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1.
Genes Chromosomes Cancer ; 62(9): 501-509, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-36965130

RESUMEN

The role of cancer relevant translocations in tumorigenesis has been historically hampered by the lack of faithful in vitro and in vivo models. The development of the latest genome editing tools (e.g., CRISPR-Cas9) allowed modeling of various chromosomal translocations with different effects on proliferation and transformation capacity depending on the cell line used and secondary genetic alterations. The cellular context is particularly relevant in the case of oncogenic fusions expressed in sarcomas whose histogenesis remain uncertain. Moreover, recent studies have emphasized the increased frequency of gene fusion promiscuity across different mesenchymal tumor entities, which are clinicopathologically unrelated. This review provides a summary of different strategies utilized to generate cancer models with a focus on fusion-driven mesenchymal neoplasia.


Asunto(s)
Sarcoma , Translocación Genética , Humanos , Sistemas CRISPR-Cas , Sarcoma/genética , Edición Génica , Reordenamiento Génico
2.
Mol Cell ; 57(5): 812-823, 2015 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-25661486

RESUMEN

Chromosomal replication is entwined with DNA damage tolerance (DDT) and chromatin structure establishment via elusive mechanisms. Here we examined how specific replication conditions affecting replisome architecture and repriming impact on DDT. We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion. The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing. Notably, the DDT defects of Polα/Primase/Ctf4 mutants are not the consequence of increased sister chromatid distance, but are instead caused by altered single-stranded DNA metabolism and abnormal replication fork topology. We propose that error-free TS is driven by timely replicative helicase-coupled re-priming. Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.


Asunto(s)
Cromátides/genética , Daño del ADN , ADN Polimerasa I/metabolismo , ADN Primasa/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN Polimerasa I/genética , ADN Primasa/genética , Reparación del ADN/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/ultraestructura , Proteínas de Unión al ADN/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Microscopía Electrónica , Modelos Genéticos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Factores de Tiempo
3.
Mod Pathol ; 35(8): 1055-1065, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35347249

RESUMEN

To elucidate the mechanisms underlying the divergent clinicopathologic spectrum of EWSR1/FUS::CREB translocation-associated tumors, we performed a comprehensive genomic analysis of fusion transcript variants, recurrent genetic alterations (mutations, copy number alterations), gene expression, and methylation profiles across a large cohort of tumor types. The distribution of the EWSR1/FUS fusion partners-ATF1, CREB1, and CREM-and exon involvement was significantly different across different tumor types. Our targeted sequencing showed that secondary genetic events are associated with tumor type rather than fusion type. Of the 39 cases that underwent targeted NGS testing, 18 (46%) had secondary OncoKB mutations or copy number alterations (29 secondary genetic events in total), of which 15 (52%) were recurrent. Secondary recurrent, but mutually exclusive, TERT promoter and CDKN2A mutations were identified only in clear cell sarcoma (CCS) and associated with worse overall survival. CDKN2A/B homozygous deletions were recurrent in angiomatoid fibrous histiocytoma (AFH) and restricted to metastatic cases. mRNA upregulation of MITF, CDH19, PARVB, and PFKP was found in CCS, compared to AFH, and correlated with a hypomethylated profile. In contrast, S100A4 and XAF1 were differentially upregulated and hypomethylated in AFH but not CCS. Unsupervised clustering of methylation profiles revealed that CREB family translocation-associated tumors form neighboring but tight, distinct clusters. A sarcoma methylation classifier was able to accurately match 100% of CCS cases to the correct methylation class; however, it was suboptimal when applied to other histologies. In conclusion, our comprehensive genomic profiling of EWSR1/FUS::CREB translocation-associated tumors uncovered mostly histotype, rather than fusion-type associated correlations in transcript variants, prognostically significant secondary genetic alterations, and gene expression and methylation patterns.


Asunto(s)
Histiocitoma Fibroso Maligno , Proteínas de Fusión Oncogénica , Genómica , Histiocitoma Fibroso Maligno/patología , Humanos , Metilación , Mutación , Proteínas de Fusión Oncogénica/genética , Proteína EWS de Unión a ARN/genética , Proteína FUS de Unión a ARN/genética , Translocación Genética
4.
Mol Cell ; 49(3): 536-46, 2013 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-23260657

RESUMEN

Damaged DNA is an obstacle during DNA replication and a cause of genome instability and cancer. To bypass this problem, eukaryotes activate DNA damage tolerance (DDT) pathways that involve ubiquitylation of the DNA polymerase clamp proliferating cell nuclear antigen (PCNA). Monoubiquitylation of PCNA mediates an error-prone pathway by recruiting translesion polymerases, whereas polyubiquitylation activates an error-free pathway that utilizes undamaged sister chromatids as templates. The error-free pathway involves recombination-related mechanisms; however, the factors that act along with polyubiquitylated PCNA remain largely unknown. Here we report that the PCNA-related 9-1-1 complex, which is typically linked to checkpoint signaling, participates together with Exo1 nuclease in error-free DDT. Notably, 9-1-1 promotes template switching in a manner that is distinct from its canonical checkpoint functions and uncoupled from the replication fork. Our findings thus reveal unexpected cooperation in the error-free pathway between the two related clamps and indicate that 9-1-1 plays a broader role in the DNA damage response than previously assumed.


Asunto(s)
Daño del ADN , Complejos Multiproteicos/metabolismo , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Exodesoxirribonucleasas/metabolismo , Fase G2 , Pruebas Genéticas , Mitosis , Modelos Biológicos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Moldes Genéticos
5.
Proc Natl Acad Sci U S A ; 114(14): 3696-3701, 2017 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-28325870

RESUMEN

Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.


Asunto(s)
Edición Génica/métodos , Proteína EWS de Unión a ARN/genética , Translocación Genética , Proteínas WT1/genética , Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteínas de Fusión Oncogénica/genética , Regiones Promotoras Genéticas
6.
Methods ; 121-122: 138-145, 2017 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-28522325

RESUMEN

Recurrent chromosomal translocations often lead to expression of fusion proteins associated with oncogenic transformation. To study translocations and downstream events, genome editing techniques have been developed to generate chromosomal translocations through non-homologous end joining of DNA double-strand breaks introduced at the two participating endogenous loci. However, the frequencies at which these events occur is usually too low to efficiently clone cells carrying the translocation. This article provides a detailed method using CRISPR-Cas9 technology and homology-directed repair to efficiently isolate cells harboring a chromosomal translocation. For an additional level of control, the resulting fusion protein is conditionally expressed to allow early events in oncogenic transformation to be studied. We focus on the generation of the EWSR1-WT1 fusion using human mesenchymal cells, which is associated with the translocation found in desmoplastic small round cell tumors.


Asunto(s)
Neoplasias Abdominales/genética , Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Tumor Desmoplásico de Células Pequeñas Redondas/genética , Endonucleasas/genética , ARN Guía de Kinetoplastida/genética , Neoplasias Abdominales/metabolismo , Neoplasias Abdominales/patología , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 22 , Roturas del ADN de Doble Cadena , Tumor Desmoplásico de Células Pequeñas Redondas/metabolismo , Tumor Desmoplásico de Células Pequeñas Redondas/patología , Endonucleasas/metabolismo , Genoma Humano , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Mutagénesis Sitio-Dirigida/métodos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Reparación del ADN por Recombinación , Translocación Genética , Proteínas WT1/genética , Proteínas WT1/metabolismo
7.
Nucleic Acids Res ; 44(11): 5204-17, 2016 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-27001513

RESUMEN

DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing.


Asunto(s)
Roturas del ADN de Doble Cadena , Endonucleasas/metabolismo , Recombinación Homóloga , Reparación del ADN por Recombinación , Animales , Línea Celular , Daño del ADN , Reparación del ADN por Unión de Extremidades , Replicación del ADN , Técnicas de Inactivación de Genes , Humanos , Ratones , Motivos de Nucleótidos , Intercambio de Cromátides Hermanas
8.
Nature ; 471(7336): 74-79, 2011 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-21368826

RESUMEN

Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA-RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA-RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.


Asunto(s)
Autofagia , Roturas del ADN de Doble Cadena , Histona Desacetilasas/metabolismo , Saccharomyces cerevisiae , Acetilación/efectos de los fármacos , Aminopeptidasas/metabolismo , Autofagia/efectos de los fármacos , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Inestabilidad Cromosómica , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido Valproico/farmacología
9.
Methods ; 69(2): 171-178, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24929070

RESUMEN

Gene targeting - homologous recombination between transfected DNA and a chromosomal locus - is greatly stimulated by a DNA break in the target locus. Recently, the RNA-guided Cas9 endonuclease, involved in bacterial adaptive immunity, has been modified to function in mammalian cells. Unlike other site-specific endonucleases whose specificity resides within a protein, the specificity of Cas9-mediated DNA cleavage is determined by a guide RNA (gRNA) containing an ∼20 nucleotide locus-specific RNA sequence, representing a major advance for versatile site-specific cleavage of the genome without protein engineering. This article provides a detailed method using the Cas9 system to target expressed genes in mouse embryonic stem cells. In this method, a promoterless marker flanked by short homology arms to the target locus is transfected into cells together with Cas9 and gRNA expression vectors. Importantly, biallelic gene knockout is obtained at high frequency by only one round of targeting using a single marker.


Asunto(s)
Alelos , Sistemas CRISPR-Cas/genética , Células Madre Embrionarias/fisiología , Marcación de Gen/métodos , ARN Guía de Kinetoplastida/genética , Animales , Bovinos , Regulación de la Expresión Génica , Humanos , Ratones , ARN Guía de Kinetoplastida/biosíntesis
10.
Nature ; 456(7224): 915-20, 2008 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-19092928

RESUMEN

Replication by template switch is thought to mediate DNA damage-bypass and fillings of gaps. Gap-filling repair requires homologous recombination as well as Rad18- and Rad5-mediated proliferating cell nuclear antigen (PCNA) polyubiquitylation. However, it is unclear whether these processes are coordinated, and the physical evidence for Rad18-Rad5-dependent template switch at replication forks is still elusive. Here we show, using genetic and physical approaches, that in budding yeast (Saccharomyces cerevisiae) Rad18 is required for the formation of X-shaped sister chromatid junctions (SCJs) at damaged replication forks through a process involving PCNA polyubiquitylation and the ubiquitin-conjugating enzymes Mms2 and Ubc13. The Rad18-Mms2-mediated damage-bypass through SCJs requires the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 and SUMOylated PCNA, and is coordinated with Rad51-dependent recombination events. We propose that the Rad18-Rad5-Mms2-dependent SCJs represent template switch events. Altogether, our results unmask a role for PCNA ubiquitylation and SUMOylation pathways in promoting transient damage-induced replication-coupled recombination events involving sister chromatids at replication forks.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Moldes Genéticos , Adenosina Trifosfatasas/metabolismo , Daño del ADN , ADN Helicasas , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Intercambio de Cromátides Hermanas , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitinación
11.
Cancer Res ; 84(9): 1504-1516, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38335254

RESUMEN

Chromoplexy is a phenomenon defined by large-scale chromosomal chained rearrangements. A previous study observed chromoplectic events in a subset of Ewing sarcomas (ES), which was linked to an increased relapse rate. Chromoplexy analysis could potentially facilitate patient risk stratification, particularly if it could be detected with clinically applied targeted next-generation sequencing (NGS) panels. Using DELLY, a structural variant (SV) calling algorithm that is part of the MSK-IMPACT pipeline, we characterized the spectrum of SVs in EWSR1-fused round cell sarcomas, including 173 ES and 104 desmoplastic small round cell tumors (DSRCT), to detect chromoplexy and evaluate its association with clinical and genomic features. Chromoplectic events were detected in 31% of the ES cases and 19% of the DSRCT cases. EWSR1 involvement accounted for 76% to 93% of these events, being rearranged with diverse noncanonical gene partners across the genome, involving mainly translocations but also intrachromosomal deletions and inversions. A major breakpoint cluster was located on EWSR1 exons 8-13. In a subset of cases, the SVs disrupted adjacent loci, forming deletion bridges. Longitudinal sequencing and breakpoint allele fraction analysis showed that chromoplexy is an early event that remains detectable throughout disease progression and likely develops simultaneously with the driver fusion. The presence of chromoplexy was validated in an external ES patient cohort with whole exome sequencing. Chromoplexy was significantly more likely to be present in cases that were metastatic at presentation. Together, this study identifies chromoplexy as a frequent genomic alteration in diverse EWSR1-rearranged tumors that can be captured by targeted NGS panels. SIGNIFICANCE: Chromoplexy is detectable using targeted NGS in a substantial portion of EWSR1-rearranged round cell sarcomas as an early and persistent clonal event, expanding the genomic complexity of fusion-associated sarcomas.


Asunto(s)
Neoplasias Óseas , Rotura Cromosómica , Evolución Clonal , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Proteína EWS de Unión a ARN/genética , Humanos , Análisis de Secuencia de ARN
12.
JCO Precis Oncol ; 8: e2300597, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38603649

RESUMEN

PURPOSE: Alterations of the NF1 tumor suppressor gene is the second most frequent genetic event in embryonal rhabdomyosarcoma (ERMS), but its associations with clinicopathologic features, outcome, or coexisting molecular events are not well defined. Additionally, NF1 alterations, mostly in the setting of neurofibromatosis type I (NF1), drive the pathogenesis of most malignant peripheral nerve sheath tumor with divergent RMS differentiation (also known as malignant triton tumor [MTT]). Distinguishing between these entities can be challenging because of their pathologic overlap. This study aims to comprehensively analyze the clinicopathologic and molecular spectrum of NF1-mutant RMS compared with NF1-associated MTT for a better understanding of their pathogenesis. METHODS: We investigated the clinicopathologic and molecular landscape of a cohort of 22 NF1-mutant RMS and a control group of 13 NF1-associated MTT. Cases were tested on a matched tumor-normal hybridization capture-based targeted DNA next-generation sequencing. RESULTS: Among the RMS group, all except one were ERMS, with a median age of 17 years while for MTT the mean age was 39 years. Three MTTs were misdiagnosed as ERMS, having clinical impact in one. The most frequent coexisting alteration in ERMS was TP53 abnormality (36%), being mutually exclusive from NRAS mutations (14%). MTT showed coexisting CDKN2A/B and PRC2 complex alterations in 38% cases and loss of H3K27me3 expression. Patients with NF1-mutant RMS exhibited a 70% 5-year survival rate, in contrast to MTT with a 33% 5-year survival. All metastatic NF1-mutant ERMS were associated with TP53 alterations. CONCLUSION: Patients with NF1-mutant ERMS lacking TP53 alterations may benefit from dose-reduction chemotherapy. On the basis of the diagnostic challenges and significant treatment and prognostic differences, molecular profiling of challenging tumors with rhabdomyoblastic differentiation is recommended.


Asunto(s)
Neurofibromatosis 1 , Rabdomiosarcoma , Adolescente , Adulto , Humanos , Neurofibromatosis 1/diagnóstico , Neurofibromatosis 1/genética , Neurofibromatosis 1/complicaciones , Neurofibrosarcoma/diagnóstico , Neurofibrosarcoma/genética , Neurofibrosarcoma/complicaciones , Fenotipo , Rabdomiosarcoma/diagnóstico , Rabdomiosarcoma/genética
13.
JCO Precis Oncol ; 8: e2300688, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38885476

RESUMEN

PURPOSE: Targeted therapy in translocation-associated sarcomas has been limited to oncogenic activation of tyrosine kinases or ligands while gene fusions resulting in aberrant expression of transcription factors have been notoriously difficult to target. Moreover, secondary genetic alterations in sarcomas driven by translocations are uncommon, comprising mostly alterations in tumor suppressor genes (TP53, CDKN2A/B). Our study was triggered by an index patient showing a dramatic clinical response by targeting the secondary BRAF V600E mutation in a metastatic angiomatoid fibrous histiocytoma (AFH) harboring the typical EWSR1::CREB1 fusion. MATERIALS AND METHODS: The patient, a 28-year-old female, was diagnosed with an AFH of the thigh and followed a highly aggressive clinical course, with rapid multifocal local recurrence within a year and widespread distant metastases (adrenal, bone, liver, lung). The tumor showed characteristic morphologic features, with histiocytoid cells intermixed with hemorrhagic cystic spaces and lymphoid aggregates. In addition to the pathognomonic EWSR1::CREB1 fusion, targeted DNA sequencing revealed in both primary and adrenal metastatic sites a hot spot BRAF V600E mutation and a CDKN2A/B deletion. Accordingly, the patient was treated with a BRAF-MEK inhibitor combination (encorafenib/binimetinib) showing an excellent but short-lived response. RESULTS: Using a CRISPR-Cas9 approach, we introduced the BRAF c.1799 T>A point mutation in human embryonic stem (hES) cells harboring a conditional EWSR1 (exon7)::CREB1 (exon7) translocation and further differentiated to mesenchymal progenitors (hES-MP) before fusion expression. The cells maintained the fusion transcript expression and the AFH core gene signature while responding to treatment with encorafenib and binimetinib. CONCLUSION: These results highlight that additional targeted DNA NGS in chemotherapy-resistant translocation-associated sarcomas may reveal actionable oncogenic drivers occurring as secondary genetic events during disease progression.


Asunto(s)
Proteínas de Fusión Oncogénica , Humanos , Femenino , Adulto , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas B-raf/genética , Histiocitoma Fibroso Maligno/genética , Histiocitoma Fibroso Maligno/tratamiento farmacológico , Sarcoma/genética , Sarcoma/tratamiento farmacológico , Mutación
14.
PLoS Genet ; 6(11): e1001205, 2010 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-21085632

RESUMEN

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.


Asunto(s)
Daño del ADN , Replicación del ADN/genética , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Moldes Genéticos , Cromosomas Fúngicos/genética , ADN de Hongos/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Exonucleasas/metabolismo , Genoma Fúngico/genética , Modelos Biológicos , Mutación/genética , Fosforilación , Proteína de Replicación A/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética
15.
Oncogenesis ; 12(1): 8, 2023 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-36801905

RESUMEN

The discovery of neurotrophic tyrosine receptor kinase (NTRK) gene fusions as pan-tumor oncogenic drivers has led to new personalized therapies in oncology. Recent studies investigating NTRK fusions among mesenchymal neoplasms have identified several emerging soft tissue tumor entities displaying various phenotypes and clinical behaviors. Among them, tumors resembling lipofibromatosis or malignant peripheral nerve sheath tumors often harbor intra-chromosomal NTRK1 rearrangements, while most infantile fibrosarcomas are characterized by canonical ETV6::NTRK3 fusions. However, appropriate cellular models to investigate mechanisms of how kinase oncogenic activation through gene fusions drives such a wide spectrum of morphology and malignancy are lacking. Progress in genome editing has facilitated the efficient generation of chromosomal translocations in isogenic cell lines. In this study we employ various strategies to model NTRK fusions, including LMNA::NTRK1 (interstitial deletion) and ETV6::NTRK3 (reciprocal translocation) in human embryonic stem (hES) cells and mesenchymal progenitors (hES-MP). Here, we undertake various methods to model non-reciprocal, intrachromosomal deletions/translocations by induction of DNA double strand breaks (DSBs) exploiting either the repair mechanisms of homology directed repair (HDR) or non-homologous end joining (NHEJ). Expression of LMNA::NTRK1 or ETV6::NTRK3 fusions in either hES cells or hES-MP did not affect cell proliferation. However, the level of mRNA expression of the fusion transcripts was significantly upregulated in hES-MP, and phosphorylation of the LMNA::NTRK1 fusion oncoprotein was noted only in hES-MP but not in hES cells. Similarly, an NTRK1-driven transcriptional profile related to neuronal and neuroectodermal lineage was upregulated mainly in hES-MP, supporting the importance of appropriate cellular context in modeling cancer relevant aberrations. As proof of concept of the validity of our in vitro models, phosphorylation was depleted by two TRK inhibitors, Entrectinib and Larotrectinib, currently used as targeted therapy for tumors with NTRK fusions.

16.
NPJ Precis Oncol ; 7(1): 96, 2023 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-37730754

RESUMEN

The genomic spectrum of rhabdomyosarcoma (RMS) progression from primary to relapse is not fully understood. In this pilot study, we explore the sensitivity of various targeted and whole-genome NGS platforms in order to assess the best genomic approach of using liquid biopsy in future prospective clinical trials. Moreover, we investigate 35 paired primary/relapsed RMS from two contributing institutions, 18 fusion-positive (FP-RMS) and 17 fusion-negative RMS (FN-RMS) by either targeted DNA or whole exome sequencing (WES). In 10 cases, circulating tumor DNA (ctDNA) from multiple timepoints through clinical care and progression was analyzed for feasibility of liquid biopsy in monitoring treatment response/relapse. ctDNA alterations were evaluated using a targeted 36-gene custom RMS panel at high coverage for single-nucleotide variation and fusion detection, and a shallow whole-genome sequencing for copy number variation. FP-RMS have a stable genome with relapse, with common secondary alterations CDKN2A/B, MYCN, and CDK4 present at diagnosis and impacting survival. FP-RMS lacking major secondary events at baseline acquire recurrent MYCN and AKT1 alterations. FN-RMS acquire a higher number of new alterations, most commonly SMARCA2 missense mutations. ctDNA analyses detect pathognomonic variants in all RMS patients within our collection at diagnosis, regardless of type of alterations, and confirmed at relapse in 86% of FP-RMS and 100% FN-RMS. Moreover, a higher number of fusion reads is detected with increased disease burden and at relapse in patients following a fatal outcome. These results underscore patterns of tumor progression and provide rationale for using liquid biopsy to monitor treatment response.

17.
Methods Mol Biol ; 2153: 127-143, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32840777

RESUMEN

Homologous recombination is a critical mechanism for the repair of DNA double-strand breaks (DSBs). It occurs predominantly between identical sister chromatids and at lower frequency can also occur between homologs. Interhomolog homologous recombination (IH-HR) has the potential lead to substantial loss of genetic information, i.e., loss of heterozygosity (LOH), when it is accompanied by crossing over. In this chapter, we describe a system to study IH-HR induced by a defined DSB in mouse embryonic stem cells derived from F1 hybrid mice. This system is based on the placement of mutant selectable marker genes, one of which contains an I-SceI endonuclease cleavage site, on the two homologs such that repair of the I-SceI-generated DSB from the homolog leads to drug resistance. Loss of heterozygosity arising during IH-HR is analyzed using a PCR-based approach. Finally, we present a strategy to analyze the role of BLM helicase in this system.


Asunto(s)
Roturas del ADN de Doble Cadena , Células Madre Embrionarias de Ratones/citología , Reparación del ADN por Recombinación , Animales , Línea Celular , Pérdida de Heterocigocidad , Ratones , Células Madre Embrionarias de Ratones/química , RecQ Helicasas/metabolismo
18.
Oncogene ; 40(32): 5095-5104, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34193943

RESUMEN

Chromosomal translocations constitute driver mutations in solid tumors and leukemias. The mechanisms of how related or even identical gene fusions drive the pathogenesis of various tumor types remain elusive. One remarkable example is the presence of EWSR1 fusions with CREB1 and ATF1, members of the CREB family of transcription factors, in a variety of sarcomas, carcinomas and mesotheliomas. To address this, we have developed in vitro models of oncogenic fusions, in particular, EWSR1-CREB1 and EWSR1-ATF1, in human embryonic stem (hES) cells, which are capable of multipotent differentiation, using CRISPR-Cas9 technology and HDR together with conditional fusion gene expression that allows investigation into the early steps of cellular transformation. We show that expression of EWSR1-CREB1/ATF1 fusion in hES cells recapitulates the core gene signatures, respectively, of angiomatoid fibrous histiocytoma (AFH) and gastrointestinal clear cell sarcoma (GI-CCS), although both fusions lead to cell lethality. Conversely, expression of the fusions in hES cells differentiated to mesenchymal progenitors is compatible with prolonged viability while maintaining the core gene signatures. Moreover, in the context of a mesenchymal lineage, the proliferation of cells expressing the EWSR1-CREB1 fusion is further extended by deletion of the tumor suppressor TP53. We expect the generation of isogenic lines carrying oncogenic fusions in various cell lineages to expand our general understanding of how those single genetic events drive tumorigenesis while providing valuable resources for drug discovery.


Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Proteínas de Fusión Oncogénica/genética , Transducción de Señal , Biomarcadores de Tumor , Línea Celular , Perfilación de la Expresión Génica , Histiocitoma Fibroso Maligno/etiología , Histiocitoma Fibroso Maligno/metabolismo , Histiocitoma Fibroso Maligno/patología , Humanos , Mutación , Proteínas de Fusión Oncogénica/metabolismo , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
19.
Nat Commun ; 12(1): 4255, 2021 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-34253720

RESUMEN

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/genética , Complejos Multiproteicos/metabolismo , Animales , Síndrome de Bloom/genética , Síndrome de Bloom/patología , Proliferación Celular , Células HEK293 , Humanos , Meiosis , Ratones , Mitosis , Células Madre Embrionarias de Ratones/metabolismo , Mutación/genética , Fenotipo , Recombinasa Rad51/metabolismo , Intercambio de Cromátides Hermanas , Análisis de Supervivencia
20.
Toxicol Sci ; 153(1): 70-8, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27449664

RESUMEN

Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis.


Asunto(s)
Cromo/toxicidad , Reparación del ADN/efectos de los fármacos , Recombinación Homóloga/efectos de los fármacos , Pulmón/efectos de los fármacos , Línea Celular Transformada , Inestabilidad Genómica , Humanos , Pulmón/citología , Pulmón/metabolismo , Recombinasa Rad51/metabolismo
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