RESUMO
BACKGROUND AND AIMS: Chloride (Cl- ) channels in the apical membrane of biliary epithelial cells (BECs), also known as cholangiocytes, provide the driving force for biliary secretion. Although two Cl- channels have been identified on a molecular basis, the Cystic Fibrosis Transmembrane Conductance Regulator and Transmembrane Member 16A, a third Cl- channel with unique biophysical properties has been described. Leucine-Rich Repeat-Containing Protein 8, subfamily A (LRRC8A) is a newly identified protein capable of transporting Cl- in other epithelium in response to cell swelling. The aim of the present study was to determine if LRRC8A represents the volume-regulated anion channel in mouse BECs. APPROACH AND RESULTS: Studies were performed in mouse small (MSC) and large (MLC) cholangiocytes. Membrane Cl- currents were measured by whole-cell patch-clamp techniques and cell volume measurements were performed by calcein-AM fluorescence. Exposure of either MSC or MLC to hypotonicity (190 mOsm) rapidly increased cell volume and activated Cl- currents. Currents exhibited outward rectification, time-dependent inactivation at positive membrane potentials, and reversal potential at 0 mV (ECl ). Removal of extracellular Cl- or specific pharmacological inhibition of LRRC8A abolished currents. LRRC8A was detected in both MSC and MLC by reverse transcription polymerase chain reaction and confirmed by western blot. Transfection with LRRC8A small interfering RNA decreased protein levels by >70% and abolished volume-stimulated Cl- currents. CONCLUSION: These results demonstrate that LRRC8A is functionally present in mouse BECs, contributes to volume-activated Cl- secretion, and, therefore, may be a target to modulate bile formation in the treatment of cholestatic liver disorders.
Assuntos
Canais de Cloreto , Regulador de Condutância Transmembrana em Fibrose Cística , Camundongos , Animais , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Leucina , Proteínas de Repetições Ricas em Leucina , RNA Interferente Pequeno/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/genéticaRESUMO
Mammalian nephrons originate from a population of nephron progenitor cells, and changes in these cells' transcriptomes contribute to the cessation of nephrogenesis, an important determinant of nephron number. To characterize microRNA (miRNA) expression and identify putative cis-regulatory regions, we collected nephron progenitor cells from mouse kidneys at embryonic day 14.5 and postnatal day zero and assayed small RNA expression and transposase-accessible chromatin. We detect expression of 1104 miRNA (114 with expression changes), and 46,374 chromatin accessible regions (2103 with changes in accessibility). Genome-wide, our data highlight processes like cellular differentiation, cell migration, extracellular matrix interactions, and developmental signaling pathways. Furthermore, they identify new candidate cis-regulatory elements for Eya1 and Pax8, both genes with a role in nephron progenitor cell differentiation. Finally, we associate expression-changing miRNAs, including let-7-5p, miR-125b-5p, miR-181a-2-3p, and miR-9-3p, with candidate cis-regulatory elements and target genes. These analyses highlight new putative cis-regulatory loci for miRNA in nephron progenitors.
Assuntos
Cromatina , MicroRNAs , Animais , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Rim/metabolismo , Mamíferos/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Néfrons/metabolismo , Células-TroncoRESUMO
TMEM16A is a Ca2+-activated Cl- channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl- currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl- channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.
Assuntos
Anoctamina-1/metabolismo , Regulação da Expressão Gênica/fisiologia , Fígado/metabolismo , Receptores de Interleucina-13/metabolismo , Receptores de Interleucina-4/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Anoctamina-1/genética , Ácidos e Sais Biliares , Ductos Biliares/metabolismo , Linhagem Celular , Cloretos , Fenômenos Eletrofisiológicos , Humanos , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Masculino , Camundongos , Técnicas de Patch-Clamp , Ratos , Receptores de Interleucina-13/genética , Receptores de Interleucina-4/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismoRESUMO
Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.
Assuntos
Ductos Biliares/metabolismo , Bile/metabolismo , Células Epiteliais/metabolismo , Canais de Cátion TRPV/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ductos Biliares/citologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Mecanorreceptores/efeitos dos fármacos , Mecanorreceptores/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Canais de Cátion TRPV/efeitos adversosRESUMO
The original article can be found online.
RESUMO
Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.
Assuntos
Neoplasias do Tronco Encefálico/genética , Diferenciação Celular/genética , Glioma Pontino Intrínseco Difuso/genética , Histonas/genética , Mutação , Animais , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Glioma Pontino Intrínseco Difuso/patologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , CamundongosRESUMO
Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias Musculares/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Rabdomiossarcoma/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Criança , Epigenômica , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genômica , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular/métodos , Neoplasias Musculares/genética , Neoplasias Musculares/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Medicina de Precisão/métodos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteômica , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The recent identification of compounds that interact with the spliceosome (sudemycins, spliceostatin A, and meayamycin) indicates that these molecules modulate aberrant splicing via SF3B1 inhibition. Through whole transcriptome sequencing, we have demonstrated that treatment of Rh18 cells with sudemycin leads to exon skipping as the predominant aberrant splicing event. This was also observed following reanalysis of published RNA-seq data sets derived from HeLa cells after spliceostatin A exposure. These results are in contrast to previous reports that indicate that intron retention was the major consequence of SF3B1 inhibition. Analysis of the exon junctions up-regulated by these small molecules indicated that these sequences were absent in annotated human genes, suggesting that aberrant splicing events yielded novel RNA transcripts. Interestingly, the length of preferred downstream exons was significantly longer than the skipped exons, although there was no difference between the lengths of introns flanking skipped exons. The reading frame of the aberrantly skipped exons maintained a ratio of 2:1:1, close to that of the cassette exons (3:1:1) present in naturally occurring isoforms, suggesting negative selection by the nonsense-mediated decay (NMD) machinery for out-of-frame transcripts. Accordingly, genes involved in NMD and RNAs encoding proteins involved in the splicing process were enriched in both data sets. Our findings, therefore, further elucidate the mechanisms by which SF3B1 inhibition modulates pre-mRNA splicing.
Assuntos
Compostos de Epóxi/farmacologia , Éxons/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Biossíntese de Proteínas/genética , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/genética , Splicing de RNA/genética , Compostos de Espiro/farmacologia , Spliceossomos/genética , Sequência de Bases , Linhagem Celular Tumoral , Células HCT116 , Células HeLa , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Fases de Leitura/genética , Análise de Sequência de RNA , Transcriptoma/genéticaRESUMO
It is well known that pancreatic recovery after a single episode of injury such as an isolated bout of pancreatitis occurs rapidly. It is unclear, however, what changes are inflicted in such conditions to the molecular landscape of the pancreas. In the caerulein hyperstimulation model of pancreatitis, the murine pancreas has the ability to recover within one week based on histological appearance. In this study, we sought to characterize by RNA-sequencing (RNA-seq) the transcriptional profile of the recovering pancreas up to two weeks post-injury. We found that one week after injury there were 319 differentially expressed genes (DEGs) compared with baseline and that after two weeks there were 53 DEGs. Forty (12.5%) of the DEGs persisted from week one to week two, and another 13 DEGs newly emerged in the second week. Amongst the top up-regulated DEGs were several trypsinogen genes (trypsinogen 4, 5, 12, 15, and 16). To our knowledge, this is the first characterization of the transcriptome during pancreatic recovery by deep sequencing, and it reveals on a molecular basis that there is an ongoing recovery of the pancreas even after apparent histological resolution. The findings also raise the possibility of an emerging novel transcriptome upon pancreatic recovery.
Assuntos
Ceruletídeo/efeitos adversos , Perfilação da Expressão Gênica/métodos , Pancreatite/genética , Regeneração , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos , Pancreatite/induzido quimicamente , Análise de Sequência de RNA/métodosRESUMO
BACKGROUND AND AIMS: There is a pressing need to develop effective preventative therapies for post-ERCP pancreatitis (PEP). We demonstrated that early PEP events are induced through the calcium-activated phosphatase calcineurin and that global calcineurin deletion abolishes PEP in mice. A crucial question is whether acinar cell calcineurin controls the initiation of PEP in vivo. METHODS: We used a mouse model of PEP and examined the effects of in vivo acinar cell-specific calcineurin deletion by either generating a conditional knockout line or infusing a novel AAV-Ela-iCre into the pancreatic duct of a calcineurin floxed line. RESULTS: We found that PEP is dependent on acinar cell calcineurin in vivo, and this led us to determine that calcineurin inhibitors, infused within the radiocontrast, can largely prevent PEP. CONCLUSIONS: These results provide impetus for launching clinical trials to test the efficacy of intraductal calcineurin inhibitors to prevent PEP.
RESUMO
Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.
Assuntos
Transformação Celular Neoplásica/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Proteínas de Homeodomínio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Transformação Celular Neoplásica/patologia , Perfilação da Expressão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Isoformas de Proteínas , Regulador Transcricional ERG/genética , Adulto JovemRESUMO
Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.
Assuntos
Biomarcadores Tumorais/genética , Fatores de Ligação ao Core/genética , Genômica/métodos , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas de Fusão Oncogênica/genética , Adulto , Criança , HumanosRESUMO
Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n = 22), diffuse oligodendroglial tumors (d-OTs; n = 20), diffuse astrocytomas (DAs; n = 17), angiocentric gliomas (n = 15), and gangliogliomas (n = 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87 %), and MYB fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78 % of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.
Assuntos
Neoplasias Encefálicas/patologia , Genes myb , Predisposição Genética para Doença , Glioma/genética , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Adolescente , Adulto , Astrocitoma/genética , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Criança , Pré-Escolar , Proteínas de Ligação a DNA , Feminino , Ganglioglioma/genética , Ganglioglioma/patologia , Glioma/patologia , Humanos , Lactente , Masculino , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA , Transativadores/genética , Fatores de Transcrição , Adulto JovemRESUMO
Paediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here we analyse 37 adrenocortical tumours (ACTs) by whole-genome, whole-exome and/or transcriptome sequencing. Most cases (91%) show loss of heterozygosity (LOH) of chromosome 11p, with uniform selection against the maternal chromosome. IGF2 on chromosome 11p is overexpressed in 100% of the tumours. TP53 mutations and chromosome 17 LOH with selection against wild-type TP53 are observed in 28 ACTs (76%). Chromosomes 11p and 17 undergo copy-neutral LOH early during tumorigenesis, suggesting tumour-driver events. Additional genetic alterations include recurrent somatic mutations in ATRX and CTNNB1 and integration of human herpesvirus-6 in chromosome 11p. A dismal outcome is predicted by concomitant TP53 and ATRX mutations and associated genomic abnormalities, including massive structural variations and frequent background mutations. Collectively, these findings demonstrate the nature, timing and potential prognostic significance of key genetic alterations in paediatric ACT and outline a hypothetical model of paediatric adrenocortical tumorigenesis.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 17/genética , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Sequência de Bases , Criança , DNA Helicases/genética , Perfilação da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Perda de Heterozigosidade , Dados de Sequência Molecular , Proteínas Nucleares/genética , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/metabolismo , Proteína Nuclear Ligada ao X , beta Catenina/genéticaRESUMO
Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.
Assuntos
Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Desequilíbrio Alélico/genética , Estudos de Coortes , Análise Mutacional de DNA , Frequência do Gene , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Proteínas de Fusão Oncogênica/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
BACKGROUND: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS: We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS: Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS: Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Inibidores de Proteínas Quinases/uso terapêutico , Adolescente , Adulto , Animais , Criança , Pré-Escolar , DNA de Neoplasias/análise , Feminino , Genoma Humano , Xenoenxertos , Humanos , Lactente , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Cromossomo Filadélfia , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Transdução de Sinais/genética , Análise de Sobrevida , Adulto JovemRESUMO
Pediatric high-grade glioma (HGG) is a devastating disease with a less than 20% survival rate 2 years after diagnosis. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs), by whole-genome, whole-exome and/or transcriptome sequencing. We identified recurrent somatic mutations in ACVR1 exclusively in DIPGs (32%), in addition to previously reported frequent somatic mutations in histone H3 genes, TP53 and ATRX, in both DIPGs and NBS-HGGs. Structural variants generating fusion genes were found in 47% of DIPGs and NBS-HGGs, with recurrent fusions involving the neurotrophin receptor genes NTRK1, NTRK2 and NTRK3 in 40% of NBS-HGGs in infants. Mutations targeting receptor tyrosine kinase-RAS-PI3K signaling, histone modification or chromatin remodeling, and cell cycle regulation were found in 68%, 73% and 59% of pediatric HGGs, respectively, including in DIPGs and NBS-HGGs. This comprehensive analysis provides insights into the unique and shared pathways driving pediatric HGG within and outside the brainstem.
Assuntos
Receptores de Ativinas Tipo I/genética , Neoplasias do Tronco Encefálico/genética , Glioma/genética , Transdução de Sinais/genética , Animais , Criança , Estudos de Coortes , Biologia Computacional , Perfilação da Expressão Gênica , Fusão Gênica/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Análise em Microsséries , Receptor trkA/genética , Receptor trkB/genética , Receptor trkC/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Estatísticas não Paramétricas , Peixe-ZebraRESUMO
Pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). To define the landscape of somatic mutations in pediatric osteosarcoma, we performed whole-genome sequencing of DNA from 20 osteosarcoma tumor samples and matched normal tissue in a discovery cohort, as well as 14 samples in a validation cohort. Single-nucleotide variations (SNVs) exhibited a pattern of localized hypermutation called kataegis in 50% of the tumors. We identified p53 pathway lesions in all tumors in the discovery cohort, nine of which were translocations in the first intron of the TP53 gene. Beyond TP53, the RB1, ATRX, and DLG2 genes showed recurrent somatic alterations in 29%-53% of the tumors. These data highlight the power of whole-genome sequencing for identifying recurrent somatic alterations in cancer genomes that may be missed using other methods.