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1.
Pediatr Res ; 85(6): 856-864, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30474648

RESUMO

BACKGROUND: Langerhans cell histiocytosis (LCH) is a histiocytic disorder driven by a constitutive activation of the MAPK signaling pathway in myeloid cells. In 50-60% of cases, it is caused by the BRAFV600E mutation. There is evidence that levels of BRAFV600E in the peripheral blood of patients with LCH correlate with disease burden and could be used as marker for disease extent and response to therapy. However, there is currently no consensus on how testing for minimal disseminated disease should be performed. METHODS: Different approaches to determine the mutation load in patients with LCH were assessed and longitudinal evaluation of patient DNA during treatment with chemotherapy and/or the RAF inhibitor vemurafenib was performed. DNA was isolated from whole blood, different leukocyte subsets, and circulating cell-free DNA (ccf-DNA). RESULTS: We show that determining BRAF levels from whole blood is superior to using ccfDNA. Furthermore, it is important to identify the clinically relevant BRAF-mutated cellular subpopulations such as CD14+ monocytes or CD1c+ DCs, since other blood cells can also harbor the mutation and therefore confound whole blood or ccfDNA measurements. CONCLUSION: Our data support the view that single-agent treatment with an RAF inhibitor reduces disease activity but does not cure LCH.


Assuntos
Histiocitose de Células de Langerhans/sangue , Histiocitose de Células de Langerhans/genética , Proteínas Mutantes/sangue , Proteínas Mutantes/genética , Proteínas Proto-Oncogênicas B-raf/sangue , Proteínas Proto-Oncogênicas B-raf/genética , Substituição de Aminoácidos , Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Pré-Escolar , DNA/sangue , DNA/genética , Análise Mutacional de DNA , Feminino , Marcadores Genéticos , Histiocitose de Células de Langerhans/tratamento farmacológico , Humanos , Lactente , Estudos Longitudinais , Masculino , Proteínas Mutantes/antagonistas & inibidores , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Vemurafenib/uso terapêutico
2.
Genome Res ; 23(11): 1797-809, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23940108

RESUMO

Deregulated E2F transcription factor activity occurs in the vast majority of human tumors and has been solidly implicated in disturbances of cell cycle control, proliferation, and apoptosis. Aberrant E2F regulatory activity is often caused by impairment of control through pRB function, but little is known about the interplay of other oncoproteins with E2F. Here we show that ETS transcription factor fusions resulting from disease driving rearrangements in Ewing sarcoma (ES) and prostate cancer (PC) are one such class of oncoproteins. We performed an integrative study of genome-wide DNA-binding and transcription data in EWSR1/FLI1 expressing ES and TMPRSS2/ERG containing PC cells. Supported by promoter activity and mutation analyses, we demonstrate that a large fraction of E2F3 target genes are synergistically coregulated by these aberrant ETS proteins. We propose that the oncogenic effect of ETS fusion oncoproteins is in part mediated by the disruptive effect of the E2F-ETS interaction on cell cycle control. Additionally, a detailed analysis of the regulatory targets of the characteristic EWSR1/FLI1 fusion in ES identifies two functionally distinct gene sets. While synergistic regulation in concert with E2F in the promoter of target genes has a generally activating effect, EWSR1/FLI1 binding independent of E2F3 is predominantly associated with repressed differentiation genes. Thus, EWSR1/FLI1 appears to promote oncogenesis by simultaneously promoting cell proliferation and perturbing differentiation.


Assuntos
Fator de Transcrição E2F3/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias da Próstata/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F3/metabolismo , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Ligação Proteica , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transativadores/genética , Transativadores/metabolismo , Regulador Transcricional ERG
3.
Blood ; 120(26): 5199-208, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23074278

RESUMO

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of Langerhans cell-like dendritic cells (DCs). In the present study, we demonstrate that LCH cells exhibit a unique transcription profile that separates them not only from plasmacytoid and myeloid DCs, but also from epidermal Langerhans cells, indicating a distinct DC entity. Molecular analysis revealed that isolated and tissue-bound LCH cells selectively express the Notch ligand Jagged 2 (JAG2) and are the only DCs that express both Notch ligand and its receptor. We further show that JAG2 signaling induces key LCH-cell markers in monocyte-derived DCs, suggesting a functional role of Notch signaling in LCH ontogenesis. JAG2 also induced matrix-metalloproteinases 1 and 12, which are highly expressed in LCH and may account for tissue destruction in LCH lesions. This induction was selective for DCs and was not recapitulated in monocytes. The results of the present study suggest that JAG2-mediated Notch activation confers phenotypic and functional aspects of LCH to DCs; therefore, interference with Notch signaling may be an attractive strategy to combat this disease.


Assuntos
Células Dendríticas/patologia , Histiocitose de Células de Langerhans/genética , Receptor Notch1/fisiologia , Adolescente , Diferenciação Celular/fisiologia , Células Cultivadas , Criança , Pré-Escolar , Células Dendríticas/metabolismo , Feminino , Histiocitose de Células de Langerhans/metabolismo , Histiocitose de Células de Langerhans/patologia , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-2 , Células de Langerhans/citologia , Células de Langerhans/metabolismo , Células de Langerhans/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transcriptoma
4.
J Leukoc Biol ; 105(1): 101-111, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30296338

RESUMO

Langerhans cell histiocytosis (LCH) is a MAPK pathway-driven disease characterized by the accumulation of CD1a+ langerin+ cells of unknown origin. We have previously reported that the Notch signaling pathway is active in LCH lesions and that the Notch ligand Jagged2 (JAG2) induces CD1a and langerin expression in monocytes in vitro. Here we show that Notch signaling induces monocytes to acquire an LCH gene signature and that Notch inhibition suppresses the LCH phenotype. In contrast, while also CD1c+ dendritic cells or IL-4-stimulated CD14+ monocytes acquire CD1a and langerin positivity in culture, their gene expression profiles and surface phenotypes are more different from primary LCH cells. We propose a model where CD14+ monocytes serve as LCH cell precursor and JAG2-mediated activation of the Notch signaling pathway initiates a differentiation of monocytes toward LCH cells in selected niches and thereby contributes to LCH pathogenesis.


Assuntos
Diferenciação Celular , Histiocitose de Células de Langerhans/metabolismo , Proteína Jagged-2/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Antígenos CD/metabolismo , Proliferação de Células , Histiocitose de Células de Langerhans/patologia , Humanos , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Fenótipo , Receptores Notch/metabolismo , Transcrição Gênica
5.
Cancer Discov ; 9(10): 1406-1421, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31345789

RESUMO

Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a hybrid position between cancers and inflammatory diseases, which makes it an attractive model for studying cancer development. To explore the molecular mechanisms underlying the pathophysiology of LCH and its characteristic clinical heterogeneity, we investigated the transcriptomic and epigenomic diversity in primary LCH lesions. Using single-cell RNA sequencing, we identified multiple recurrent types of LCH cells within these biopsies, including putative LCH progenitor cells and several subsets of differentiated LCH cells. We confirmed the presence of proliferative LCH cells in all analyzed biopsies using IHC, and we defined an epigenomic and gene-regulatory basis of the different LCH-cell subsets by chromatin-accessibility profiling. In summary, our single-cell analysis of LCH uncovered an unexpected degree of cellular, transcriptomic, and epigenomic heterogeneity among LCH cells, indicative of complex developmental hierarchies in LCH lesions. SIGNIFICANCE: This study sketches a molecular portrait of LCH lesions by combining single-cell transcriptomics with epigenome profiling. We uncovered extensive cellular heterogeneity, explained in part by an intrinsic developmental hierarchy of LCH cells. Our findings provide new insights and hypotheses for advancing LCH research and a starting point for personalizing therapy.See related commentary by Gruber et al., p. 1343.This article is highlighted in the In This Issue feature, p. 1325.


Assuntos
Epigênese Genética , Epigenômica , Histiocitose de Células de Langerhans/genética , Biomarcadores , Biópsia , Suscetibilidade a Doenças , Epigenômica/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histiocitose de Células de Langerhans/diagnóstico , Histiocitose de Células de Langerhans/metabolismo , Humanos , Imuno-Histoquímica , Radiografia , Análise de Célula Única
6.
Blood Adv ; 1(6): 352-356, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-29296950

RESUMO

Single-agent vemurafenib leads to a rapid and sustained clinical response in severe multisystem LCH but does not eradicate the disease.Longitudinal assessment of BRAF V600E during treatment shows that clinical remission can occur despite significant amounts of mutated BRAF.

7.
Oncogene ; 22(21): 3193-204, 2003 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-12761489

RESUMO

The EWS-FLI1 transcription factor is consistently expressed in 85% of Ewing tumors (EFT). In heterologous cells, EWS-FLI1 induces p53-dependent cell cycle arrest or apoptosis. It has been speculated that the p53 tumor suppressor pathway may be generally compromised in EFT despite only rare p53 mutations. In order to test for functional integrity of this pathway, we have investigated a series of EFT cell lines that differ from each other with respect to their endogenous p53 and INK4A gene status for their response to ectopic p53 expression and to stimulation of endogenous p53 activity by X-ray treatment. Significant interindividual and intratumoral variations in the apoptotic propensity of EFT cell lines to transient expression of ectopic p53 were observed, which was independent of the level of p53 expression. In cell lines with a low apoptotic incidence, apoptosis was delayed and the surviving fraction showed a prolonged growth arrest. Complete resistance to p53-induced apoptosis in two cell lines established from the same patient was associated with a high BCL2/BAX ratio and low levels of APAF1. Sensitivity to X-rays showed a trend towards a higher apoptotic rate in wild-type (wt) p53 expressing than in p53 mutant cells. However, one wt p53-expressing EFT cell line was completely refractory to irradiation-stimulated cell death despite high apoptotic responsiveness to ectopic p53. No difference in Ser15 phosphorylation and the transcriptional activation of p53 targets was observed in wt p53 EFT cell lines irrespective of the induction of cell death or growth arrest. All together, our results demonstrate that despite significant variability in the outcome, cell death or cell cycle arrest, the p53 downstream pathway and the DNA damage signaling pathway are functionally intact in EFT.


Assuntos
Apoptose , Neoplasias Ósseas/metabolismo , Sarcoma de Ewing/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Ósseas/patologia , Dano ao DNA , Genes p16 , Humanos , Mutação , Sarcoma de Ewing/patologia , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Raios X
8.
Mol Cancer Res ; 2(5): 296-304, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15192123

RESUMO

The p53 gene is compromised in most human cancers by point mutation. Evidence is accumulating that these alterations frequently do not result in a complete loss of the sequence-specific transcriptional regulatory function of p53. Here, we describe the transcriptional activity of the p53 mutant C277Y isolated from a Ewing's sarcoma with high constitutive pig3 expression. Transient transfection of this mutant into a p53 null cell line resulted in activation not only of the pig3 but also of the MDM2 gene compatible with the presence of constitutively expressed MDM2 transcripts initiated from the P2 promoter in the p53-C277Y hemizygous Ewing's sarcoma cell line. Expression of endogenous pig3 and MDM2 genes was further enhanced on irradiation of this cell line. Here, suppression of p53-C277Y by RNAi reduced pig3 promoter activity, RNA, and protein expression. Reporter gene assays revealed that the potential of p53-C277Y to up-regulate MDM2 expression was similar to wild-type p53, whereas activation of the pig3 promoter was at least 5-fold increased over wild-type p53. The pentanucleotide microsatellite sequence present in exon 1 of the pig3 gene was found to be responsible for p53-C277Y-mediated activation. In concordance with a role of PIG3 protein for cell death, we showed residual apoptotic activity of p53-C277Y to which the described Ewing's sarcoma cell line was found to be resistant. p53-C277Y has previously been reported to bind to DNA with altered sequence specificity and to be unable to activate generic p53 target genes in yeast-based functional assays. Our results, therefore, show that a p53 mutant may behave differently when tested in its authentic cellular context.


Assuntos
Cisteína/genética , Dano ao DNA , Mutação/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Cisteína/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética
9.
Cancer Res ; 68(17): 7100-9, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18757425

RESUMO

Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21(WAF1/CIP1) levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21(WAF1/CIP1) induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21(WAF1/CIP1). These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53.


Assuntos
Genes p53 , Proteínas de Fusão Oncogênica/fisiologia , Receptores Notch/fisiologia , Sarcoma de Ewing/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Primers do DNA , Imunofluorescência , Inativação Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética
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