RESUMO
Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.
Assuntos
Replicação do DNA , DNA Mitocondrial/metabolismo , Deleção de Sequência , Imagem Individual de Molécula/métodos , Envelhecimento/genética , DNA Helicases/genética , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
BACKGROUND: Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. METHODS: We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. RESULTS: Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. CONCLUSIONS: In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.
Assuntos
Linfoma Anaplásico de Células Grandes , Quinase do Linfoma Anaplásico/genética , Animais , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Fenótipo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Translocação GenéticaRESUMO
PURPOSE: X-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants in XIAP including missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons of XIAP was identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression. METHODS: Targeted next-generation sequencing (NGS) of the entire locus of XIAP was performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells. RESULTS: NGS of XIAP identified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839 bp that encompassed the first non-coding exon of XIAP and contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839 bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression. CONCLUSIONS: These results identify a key promoter sequence contained in the first non-coding exon of XIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Transtornos Linfoproliferativos , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Células Germinativas/metabolismo , Humanos , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Masculino , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligases/genética , Proteínas de Ligação a DNA/genética , Desoxirribonucleases/fisiologia , Translocação Genética/genética , Animais , Cromossomos Humanos , DNA Ligase Dependente de ATP , Humanos , Camundongos , Deleção de Sequência , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
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.
Assuntos
Edição de Genes/métodos , Proteína EWS de Ligação a RNA/genética , Translocação Genética , Proteínas WT1/genética , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras GenéticasRESUMO
Chromosomal translocations are associated with several tumor types, including hematopoietic malignancies, sarcomas, and solid tumors of epithelial origin, due to their activation of a proto-oncogene or generation of a novel fusion protein with oncogenic potential. In many cases, the availability of suitable human models has been lacking because of the difficulty in recapitulating precise expression of the fusion protein or other reasons. Further, understanding how translocations form mechanistically has been a goal, as it may suggest ways to prevent their occurrence. Chromosomal translocations arise when DNA ends from double-strand breaks (DSBs) on two heterologous chromosomes are improperly joined. This review provides a summary of DSB repair mechanisms and their contribution to translocation formation, the various programmable nuclease platforms that have been used to generate translocations, and the successes that have been achieved in this area.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , Endonucleases , Translocação Genética , Animais , Quebras de DNA de Cadeia Dupla , Humanos , Camundongos , Proto-Oncogene MasRESUMO
Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme, or Escherichia coli LigA, an NAD(+)-dependent ligase. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.
Assuntos
DNA Ligases/metabolismo , Reparo do DNA , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/genética , Animais , Biocatálise , Sobrevivência Celular , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/química , DNA Ligases/deficiência , DNA Ligases/genética , Células-Tronco Embrionárias/metabolismo , Genes Essenciais , Teste de Complementação Genética , Humanos , Camundongos , Mitocôndrias/patologia , Proteínas de Ligação a Poli-ADP-Ribose , Estrutura Terciária de Proteína , Troca de Cromátide Irmã/efeitos dos fármacos , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.
Assuntos
Endonucleases/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Translocação Genética , Dedos de Zinco , Linhagem Celular , Pontos de Quebra do Cromossomo , Humanos , Nucleofosmina , Proteínas Tirosina Quinases/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismoRESUMO
Chromosomal translocations in lymphoid tumours can involve antigen-receptor loci undergoing V(D)J recombination. Here, we show that translocations are recovered from the joining of RAG-generated double-strand breaks (DSBs) on one chromosome to an endonuclease-generated DSB on a second chromosome, providing evidence for the participation of non-RAG DSBs in some lymphoid translocations. Surprisingly, translocations are increased in cells deficient for the nonhomologous end-joining (NHEJ) protein Ku70, implicating non-canonical joining pathways in their etiology.
Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética , Translocação Genética , Animais , Antígenos Nucleares/genética , Sequência de Bases , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Genes Reporter , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Autoantígeno Ku , Camundongos , Homologia de Sequência do Ácido NucleicoRESUMO
Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.
Assuntos
DNA Ligases/metabolismo , Translocação Genética , Animais , Sequência de Bases , Núcleo Celular/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas de Ligação a Poli-ADP-Ribose , Transfecção , Proteínas de XenopusRESUMO
Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm with male dominance and a poor prognosis. A better understanding of the genetic alterations and their functional roles in ENKTCL could help improve patient stratification and treatments. In this study, we performed a comprehensive genetic analysis of 178 ENKTCL cases to delineate the landscape of mutations, copy number alterations (CNA), and structural variations, identifying 34 driver genes including six previously unappreciated ones, namely, HLA-B, HLA-C, ROBO1, CD58, POT1, and MAP2K1. Among them, CD274 (24%) was the most frequently altered, followed by TP53 (20%), CDKN2A (19%), ARID1A (15%), HLA-A (15%), BCOR (14%), and MSN (14%). Chromosome X losses were the most common arm-level CNAs in females (â¼40%), and alterations of four X-linked driver genes (MSN, BCOR, DDX3X, and KDM6A) were more frequent in males and females harboring chromosome X losses. Among X-linked drivers, MSN was the most recurrently altered, and its expression was lost in approximately one-third of cases using immunohistochemical analysis. Functional studies of human cell lines showed that MSN disruption promoted cell proliferation and NF-κB activation. Moreover, MSN inactivation increased sensitivity to NF-κB inhibition in vitro and in vivo. In addition, recurrent deletions were observed at the origin of replication in the EBV genome (6%). Finally, by integrating the 34 drivers and 19 significant arm-level CNAs, nonnegative matrix factorization and consensus clustering identified two molecular groups with different genetic features and prognoses irrespective of clinical prognostic factors. Together, these findings could help improve diagnostic and therapeutic strategies in ENKTCL. Significance: Integrative genetic analyses and functional studies in extranodal NK/T-cell lymphoma identify frequent disruptions of X-linked drivers, reveal prognostic molecular subgroups, and uncover recurrent MSN alterations that confer sensitivity to NF-κB inhibition.
Assuntos
Cromossomos Humanos X , Linfoma Extranodal de Células T-NK , Humanos , Masculino , Feminino , Cromossomos Humanos X/genética , Linfoma Extranodal de Células T-NK/genética , Linfoma Extranodal de Células T-NK/virologia , Linfoma Extranodal de Células T-NK/patologia , Linfoma Extranodal de Células T-NK/metabolismo , Variações do Número de Cópias de DNA , Mutação , Pessoa de Meia-Idade , Animais , Adulto , Camundongos , Prognóstico , Idoso , Perfilação da Expressão Gênica , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Adulto Jovem , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/complicaçõesRESUMO
The precise genetic manipulation of stem and precursor cells offers extraordinary potential for the analysis, prevention, and treatment of human malignancies. Chromosomal translocations are hallmarks of several tumor types where they are thought to have arisen in stem or precursor cells. Although approaches exist to study factors involved in translocation formation in mouse cells, approaches in human cells have been lacking, especially in relevant cell types. The technology of zinc finger nucleases (ZFNs) allows DNA double-strand breaks (DSBs) to be introduced into specified chromosomal loci. We harnessed this technology to induce chromosomal translocations in human cells by generating concurrent DSBs at 2 endogenous loci, the PPP1R12C/p84 gene on chromosome 19 and the IL2Rgamma gene on the X chromosome. Translocation breakpoint junctions for t(19;X) were detected with nested quantitative PCR in a high throughput 96-well format using denaturation curves and DNA sequencing in a variety of human cell types, including embryonic stem (hES) cells and hES cell-derived mesenchymal precursor cells. Although readily detected, translocations were less frequent than repair of a single DSB by gene targeting or nonhomologous end-joining, neither of which leads to gross chromosomal rearrangements. While previous studies have relied on laborious genetic modification of cells and extensive growth in culture, the approach described in this report is readily applicable to primary human cells, including multipotent and pluripotent cells, to uncover both the underlying mechanisms and phenotypic consequences of targeted translocations and other genomic rearrangements.
Assuntos
Dano ao DNA , Células-Tronco/metabolismo , Translocação Genética , Sequência de Bases , Linhagem Celular , Quebra Cromossômica , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos X/genética , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Rearranjo Gênico , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hibridização in Situ Fluorescente , Subunidade gama Comum de Receptores de Interleucina/genética , Reação em Cadeia da Polimerase , Células-Tronco/citologiaRESUMO
The inefficiency of gene modification by homologous recombination can be overcome by the introduction of a double-strand break (DSB) in the target. Engineering the endonucleases needed, however, remains a challenging task that limits widespread application of nuclease-driven gene modification. We report here that conjugates of orthophenanthroline (OP), a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences, are synthetic nucleases efficient at stimulating targeted genome modification. We show that in cultured cells, OP-TFO conjugates induce targeted DSBs. An OP-TFO with a unique target was highly efficient, and mutations at the target site were found in approximately 10% of treated cells, including small deletions most likely introduced during DSB repair by nonhomologous end joining. Importantly, we found that when homologous donor DNA was cotransfected, targeted gene modification took place in >1.5% of treated cells. Because triplex-forming sequences are frequent in human and mouse genes, OP-TFO conjugates therefore constitute an important class of site-specific nucleases for targeted gene modification. Harnessing DNA-damaging molecules to predetermined genomic sites, as achieved here, should also provide inroads into mechanisms of DNA repair and cancer.
Assuntos
Marcação de Genes/métodos , Mutagênese Sítio-Dirigida/métodos , Oligonucleotídeos/farmacologia , Fenantrolinas/farmacologia , Animais , Células Cultivadas , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Endonucleases , Engenharia Genética , Humanos , Mimetismo Molecular , Oligonucleotídeos/química , Fenantrolinas/químicaRESUMO
Circular RNAs (circRNAs) represent a type of endogenous noncoding RNA generated by back-splicing events. Unlike the majority of RNAs, circRNAs are covalently closed, without a 5' end or a 3' poly(A) tail. A few circRNAs can be associated with polysomes, suggesting a protein-coding potential. CircRNAs are not degraded by RNA exonucleases or ribonuclease R and are enriched in exosomes. Recent developments in experimental methods coupled with evolving bioinformatic approaches have accelerated functional investigation of circRNAs, which exhibit a stable structure, a long half-life, and tumor specificity and can be extracted from body fluids and used as potential biological markers for tumors. Moreover, circRNAs may regulate the occurrence and development of cancers and contribute to drug resistance through a variety of molecular mechanisms. Despite the identification of a growing number of circRNAs, their effects in hematological cancers remain largely unknown. Recent studies indicate that circRNAs could also originate from fusion genes (fusion circRNAs, f-circRNAs) next to chromosomal translocations, which are considered the primary cause of various cancers, notably hematological malignancies. This Review will focus on circRNAs and f-circRNAs in hematological cancers.
Assuntos
Neoplasias Hematológicas/genética , RNA Circular/genética , HumanosRESUMO
STAG2, a cohesin family gene, is among the most recurrently mutated genes in cancer. STAG2 loss of function (LOF) is associated with aggressive behavior in Ewing sarcoma, a childhood cancer driven by aberrant transcription induced by the EWSR1-FLI1 fusion oncogene. Here, using isogenic Ewing cells, we show that, while STAG2 LOF profoundly changes the transcriptome, it does not significantly impact EWSR1-FLI1, CTCF/cohesin, or acetylated H3K27 DNA binding patterns. In contrast, it strongly alters the anchored dynamic loop extrusion process at boundary CTCF sites and dramatically decreases promoter-enhancer interactions, particularly affecting the expression of genes regulated by EWSR1-FLI1 at GGAA microsatellite neo-enhancers. Down-modulation of cis-mediated EWSR1-FLI1 activity, observed in STAG2-LOF conditions, is associated with enhanced migration and invasion properties of Ewing cells previously observed in EWSR1-FLI1low cells. Our study illuminates a process whereby STAG2-LOF fine-tunes the activity of an oncogenic transcription factor through altered CTCF-anchored loop extrusion and cis-mediated enhancer mechanisms.
Assuntos
Neoplasias Ósseas/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Fusão Oncogênica/genética , Sarcoma de Ewing/genética , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Mutação com Perda de Função , Lisina/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Sarcoma de Ewing/mortalidade , Sarcoma de Ewing/patologia , CoesinasRESUMO
Ewing sarcoma is characterized by pathognomonic translocations, most frequently fusing EWSR1 with FLI1. An estimated 30% of Ewing sarcoma tumors also display genetic alterations in STAG2, TP53, or CDKN2A (SPC). Numerous attempts to develop relevant Ewing sarcoma models from primary human cells have been unsuccessful in faithfully recapitulating the phenotypic, transcriptomic, and epigenetic features of Ewing sarcoma. In this study, by engineering the t(11;22)(q24;q12) translocation together with a combination of SPC mutations, we generated a wide collection of immortalized cells (EWIma cells) tolerating EWSR1-FLI1 expression from primary mesenchymal stem cells (MSC) derived from a patient with Ewing sarcoma. Within this model, SPC alterations strongly favored Ewing sarcoma oncogenicity. Xenograft experiments with independent EWIma cells induced tumors and metastases in mice, which displayed bona fide features of Ewing sarcoma. EWIma cells presented balanced but also more complex translocation profiles mimicking chromoplexy, which is frequently observed in Ewing sarcoma and other cancers. Collectively, these results demonstrate that bone marrow-derived MSCs are a source of origin for Ewing sarcoma and also provide original experimental models to investigate Ewing sarcomagenesis. SIGNIFICANCE: These findings demonstrate that Ewing sarcoma can originate from human bone-marrow-derived mesenchymal stem cells and that recurrent mutations support EWSR1-FLI1 translocation-mediated transformation.
Assuntos
Transformação Celular Neoplásica , Suscetibilidade a Doenças , Células-Tronco Mesenquimais/metabolismo , Sarcoma de Ewing/etiologia , Sarcoma de Ewing/metabolismo , Animais , Biomarcadores , Sistemas CRISPR-Cas , Células Cultivadas , Biologia Computacional/métodos , Modelos Animais de Doenças , Edição de Genes , Perfilação da Expressão Gênica , Rearranjo Gênico , Marcação de Genes , Xenoenxertos , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Células-Tronco Mesenquimais/patologia , Camundongos , Mutação , Sarcoma de Ewing/patologia , Translocação GenéticaRESUMO
The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.
Assuntos
Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Camundongos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Stromal Antigen 1 and 2 (STAG1/2) are key subunits of the cohesin complex that mediate sister chromatid cohesion, DNA repair, transcriptional regulation, and genome topology. Genetic alterations comprising any of the 11 cohesin-associated genes possibly occur in up to 26% of patients included in The Cancer Genome Atlas (TCGA) studies. STAG2 shows the highest number of putative driver truncating mutations. We provide a comprehensive review of the function of STAG1/2 in human physiology and disease and an integrative analysis of available omics data on STAG alterations in a wide array of cancers, comprising 53 691 patients and 1067 cell lines. Lastly, we discuss opportunities for therapeutic intervention.
Assuntos
Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias/genética , Proteínas Nucleares/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Metilação de DNA , Reparo do DNA/efeitos dos fármacos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Taxa de Mutação , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Regiões Promotoras Genéticas , Mutações Sintéticas Letais/efeitos dos fármacos , CoesinasRESUMO
Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631.
Assuntos
Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/genética , Adolescente , Fatores Etários , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Leucemia Mieloide Aguda/genética , Camundongos , Transplante de Neoplasias , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
Reciprocal chromosomal translocations are early and essential events in the malignant transformation of several tumor types, yet the precise mechanisms that mediate translocation formation are poorly understood. We review here the development of approaches to induce and recover translocations between two targeted DNA double-strand breaks (DSBs) in mammalian chromosomes. Using mouse cells, we find that nonhomologous end-joining readily mediates translocation formation between two DSBs generated by site-specific endonucleases. Translocations occur much less frequently, however, than intrachromosomal repair of a single DSB. Translocation junctions obtained with this approach have similar end modifications to translocation junctions found in human tumors, including deletions, insertions, and repair at short stretches of homology. These modifications are more extensive than repair junctions at a single DSB, suggesting that different factors may be involved in translocation formation and repair of a single DSB. Finally, we describe a novel approach to induce translocations in human cells. Translocation model systems provide an opportunity to study the involvement of mammalian DNA repair and signaling factors in the etiology of chromosomal rearrangements.