RESUMEN
Sézary syndrome (SS) is an aggressive, leukemic cutaneous T-cell lymphoma variant. Molecular pathogenesis of SS is still unclear despite many studies on genetic alterations, gene expression and epigenetic regulations. Through whole genome and transcriptome next generation sequencing nine Sézary syndrome patients were analyzed in terms of copy number variations and rearrangements affecting gene expression. Recurrent copy number variations were detected within 8q (MYC, TOX), 17p (TP53, NCOR1), 10q (PTEN, FAS), 2p (DNMT3A), 11q (USP28), 9p (CAAP1), but no recurrent rearrangements were identified. However, expression of five genes involved in rearrangements (TMEM244, EHD1, MTMR2, RNF123 and TOX) was altered in all patients. Fifteen rearrangements detected in Sézary syndrome patients and SeAx resulted in an expression of new fusion transcripts, nine of them were in frame (EHD1-CAPN12, TMEM66-BAIAP2, MBD4-PTPRC, PTPRC-CPN2, MYB-MBNL1, TFG-GPR128, MAP4K3-FIGLA, DCP1A-CCL27, MBNL1-KIAA2018) and five resulted in ectopic expression of fragments of genes not expressed in normal T-cells (BAIAP2, CPN2, GPR128, CAPN12, FIGLA). Our results not only underscored the genomic complexity of the Sézary cancer cell genome but also showed an unpreceded large variety of novel gene rearrangements resulting in fusions transcripts and ectopically expressed genes.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Proteínas de Fusión Oncogénica/genética , Síndrome de Sézary/genética , Neoplasias Cutáneas/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Cromosomas Humanos , Variaciones en el Número de Copia de ADN , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Síndrome de Sézary/patología , Neoplasias Cutáneas/patologíaRESUMEN
A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.
Asunto(s)
Cisteína Endopeptidasas/efectos de los fármacos , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , ARN Interferente Pequeño/farmacología , Receptor Toll-Like 9/efectos de los fármacos , Animales , Western Blotting , Línea Celular Tumoral , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Femenino , Inmunidad Humoral/efectos de los fármacos , Inmunidad Humoral/fisiología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Terapia Molecular Dirigida , FN-kappa B/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
The 6q23-27 region, recurrently deleted in Sézary syndrome (SS), was characterized at the molecular level in 13 SS patients and SS cell line SeAx. Using fine-tiling comparative genomic hybridization, deletions within the 6q23-27 region were detected in half of the samples (six patients and SeAx). All samples with deletions were further analyzed by ligation-mediated PCR. In addition, in one patient sample and in SeAx, paired-end next-generation sequencing was performed on the HiSeq2000 Illumina platform. Using those techniques, 23 rearrangements associated with the deletions were identified. The majority of rearrangements showed enormous complexity and diversity, including eight inversions, three transpositions, and four translocations (with chromosomes 3, 17, 10, and 12). Fifteen genes were disrupted by those rearrangements, the MYB proto-oncogene three times and the interleukin-22 receptor subunit alpha-2 gene (IL22RA2) twice. All three patients with MYB alterations showed low MYB expression, whereas seven of the remaining patients showed overexpression. Most patients overexpressing MYB also presented increased expression of MYC, HSPA8, and BCL2. Five gene fusions were identified, of which two, CCDC28A-IL22RA2 and AIG1-GOSR1, both in SeAx, were in the same orientation and were expressed at the messenger RNA level.