealed two inherited tumor predisposition syndromes in all three family members: Birt-Hogg-Dubé syndrome and Li-Fraumeni syndrome. The corresponding genes (FLCN and TP53) are both located on the short arm of chromosome 17. METHODS: We describe the phenotype and performed single nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) analysis of the tumors. RESULTS: All examined tumors showed somatic loss of the wild-type alleles of both FLCN and TP53. CONCLUSIONS: We hypothesize that a synergistic effect of both mutations caused the unusual phenotype of childhood renal cell carcinoma in this family. This family emphasizes the importance of further genetic testing if a tumor develops at an unexpected young age in an inherited cancer predisposition syndrome.
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Neoplasias Encefálicas , Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Proteínas Supresoras de Tumor/genética , Predisposición Genética a la Enfermedad , Células Germinativas/metabolismo , Células Germinativas/patología , Proteína p53 Supresora de Tumor/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder characterized by fibrofolliculomas, pulmonary cysts, pneumothoraces and renal cell carcinomas. Here, we reveal a novel hereditary disorder in a family with skin and mucosal lesions, extensive lipomatosis and renal cell carcinomas. The proband was initially diagnosed with BHD based on the presence of fibrofolliculomas, but no pathogenic germline variant was detected in FLCN, the gene associated with BHD. By whole exome sequencing we identified a heterozygous missense variant (p.(Cys677Tyr)) in a zinc-finger encoding domain of the PRDM10 gene which co-segregated with the phenotype in the family. We show that PRDM10Cys677Tyr loses affinity for a regulatory binding motif in the FLCN promoter, abrogating cellular FLCN mRNA and protein levels. Overexpressing inducible PRDM10Cys677Tyr in renal epithelial cells altered the transcription of multiple genes, showing overlap but also differences with the effects of knocking out FLCN. We propose that PRDM10 controls an extensive gene program and acts as a critical regulator of FLCN gene transcription in human cells. The germline variant PRDM10Cys677Tyr curtails cellular folliculin expression and underlies a distinguishable syndrome characterized by extensive lipomatosis, fibrofolliculomas and renal cell carcinomas.
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Síndrome de Birt-Hogg-Dubé , Carcinoma de Células Renales , Neoplasias Renales , Lipomatosis , Neoplasias Cutáneas , Humanos , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patología , Carcinoma de Células Renales/genética , Genes Supresores de Tumor , Neoplasias Cutáneas/genética , Lipomatosis/genética , Neoplasias Renales/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
In Birt-Hogg-Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCNPOS and FLCNNEG human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins. INKA analysis revealed that FLCN loss alters the activity of numerous kinases, including tyrosine kinases EGFR, MET, and the Ephrin receptor subfamily (EPHA2 and EPHB1), as well their downstream targets MAPK1/3. Validation experiments in the BHD renal tumor cell line UOK257 confirmed that FLCN loss contributes to enhanced MAPK1/3 and downstream RPS6K1/3 signaling. The clinically available MAPK inhibitor Ulixertinib showed enhanced toxicity in FLCNNEG cells. Interestingly, FLCN inactivation induced the phosphorylation of PIK3CD (Tyr524) without altering the phosphorylation of canonical Akt1/Akt2/mTOR/EIF4EBP1 phosphosites. Also, we identified that FLCN inactivation resulted in dephosphorylation of TFEB Ser109, Ser114, and Ser122, which may be linked to increased oxidative stress levels in FLCNNEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCNNEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.
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Síndrome de Birt-Hogg-Dubé , Neoplasias Renales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/metabolismo , Síndrome de Birt-Hogg-Dubé/patología , Efrinas , Receptores ErbB , Humanos , Neoplasias Renales/genética , Fosfoserina , Proteínas Proto-Oncogénicas , Serina-Treonina Quinasas TOR , Proteínas Supresoras de Tumor , TirosinaRESUMEN
Budding uninhibited by benzimidazoles (BUB1) contributes to multiple mitotic processes. Here, we describe the first two patients with biallelic BUB1 germline mutations, who both display microcephaly, intellectual disability, and several patient-specific features. The identified mutations cause variable degrees of reduced total protein level and kinase activity, leading to distinct mitotic defects. Both patients' cells show prolonged mitosis duration, chromosome segregation errors, and an overall functional spindle assembly checkpoint. However, while BUB1 levels mostly affect BUBR1 kinetochore recruitment, impaired kinase activity prohibits centromeric recruitment of Aurora B, SGO1, and TOP2A, correlating with anaphase bridges, aneuploidy, and defective sister chromatid cohesion. We do not observe accelerated cohesion fatigue. We hypothesize that unresolved DNA catenanes increase cohesion strength, with concomitant increase in anaphase bridges. In conclusion, BUB1 mutations cause a neurodevelopmental disorder, with clinical and cellular phenotypes that partially resemble previously described syndromes, including autosomal recessive primary microcephaly, mosaic variegated aneuploidy, and cohesinopathies.
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Segregación Cromosómica , Microcefalia , Aneuploidia , Segregación Cromosómica/genética , Humanos , Microcefalia/genética , Mutación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
In 2020, the Nobel Prize in Chemistry was awarded to American molecular biologist Jennifer Doudna and her French colleague Emmanuelle Charpentier for their fundamental research on CRISPR, an ingenious bacterial immune system. Studies into the working mechanism of CRISPR led to many Eureka moments. Through smart biotechnological engineering, CRISPR became suitable for applications in 'DNA surgery': the targeted editing of the genetic code. Here, we discuss emerging medical CRISPR applications for the treatment of human genetic disorders, including in vivo therapy. This Nobel Prize-winning discovery is powerful, adaptable and accurate, and clinical trials are being launched at an amazing pace. However, extensive research is needed on safe clinical use and possible side effects of CRISPR. In addition, the regulations on market authorization and reimbursement are not yet tailored to this very personal and potentially expensive therapy. Whereas challenges remain, CRISPR gene therapy will continue to rapidly mature as a clinical reality.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Sistemas CRISPR-Cas , ADN , Femenino , Terapia Genética , HumanosRESUMEN
Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.
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Daño del ADN , Reparación del ADN , Factor 1 de Elongación Peptídica/metabolismo , ARN Polimerasa II/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Sistemas CRISPR-Cas , Línea Celular Tumoral , ADN Helicasas/genética , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Humanos , Factor 1 de Elongación Peptídica/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/genética , Elongación de la Transcripción Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction.
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Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Riñón/patología , Riñón/fisiopatología , Mutación/genética , Alelos , Sustitución de Aminoácidos , Secuencia de Bases , Línea Celular , Citoplasma/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Endonucleasas/deficiencia , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Luz , Hígado/patología , Hígado/fisiopatología , Proteínas Mutantes/metabolismo , Mutación Missense/genética , Estabilidad Proteica , HermanosRESUMEN
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.
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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
With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.
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Técnicas Biosensibles , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Ácidos Nucleicos/genética , Sistemas de Atención de PuntoRESUMEN
Premature loss of sister chromatid cohesion at metaphase is a diagnostic marker for different cohesinopathies. Here, we report that metaphase spreads of many cancer cell lines also show premature loss of sister chromatid cohesion. Cohesion loss occurs independently of mutations in cohesion factors including SA2, a cohesin subunit frequently inactivated in cancer. In untransformed cells, induction of DNA replication stress by activation of oncogenes or inhibition of DNA replication is sufficient to trigger sister chromatid cohesion loss. Importantly, cell growth under conditions of replication stress requires the cohesin remover WAPL. WAPL promotes rapid RAD51-dependent repair and restart of broken replication forks. We propose that active removal of cohesin allows cancer cells to overcome DNA replication stress. This leads to oncogene-induced cohesion loss from newly synthesized sister chromatids that may contribute to genomic instability and likely represents a targetable cancer cell vulnerability.
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Proteínas Portadoras/metabolismo , Cromátides/genética , Reparación del ADN , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ras/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Células Cultivadas , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Células HEK293 , Humanos , Ratones , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , CohesinasRESUMEN
HPV-negative head and neck squamous cell carcinomas (HNSCCs) develop in precancerous changes in the mucosal lining of the upper-aerodigestive tract. These precancerous cells contain cancer-associated genomic changes and cause primary tumors and local relapses. Therapeutic strategies to eradicate these precancerous cells are very limited. Using functional genomic screens, we identified the therapeutic vulnerabilities of premalignant mucosal cells, which are shared with fully malignant HNSCC cells. We screened 319 previously identified tumor-lethal siRNAs on a panel of cancer and precancerous cell lines as well as primary fibroblasts. In total we identified 147 tumor-essential genes including 34 druggable candidates. Of these 34, 13 were also essential in premalignant cells. We investigated the variable molecular basis of the vulnerabilities in tumor and premalignant cell lines and found indications of collateral lethality. Wee1-like kinase (WEE1) was amongst the most promising targets for both tumor and precancerous cells. All four precancerous cell lines were highly sensitive to Wee1 inhibition by Adavosertib (AZD1775), while primary keratinocytes tolerated this inhibitor. Wee1 inhibition caused induction of DNA damage during S-phase followed by mitotic failure in (pre)cancer cells. In conclusion, we uncovered Wee1 inhibition as a promising chemopreventive strategy for precancerous cells, with comparable responses as fully transformed HNSCC cells.
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Biomarcadores de Tumor/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/prevención & control , Lesiones Precancerosas/prevención & control , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Proliferación Celular , Daño del ADN , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Ensayos Analíticos de Alto Rendimiento , Humanos , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales CultivadasRESUMEN
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.