RESUMO
Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia patients in chronic phase fail to respond to imatinib or to second-generation inhibitors and progress to blast crisis. Until now, improvements in the understanding of the molecular mechanisms responsible for chronic myeloid leukemia transformation from chronic phase to the aggressive blast crisis remain limited. Here we present a large parallel sequencing analysis of 10 blast crisis samples and of the corresponding autologous chronic phase controls that reveals, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A). Additional analyses on a cohort of 24 blast crisis, 41 chronic phase as well as 40 acute myeloid leukemia and 38 atypical chronic myeloid leukemia patients at onset confirmed that UBE2A mutations are specifically acquired during chronic myeloid leukemia progression, with a frequency of 16.7% in advanced phases. In vitro studies show that the mutations here described cause a decrease in UBE2A activity, leading to an impairment of myeloid differentiation in chronic myeloid leukemia cells.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/genética , Mutação , Enzimas de Conjugação de Ubiquitina/genética , Crise Blástica/genética , Diferenciação Celular , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células HEK293 , Humanos , Mesilato de Imatinib/uso terapêutico , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/patologia , Masculino , Inibidores de Proteínas Quinases/farmacologia , Análise de Sequência de DNA , Sequenciamento do ExomaRESUMO
Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples (P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielomonocítica Crônica/genética , Mutação/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Estudos de Casos e Controles , Seguimentos , Humanos , Dados de Sequência Molecular , Prognóstico , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Chronic Myeloid Leukaemia (CML) is caused by the BCR/ABL1 fusion gene. Both the presence and the levels of BCR/ABL1 expression seem to be critical for CML progression from chronic phase (CP) to blast crisis (BC). After the oncogenic translocation, the BCR/ABL1 gene is under the transcriptional control of BCR promoter but the molecular mechanisms involved in the regulation of oncogene expression are mostly unknown. METHODS: A region of 1443bp of the functional BCR promoter was studied for transcription factor binding sites through in-silico analysis and Chromatin Immunoprecipitation experiments. BCR and BCR/ABL1 expression levels were analysed in CML cell lines after over-expression or silencing of MYC transcription factor. A luciferase reporter assay was used to confirm its activity on BCR promoter. RESULTS: In the present study we demonstrate that MYC and its partner MAX bind to the BCR promoter, leading to up-regulation of BCR and BCR/ABL1 at both transcriptional and protein levels. Accordingly, silencing of MYC expression in various BCR/ABL1 positive cell lines causes significant downregulation of BCR and BCR/ABL1, which consequently leads to decreased proliferation and induction of cell death. CONCLUSIONS: Here we describe a regulatory pathway modulating BCR and BCR/ABL1 expression, showing that the BCR promoter is under the transcriptional control of the MYC/MAX heterodimer. Since MYC is frequently over-expressed in BC, this phenomenon could play a critical role in BCR/ABL1 up-regulation and blast aggressiveness acquired during CML evolution.
Assuntos
Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica , Proteínas Proto-Oncogênicas c-bcr/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transcrição Gênica , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcr/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2-ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data.
Assuntos
Fusão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/genética , Software , Translocação Genética , Sequência de Bases , Gráficos por Computador , Genômica/métodos , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , TranscriptomaRESUMO
Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor. Although a comprehensive assessment of BIM CpG island methylation revealed no consistent changes, glucocorticoid resistance in xenografts and patient biopsies significantly correlated with decreased histone H3 acetylation. Moreover, the histone deacetylase inhibitor vorinostat relieved BIM repression and exerted synergistic antileukemic efficacy with dexamethasone in vitro and in vivo. These findings provide a novel therapeutic strategy to reverse glucocorticoid resistance and improve outcome for high-risk pediatric ALL.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/genética , Resistencia a Medicamentos Antineoplásicos , Inativação Gênica , Glucocorticoides/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Proteínas de Membrana/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Proteína 11 Semelhante a Bcl-2 , Criança , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Loci Gênicos , Glucocorticoides/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Camundongos , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , VorinostatRESUMO
BCR/ABL (Breakpoint Cluster Region protein/Abelson tyrosine-protein kinase 1) kinase domain (KD) mutations represent the most frequently described mechanism of resistance to the treatment with tyrosine kinase inhibitors (TKI) in patients with chronic myeloid leukemia (CML). Mutations may impair TKI activity by directly or indirectly impairing the drug binding to the protein. We report the discovery of three new BCR/ABL mutations, L248R, T315V, and F317R identified in two patients with CML (L248R and T315V) and in one patient with Ph+ acute lymphoblastic leukemia (ALL) (F317R). Mutations were screened against second-generation (bosutinib, nilotinib, and dasatinib), as well as third-generation TKIs (ponatinib/AP-24534 and DCC-2036). Furthermore, the activity profile of ponatinib and DCC-2036 against a panel of 24 clinically relevant BCR/ABL mutants is presented and compared to the other TKIs. The IC50 values for each TKI against the mutants and the IC50 increase over wild type BCR/ABL (relative resistance, RR) were calculated to define four resistance levels: sensitive (RR ≤ 2), moderately resistant (2 < RR ≤ 4), resistant (4 < RR ≤ 10), or highly resistant (RR > 10). L248R and T315V showed high resistance to imatinib, bosutinib, dasatinib, and nilotinib, intermediate resistance to ponatinib, but were sensitive to DCC-2036. Interestingly, F317R showed a moderate resistance to imatinib and nilotinib, but is resistant/highly resistant to dasatinib, bosutinib, ponatinib, and DCC-2036. The availability of drugs activity profiles may become a useful tool for clinicians dealing with the treatment of drug-resistant CML patients.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/genética , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Técnicas de Cultura de Células , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/química , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Mutagênese Sítio-Dirigida , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Transfecção , Células Tumorais CultivadasRESUMO
Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma characterized by expression of the oncogenic NPM/ALK fusion protein. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. In these patients, the ALK inhibitor crizotinib achieves high response rates, however 30-40% of them develop further resistance to crizotinib monotherapy, indicating that new therapeutic approaches are needed in this population. We here investigated the efficacy of upfront rational drug combinations to prevent the rise of resistant ALCL, in vitro and in vivo. Different combinations of crizotinib with CHOP chemotherapy, decitabine and trametinib, or with second-generation ALK inhibitors, were investigated. We found that in most cases combined treatments completely suppressed the emergence of resistant cells and were more effective than single drugs in the long-term control of lymphoma cells expansion, by inducing deeper inhibition of oncogenic signaling and higher rates of apoptosis. Combinations showed strong synergism in different ALK-dependent cell lines and better tumor growth inhibition in mice. We propose that drug combinations that include an ALK inhibitor should be considered for first-line treatments in ALK+ ALCL.
RESUMO
Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.
RESUMO
Unbalanced histone deacetylase (HDAC) hyperactivity is a common feature of tumor cells. Inhibition of HDAC activity is often associated with cancer cell growth impairment and death. Valproic acid (VPA) is a HDAC inhibitor used for the treatment of epilepsy. It has recently been recognized as a promising anticancer drug. We investigated the effects of VPA on growth and survival of colon cancer cells. VPA caused growth inhibition and programmed cell death that correlated with histone hyperacetylation. VPA modulated the expression of various factors involved in cell cycle control and apoptosis and induced caspase activation. Interestingly, VPA induced downregulation of c-Src and potentiated the cytotoxic effects of the c-Src inhibitor bosutinib, both in vitro and in vivo. The combination of sublethal doses of VPA and bosutinib led to massive apoptosis of colon cancer cells, irrespective of their genetic background. These results suggest that VPA may be employed as a positive modulator of bosutinib antitumor activity in colorectal cancer.
Assuntos
Compostos de Anilina/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Nitrilas/administração & dosagem , Quinolinas/administração & dosagem , Ácido Valproico/administração & dosagem , Acetilação , Animais , Apoptose , Linhagem Celular Tumoral , Feminino , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Quinases da Família src/metabolismoRESUMO
Resistance to imatinib represents an important scientific and clinical issue in chronic myelogenous leukemia. In the present study, the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 proved to be an active inhibitor of Bcr-Abl in several chronic myelogenous leukemia cell lines and transfectants, with IC(50) values in the low nanomolar range, 1 to 2 logs lower than those obtained with imatinib. Cells expressing activated forms of KIT or platelet-derived growth factor receptor (PDGFR), two additional targets of imatinib, were unaffected by SKI-606, whereas activity was found against PIM2. SKI-606 retained activity in cells where resistance to imatinib was caused by BCR-ABL gene amplification and in three of four Bcr-Abl point mutants tested. In vivo experiments confirmed SKI-606 activity in models where resistance was not caused by mutations as well as in cells carrying the Y253F, E255K, and D276G mutations. Modeling considerations attribute the superior activity of SKI-606 to its ability to bind a conformation of Bcr-Abl different from imatinib.
Assuntos
Compostos de Anilina/farmacologia , Neoplasias/tratamento farmacológico , Nitrilas/farmacologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Compostos de Anilina/química , Animais , Benzamidas , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Genótipo , Humanos , Mesilato de Imatinib , Células K562 , Camundongos , Camundongos Nus , Modelos Moleculares , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Nitrilas/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/química , Análise de Sobrevida , Tiazóis/farmacologia , Células U937 , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel-Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel-Giedion syndrome caused by SETBP1 mutations.
Assuntos
Proteínas de Transporte/genética , Epigênese Genética , Perfilação da Expressão Gênica , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Anormalidades Múltiplas/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Anormalidades Craniofaciais/genética , Ontologia Genética , Células HEK293 , Deformidades Congênitas da Mão/genética , Humanos , Deficiência Intelectual/genética , Leucemia/genética , Leucemia/patologia , Camundongos , Unhas Malformadas/genética , Neurogênese/genética , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
PIM1 is over-expressed in multiple tumors, including prostate cancer (PCa). PIM1 upregulation is mediated by direct binding of the ERG transcription factor to its promoter. About 50% of PCa cases are characterized by the presence of the TMPRSS2/ERG fusion, leading to ERG over-expression and thus to PIM1 transcriptional activation. PIM kinases are considered as weak oncogenes, but when combined with additional genetic alterations can induce strong transforming effects. Here we show anti-proliferative activity of the newly described PIM1 inhibitor NMS-P645 in combination with the PI3K inhibitor GDC-0941 in TMPRSS2/ERG positive and negative PCa cells. Treatment with NMS-P645 alone can reverse PIM1-mediated pro-survival signals in prostate cells, such as activation of STAT3 through Tyr705 phosphorylation and resistance to taxane-based treatments, but does not exert a strong anti-tumoral effect. However, the simultaneous treatment with NMS-P645 and GDC-0941 induces a significant anti-proliferative response in PCa cells. These results support the use of combination strategies with PIM and PI3K inhibitors as effective treatment for PCa cases.
RESUMO
The complicated, evolving landscape of cancer mutations poses a formidable challenge to identify cancer genes among the large lists of mutations typically generated in NGS experiments. The ability to prioritize these variants is therefore of paramount importance. To address this issue we developed OncoScore, a text-mining tool that ranks genes according to their association with cancer, based on available biomedical literature. Receiver operating characteristic curve and the area under the curve (AUC) metrics on manually curated datasets confirmed the excellent discriminating capability of OncoScore (OncoScore cut-off threshold = 21.09; AUC = 90.3%, 95% CI: 88.1-92.5%), indicating that OncoScore provides useful results in cases where an efficient prioritization of cancer-associated genes is needed.
Assuntos
Genes Neoplásicos , Neoplasias/genética , Software , Humanos , MutaçãoRESUMO
This corrects the article DOI: 10.1038/srep46290.
RESUMO
Identifying patients who may benefit from targeted therapy is an urgent clinical issue in prostate cancer (PCa). We investigated the molecular relationship between TMPRSS2-ERG (T2E) fusion gene and insulin-like growth factor receptor (IGF-1R) to optimize the use of IGF-1R inhibitors.IGF-1R was analyzed in cell lines and in radical prostatectomy specimens in relation to T2E status. ERG binding to IGF-1R promoter was evaluated by chromatin immunoprecipitation (ChIP). Sensitivity to anti-IGF-1R agents was evaluated alone or in combination with anti-androgen abiraterone acetate in vitro at basal levels or upon ERG modulation.IGF-1R analysis performed in PCa cells or clinical samples showed that T2E expression correlated with higher IGF-1R expression at mRNA and protein levels. Genetic modulation of ERG directly affected IGF-1R protein levels in vitro. ChIP analysis showed that ERG binds IGF-1R promoter and that promoter occupancy is higher in T2E-positive cells. IGF-1R inhibition was more effective in cell lines expressing the fusion gene and combination of IGF-1R inhibitors with abiraterone acetate produced synergistic effects in T2E-expressing cells.Here, we provide the rationale for use of T2E fusion gene to select PCa patients for anti-IGF-1R treatments. The combination of anti-IGF-1R-HAbs with an anti-androgen therapy is strongly advocated for patients expressing T2E.
Assuntos
Antagonistas de Androgênios/farmacologia , Neoplasias da Próstata/patologia , Receptor IGF Tipo 1/antagonistas & inibidores , Serina Endopeptidases/genética , Transativadores/genética , Acetato de Abiraterona/farmacologia , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Regiões Promotoras Genéticas/genética , Próstata/patologia , Próstata/cirurgia , Ligação Proteica , Receptor IGF Tipo 1/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismo , Transativadores/metabolismo , Regulador Transcricional ERGRESUMO
Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC 3 (NM_003786.3:c.1783-1G>A), KLHDC 1 (NM_172193.1:c.568-2A>G), HOOK 1 (NM_015888.4:c.1662-1G>A), SMAD 9 (NM_001127217.2:c.1004-1C>T), and DNAH 9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC 3, HOOK 1. In ABCC 3 and HOOK 1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK 1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4-6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis.
RESUMO
Copy number alterations (CNA) are common events occurring in leukaemias and solid tumors. Comparative Genome Hybridization (CGH) is actually the gold standard technique to analyze CNAs; however, CGH analysis requires dedicated instruments and is able to perform only low resolution Loss of Heterozygosity (LOH) analyses. Here we present CEQer (Comparative Exome Quantification analyzer), a new graphical, event-driven tool for CNA/allelic-imbalance (AI) coupled analysis of exome sequencing data. By using case-control matched exome data, CEQer performs a comparative digital exonic quantification to generate CNA data and couples this information with exome-wide LOH and allelic imbalance detection. This data is used to build mixed statistical/heuristic models allowing the identification of CNA/AI events. To test our tool, we initially used in silico generated data, then we performed whole-exome sequencing from 20 leukemic specimens and corresponding matched controls and we analyzed the results using CEQer. Taken globally, these analyses showed that the combined use of comparative digital exon quantification and LOH/AI allows generating very accurate CNA data. Therefore, we propose CEQer as an efficient, robust and user-friendly graphical tool for the identification of CNA/AI in the context of whole-exome sequencing data.
Assuntos
Desequilíbrio Alélico/genética , Exoma/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Éxons/genética , HumanosRESUMO
BIM is a proapoptotic member of the Bcl-2 family. Here, we investigated the epigenetic status of the BIM locus in NPM/ALK+ anaplastic large cell lymphoma (ALCL) cell lines and in lymph node biopsies from NPM/ALK+ ALCL patients. We show that BIM is epigenetically silenced in cell lines and lymph node specimens and that treatment with the deacetylase inhibitor trichostatin A restores the histone acetylation, strongly upregulates BIM expression, and induces cell death. BIM silencing occurs through recruitment of MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2) corepressor complex. This event requires BIM CpG methylation/demethylation with 5-azacytidine that leads to detachment of the MeCP2 corepressor complex and reacetylation of the histone tails. Treatment with the ALK inhibitor PF2341066 or with an inducible shRNA targeting NPM/ALK does not restore BIM locus reacetylation; however, enforced expression of NPM/ALK in an NPM/ALK-negative cell line significantly increases the methylation at the BIM locus. This study demonstrates that BIM is epigenetically silenced in NPM/ALK-positive cells through recruitment of the SIN3a/HDAC1/2 corepressor complex and that NPM/ALK is dispensable to maintain BIM epigenetic silencing but is able to act as an inducer of BIM methylation.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Inativação Gênica , Linfoma Anaplásico de Células Grandes/genética , Proteínas de Membrana/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/metabolismo , Regiões 5' não Traduzidas , Acetilação , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Linfoma Anaplásico de Células Grandes/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.