RESUMEN
BACKGROUND: Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression. RESULTS: Exon expression analysis shows that patients' untransformed and malignant HTLV-1(+) CD4(+) T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4(+) T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL. CONCLUSIONS: Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Exones , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/fisiología , Leucemia-Linfoma de Células T del Adulto/patología , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Humanos , Transcripción GenéticaRESUMEN
BACKGROUND: Adult T cell leukemia results from the malignant transformation of a CD4+ lymphoid clone carrying an integrated HTLV-1 provirus that has undergone several oncogenic events over a 30-60 year period of persistent clonal expansion. Both CD4+ and CD8+ lymphocytes are infected in vivo; their expansion relies on CD4+ cell cycling and on the prevention of CD8+ cell death. Cloned infected CD4+ but not CD8+ T cells from patients without malignancy also add up nuclear and mitotic defects typical of genetic instability related to the expression of the virus-encoded oncogene tax. HTLV-1 expression is cancer-prone in vitro, but in vivo numerous selection forces act to maintain T cell homeostasis and are possibly involved in clonal selection. RESULTS: Here we demonstrate that the HTLV-1 associated CD4+ preleukemic phenotype and the specific patterns of CD4+ and CD8+ clonal expansion are in vivo selected processes. By comparing the effects of recent (1 month) experimental infections performed in vitro and those observed in cloned T cells from patients infected for >6-26 years, we found that in chronically HTLV-1 infected individuals, HTLV-1 positive clones are selected for tax expression. In vivo, infected CD4+ cells are positively selected for cell cycling whereas infected CD8+ cells and uninfected CD4+ cells are negatively selected for the same processes. In contrast, the known HTLV-1-dependent prevention of CD8+ T cell death pertains to both in vivo and in vitro infected cells. CONCLUSIONS: Therefore, virus-cell interactions alone are not sufficient to initiate early leukemogenesis in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Muerte Celular , Proliferación Celular , Genes pX , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Leucemia-Linfoma de Células T del Adulto/virología , Transformación Celular Viral , Expresión Génica , Interacciones Huésped-Patógeno , HumanosRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) infects both CD4+ and CD8+ lymphocytes, yet it induces adult T cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Here we show that in vivo infected CD4+ and CD8+ T cells displayed similar patterns of clonal expansion in carriers without malignancy. Cloned infected cells from individuals without malignancy had a dramatic increase in spontaneous proliferation, which predominated in CD8+ lymphocytes and depended on the amount of tax mRNA. In fact, the clonal expansion of HTLV-1-positive CD8+ and CD4+ lymphocytes relied on 2 distinct mechanisms--infection prevented cell death in the former while recruiting the latter into the cell cycle. Cell cycling, but not apoptosis, depended on the level of viral-encoded tax expression. Infected tax-expressing CD4+ lymphocytes accumulated cellular defects characteristic of genetic instability. Therefore, HTLV-1 infection establishes a preleukemic phenotype that is restricted to CD4+ infected clones.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Adulto , Apoptosis , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Portador Sano/virología , Ciclo Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Preleucemia/metabolismo , Preleucemia/virología , Factores de TiempoRESUMEN
In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p<10-4). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.
Asunto(s)
Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/genética , Factores de Edad , Citogenética , Dioxigenasas , Supervivencia sin Enfermedad , Exones , Femenino , Humanos , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Nucleofosmina , Pronóstico , Medición de Riesgo , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Chromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Diferenciación Celular/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica/genética , Metionina Sulfóxido Reductasas/genética , Células Madre/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Inmunoprecipitación de Cromatina , Daño del ADN , Células Epiteliales/citología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Immunoblotting , Glándulas Mamarias Humanas/citología , Metionina Sulfóxido Reductasas/metabolismo , Ratones Endogámicos NOD , Persona de Mediana Edad , Especies Reactivas de Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Células Madre/citología , Análisis de Matrices Tisulares , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adulto Joven , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.
Asunto(s)
Empalme Alternativo/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Anciano , Antraciclinas/farmacología , Azacitidina/farmacología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Citarabina/farmacología , Doxorrubicina/farmacología , Exones/genética , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas/metabolismo , Oncogenes/genética , Proteínas de Unión a Poli-ADP-Ribosa , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas WT1/metabolismoRESUMEN
Viruses disrupt the host cell microRNA (miRNA) network to facilitate their replication. Human T-cell leukemia virus type I (HTLV-1) replication relies on the clonal expansion of its host CD4(+) and CD8(+) T cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4(+) phenotype. The viral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells and is involved in sustaining cell proliferation and silencing virus expression. Here, we show that an HBZ-miRNA axis promotes cell proliferation and genetic instability, as indicated by comet assays that showed increased numbers of DNA-strand breaks. Expression profiling of miRNA revealed that infected CD4(+) cells, but not CD8(+) T cells, overexpressed oncogenic miRNAs, including miR17 and miR21. HBZ activated these miRNAs via a posttranscriptional mechanism. These effects were alleviated by knocking down miR21 or miR17 and by ectopic expression of OBFC2A, a DNA-damage factor that is downregulated by miR17 and miR21 in HTLV-1-infected CD4(+) T cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLL cells.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proliferación Celular/genética , Inestabilidad Genómica , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas Virales/genética , Adulto , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Regulación Viral de la Expresión Génica , Genes pX/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/inmunología , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas de los Retroviridae , Proteínas Virales/metabolismoRESUMEN
Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proteínas Oncogénicas/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Células de la Médula Ósea/citología , Núcleo Celular/metabolismo , Perfilación de la Expresión Génica , Productos del Gen tax/metabolismo , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Leucemia Mieloide Aguda/metabolismo , Hibridación de Ácido Nucleico , Proteínas Oncogénicas/genética , Proteínas de Unión a Poli-ADP-Ribosa , Regiones Promotoras GenéticasRESUMEN
Here we investigate the mechanisms by which HTLV-1 infection prevents the cell death of CD8(+) T cells in vivo. We show that upon natural infection, cloned CD8(+) but not CD4(+) cells from patients without malignancy become resistant to Fas-mediated cell death and acquire an antiapoptotic transcriptome that includes the overexpression of cIAP-2 and c-FLIP(L). CD8(+) lymphocyte-restricted cIAP-2 overexpression correlates with resistance to Fas-mediated apoptosis and depends on tax expression via NF-KappaB. In contrast, in the same CD8(+) cells, the HTLV-1-dependent overexpression of c-FLIP(L) does not correlate with resistance to Fas-mediated cell death nor with tax expression. In the present model, infected CD8(+) clones are the only cell subtype in which cIAP-2 expression correlates with resistance to cell death. These results support a role for Tax-dependent cIAP-2 expression in preventing the death of naturally infected CD8(+) cells and thereby in their clonal expansion in vivo.