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1.
Curr Issues Mol Biol ; 43(3): 2147-2156, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34940123

RESUMO

For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.


Assuntos
Biomarcadores Tumorais , Linhagem Celular Tumoral , Predisposição Genética para Doença , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Alelos , Aberrações Cromossômicas , Análise Mutacional de DNA , Progressão da Doença , Humanos , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , RNA-Seq , Sequenciamento do Exoma
2.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072771

RESUMO

Recently, we documented a hematopoietic NKL-code mapping physiological expression patterns of NKL homeobox genes in human myelopoiesis including monocytes and their derived dendritic cells (DCs). Here, we enlarge this map to include normal NKL homeobox gene expressions in progenitor-derived DCs. Analysis of public gene expression profiling and RNA-seq datasets containing plasmacytoid and conventional dendritic cells (pDC and cDC) demonstrated HHEX activity in both entities while cDCs additionally expressed VENTX. The consequent aim of our study was to examine regulation and function of VENTX in DCs. We compared profiling data of VENTX-positive cDC and monocytes with VENTX-negative pDC and common myeloid progenitor entities and revealed several differentially expressed genes encoding transcription factors and pathway components, representing potential VENTX regulators. Screening of RNA-seq data for 100 leukemia/lymphoma cell lines identified prominent VENTX expression in an acute myelomonocytic leukemia cell line, MUTZ-3 containing inv(3)(q21q26) and t(12;22)(p13;q11) and representing a model for DC differentiation studies. Furthermore, extended gene analyses indicated that MUTZ-3 is associated with the subtype cDC2. In addition to analysis of public chromatin immune-precipitation data, subsequent knockdown experiments and modulations of signaling pathways in MUTZ-3 and control cell lines confirmed identified candidate transcription factors CEBPB, ETV6, EVI1, GATA2, IRF2, MN1, SPIB, and SPI1 and the CSF-, NOTCH-, and TNFa-pathways as VENTX regulators. Live-cell imaging analyses of MUTZ-3 cells treated for VENTX knockdown excluded impacts on apoptosis or induced alteration of differentiation-associated cell morphology. In contrast, target gene analysis performed by expression profiling of knockdown-treated MUTZ-3 cells revealed VENTX-mediated activation of several cDC-specific genes including CSFR1, EGR2, and MIR10A and inhibition of pDC-specific genes like RUNX2. Taken together, we added NKL homeobox gene activities for progenitor-derived DCs to the NKL-code, showing that VENTX is expressed in cDCs but not in pDCs and forms part of a cDC-specific gene regulatory network operating in DC differentiation and function.


Assuntos
Células Dendríticas/metabolismo , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Genes Homeobox , Humanos , Imunofenotipagem , Transcriptoma
3.
Int J Cancer ; 146(2): 400-412, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31271662

RESUMO

Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP+ and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.


Assuntos
Antineoplásicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Leucemia Aguda Bifenotípica/tratamento farmacológico , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Adulto , Idoso , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desmetilação do DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Aguda Bifenotípica/patologia , Masculino , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Tretinoína/farmacologia , Tretinoína/uso terapêutico
4.
Blood ; 132(21): 2280-2285, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30282799

RESUMO

The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein "preBLL"). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.


Assuntos
Linfoma de Burkitt/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfócitos B/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Recombinação V(D)J , Adolescente , Adulto , Idoso , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/patologia , Criança , Pré-Escolar , Metilação de DNA , Feminino , Rearranjo Gênico do Linfócito B , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Precursoras de Linfócitos B/metabolismo , Estudos Retrospectivos , Translocação Genética , Adulto Jovem
5.
Int J Mol Sci ; 21(16)2020 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823535

RESUMO

Certified cell line models provide ideal experimental platforms to answer countless scientific questions. The LL-100 panel is a cohort of cell lines that are broadly representative of all leukemia-lymphoma entities (including multiple myeloma and related diseases), rigorously authenticated and validated, and comprehensively annotated. The process of the assembly of the LL-100 panel was based on evidence and experience. To expand the genetic characterization across all LL-100 cell lines, we performed whole-exome sequencing and RNA sequencing. Here, we describe the conception of the panel and showcase some exemplary applications with a focus on cancer genomics. Due diligence was paid to exclude cross-contaminated and non-representative cell lines. As the LL-100 cell lines are so well characterized and readily available, the panel will be a valuable resource for identifying cell lines with mutations in cancer genes, providing superior model systems. The data also add to the current knowledge of the molecular pathogenesis of leukemia-lymphoma. Additional efforts to expand the breadth of available high-quality cell lines are clearly warranted.


Assuntos
Pesquisa Biomédica , Leucemia/patologia , Linfoma/patologia , Linhagem Celular Tumoral , Evolução Clonal/genética , Humanos , Leucemia/genética , Linfoma/genética , Mutação/genética
6.
Biol Chem ; 399(6): 511-523, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29533902

RESUMO

Characteristic components of Hodgkin lymphoma (HL) tissue are the mono- or multinucleated Hodgkin-Reed-Sternberg (HRS) cells. Given the challenges of isolating these rare malignant cells and the difficulty in culturing cells from patients, many investigators have tried to establish cell lines in efforts to develop cellular tools for in vitro studies. A limited number of HL cell lines exist and have provided valuable insights into HL pathobiology. A literature survey indicated that 35 cell lines derived from HL patients have been published. To determine whether all these alleged HL cell lines hold up to scrutiny, we examined the available data and also put some of these cell lines to the test of hierarchical clustering, providing additional information regarding assignment to cell line type and tissue derivation. Hierarchical clustering separated the bona fide (classical) HL cell lines completely from cell lines derived from other lymphoma categories and proved conclusively that HL cell lines represent a distinct entity, irrespective of the cellular origin of the HRS cells. We conclude by pointing out the need for an intensified search for new cell culture avenues in order to develop a new generation of informative HL cell lines covering more widely the spectrum of HL stages and subtypes.


Assuntos
Doença de Hodgkin/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Doença de Hodgkin/genética , Humanos
7.
Blood ; 128(23): 2666-2670, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27670424

RESUMO

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (<2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%]). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P =022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL.


Assuntos
Biomarcadores Tumorais/genética , Deleção de Genes , Proteínas I-kappa B/genética , Linfoma de Células B , Neoplasias do Mediastino , Proteínas Proto-Oncogênicas/genética , Adolescente , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Linfoma de Células B/genética , Linfoma de Células B/mortalidade , Masculino , Neoplasias do Mediastino/genética , Neoplasias do Mediastino/mortalidade , Pessoa de Meia-Idade , Taxa de Sobrevida
8.
Genes Chromosomes Cancer ; 56(3): 243-252, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27750403

RESUMO

Leukemia cell lines have been widely used in the hematology field to unravel mechanistic insights and to test new therapeutic strategies. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases that are characterized by ineffective hematopoiesis and frequent progress to acute myeloid leukemia (AML). A few cell lines have been established from MDS patients after progression to AML but their characterization is incomplete. Here we provide a detailed description of the immunophenotypic profile of the MDS-derived cell lines SKK-1, SKM-1, F-36P; and MOLM-13. Specifically, we analyzed a comprehensive panel of markers that are currently applied in the diagnostic routine for myeloid disorders. To provide high-resolution genetic data comprising copy number alterations and losses of heterozygosity we performed whole genome single nucleotide polymorphism-based arrays and included the cell line OHN-GM that harbors the frequent chromosome arm 5q deletion. Furthermore, we assessed the mutational status of 83 disease-relevant genes. Our results provide a resource to the MDS and AML field that allows researchers to choose the best-matching cell line for their functional studies. © 2016 Wiley Periodicals, Inc.


Assuntos
Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Análise Citogenética/métodos , Imunofenotipagem/métodos , Leucemia Mieloide Aguda/genética , Mutação/genética , Síndromes Mielodisplásicas/genética , Progressão da Doença , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/patologia , Células Tumorais Cultivadas
9.
Int J Cancer ; 140(5): 1209-1214, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-27870004

RESUMO

Leukemia-lymphoma cell lines are important research tools in a variety of fields. To represent adequate model systems it is of utmost importance that cell lines faithfully model the primary tumor material and are not cross-contaminated with unrelated cell material (or contaminated with mycoplasma). As it has been previously reported that cross-contaminated cell lines represent a significant problem, it is of interest to know whether any improvement in the prevalence of such "false cell lines" had occurred since we called the alert in 1999. A retrospective review of our data archives covered 848 cell lines received from 1990 to 2014 from 290 laboratories in 23 countries spanning the spectrum of leukemia-lymphoma entities. Two variables were considered: authenticity and freedom from mycoplasma infection. Regarding provenance, we separately considered primary sources (original investigators having established the cell lines or reference repositories) and secondary sources. The percentages of mycoplasma-contaminated cell lines decreased significantly over the 25-year timespan. Among primary sourced material: mycoplasma-contamination fell from 23% to 0%; among secondary sourced: from 48% to 21%. The corresponding figures for cross-contamination declined from 15% to 6%, while among material obtained from secondary sources prevalence remained remarkably high, throughout the time periods at 14-18%. Taken together, our data indicate that using non-authenticated cell lines from secondary sources carries a risk of about 1:6 for obtaining a false cell line. The use of authentic leukemia-lymphoma cell lines holds important translational value for their model character and the reproducibility of the laboratory data in the clinical arena.


Assuntos
Linhagem Celular Tumoral/microbiologia , Leucemia/patologia , Linfoma/patologia , Mycoplasma/isolamento & purificação , Impressões Digitais de DNA , Humanos , Hibridização In Situ , Cariotipagem , Estudos Retrospectivos
10.
Haematologica ; 102(7): 1204-1214, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28411256

RESUMO

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1 We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML.


Assuntos
Diferenciação Celular/genética , Expressão Ectópica do Gene , Epigênese Genética , Proteínas de Homeodomínio/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional/métodos , DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , DNA Metiltransferase 3A , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Mutação , Nucleofosmina , Translocação Genética , Adulto Jovem
11.
BMC Genomics ; 17: 399, 2016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27225215

RESUMO

BACKGROUND: Whole exome sequencing (WES) has been proven to serve as a valuable basis for various applications such as variant calling and copy number variation (CNV) analyses. For those analyses the read coverage should be optimally balanced throughout protein coding regions at sufficient read depth. Unfortunately, WES is known for its uneven coverage within coding regions due to GC-rich regions or off-target enrichment. RESULTS: In order to examine the irregularities of WES within genes, we applied Agilent SureSelectXT exome capture on human samples and sequenced these via Illumina in 2 × 101 paired-end mode. As we suspected the sequenced insert length to be crucial in the uneven coverage of exome captured samples, we sheared 12 genomic DNA samples to two different DNA insert size lengths, namely 130 and 170 bp. Interestingly, although mean coverages of target regions were clearly higher in samples of 130 bp insert length, the level of evenness was more pronounced in 170 bp samples. Moreover, merging overlapping paired-end reads revealed a positive effect on evenness indicating overlapping reads as another reason for the unevenness. In addition, mutation analysis on a subset of the samples was performed. In these isogenic subclones, the false negative rate in the 130 bp samples was almost double to that in the 170 bp samples. Visual inspection of the discarded mutation sites exposed low coverages at the sites flanked by high amplitudes of coverage depth. CONCLUSIONS: Producing longer insert reads could be a good strategy to achieve better uniform read coverage in coding regions and hereby enhancing the effective sequencing yield to provide an improved basis for further variant calling and CNV analyses.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Exoma , Genoma Humano , Humanos
12.
Blood ; 123(8): 1187-98, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24398325

RESUMO

The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.


Assuntos
Linfócitos B/fisiologia , Linfoma de Burkitt/classificação , Linfoma de Burkitt/genética , Genes myc/genética , Translocação Genética/genética , Adolescente , Adulto , Idoso , Linfoma de Burkitt/patologia , Linhagem Celular , Criança , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 8 , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Gradação de Tumores , Recidiva , Adulto Jovem
13.
Haematologica ; 101(11): 1380-1389, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27390358

RESUMO

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project "Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing", we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.


Assuntos
Linfoma de Células B/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Adolescente , Linfoma de Burkitt/genética , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Centro Germinativo , Humanos , Lactente , Recém-Nascido , Linfoma Folicular/genética , Linfoma Difuso de Grandes Células B/genética , Masculino , MicroRNAs/genética , Mutação , Edição de RNA
14.
Genes Chromosomes Cancer ; 54(7): 418-32, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25960206

RESUMO

Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder of unclear pathogenesis. Recent studies have identified BRAF(V600E) mutations in most HCL patients, highlighting this abnormality as a molecular hallmark for this disease. Cell lines originating from HCL patients lack BRAF mutations but retain the typical piliferous morphology and the distinctive HCL immunophenotype, thus, constituting suitable tools for identifying alternative tumor genes and leukemic mechanisms in this malignancy. To this end, we integrated genomic and transcriptional profiling of the HCL cell line MLMA. The expression levels of genomically targeted genes were compared to four HCL control cell lines, thus, identifying 91 chromosomally deregulated genes. Gene set enrichment analysis of these indicted apoptosis, proliferation, and DNA damage response as altered processes. Accordingly, prominent target genes overexpressed in this cell line include ATM, BRAF, CDK6, CUTL1/CUX1, H2AFX, and REL. Treatment of MLMA with selective pharmacological inhibitors and specific siRNA-mediated gene knockdowns highlighted a central role for NFkB in their deregulation in HCL. Moreover, relevant expression profiling data from HCL and ABC-DLBCL cell lines display elevated NFkB-pathway activity when compared to GC-DLBCL equivalents. Finally, analysis of HCL patient samples in silico collectively supported the clinical significance of NFkB activation in this disease. In conclusion, we identified deregulated genes and multiple mechanisms underlying aberrantly activated NFkB-pathway in HCL. Therefore, NFkB may represent a B-cell specific hallmark of HCL and a promising novel therapeutic target, most notably in patients lacking BRAF mutations in this entity including variant HCL.


Assuntos
Leucemia de Células Pilosas/metabolismo , NF-kappa B/metabolismo , Apoptose , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética
15.
Genes Chromosomes Cancer ; 54(9): 555-64, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26173642

RESUMO

The genetic hallmark of Burkitt lymphoma is the translocation t(8;14)(q24;q32), or one of its light chain variants, resulting in IG-MYC juxtaposition. However, these translocations alone are insufficient to drive lymphomagenesis, which requires additional genetic changes for malignant transformation. Recent studies of Burkitt lymphoma using next generation sequencing approaches have identified various recurrently mutated genes including ID3, TCF3, CCND3, and TP53. Here, by using similar approaches, we show that PCBP1 is a recurrently mutated gene in Burkitt lymphoma. By whole-genome sequencing, we identified somatic mutations in PCBP1 in 3/17 (18%) Burkitt lymphomas. We confirmed the recurrence of PCBP1 mutations by Sanger sequencing in an independent validation cohort, finding mutations in 3/28 (11%) Burkitt lymphomas and in 6/16 (38%) Burkitt lymphoma cell lines. PCBP1 is an intron-less gene encoding the 356 amino acid poly(rC) binding protein 1, which contains three K-Homology (KH) domains and two nuclear localization signals. The mutations predominantly (10/12, 83%) affect the KH III domain, either by complete domain loss or amino acid changes. Thus, these changes are predicted to alter the various functions of PCBP1, including nuclear trafficking and pre-mRNA splicing. Remarkably, all six primary Burkitt lymphomas with a PCBP1 mutation expressed MUM1/IRF4, which is otherwise detected in around 20-40% of Burkitt lymphomas. We conclude that PCBP1 mutations are recurrent in Burkitt lymphomas and might contribute, in cooperation with other mutations, to its pathogenesis.


Assuntos
Linfoma de Burkitt/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Mutação , Adolescente , Adulto , Idoso , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Estudos de Coortes , Proteínas de Ligação a DNA , Feminino , Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Masculino , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA , Adulto Jovem
16.
Haematologica ; 100(6): 801-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25769544

RESUMO

Genetic heterogeneity is widespread in tumors, but poorly documented in cell lines. According to immunoglobulin hypermutation analysis, the diffuse large B-cell lymphoma cell line U-2932 comprises two subpopulations faithfully representing original tumor subclones. We set out to identify molecular causes underlying subclone-specific expression affecting 221 genes including surface markers and the germinal center oncogenes BCL6 and MYC. Genomic copy number variations explained 58/221 genes differentially expressed in the two U-2932 clones. Subclone-specific expression of the aryl-hydrocarbon receptor (AhR) and the resulting activity of the AhR/ARNT complex underlaid differential regulation of 11 genes including MEF2B. Knock-down and inhibitor experiments confirmed that AhR/ARNT regulates MEF2B, a key transcription factor for BCL6. AhR, MEF2B and BCL6 levels correlated not only in the U-2932 subclones but in the majority of 23 cell lines tested, indicting overexpression of AhR as a novel mechanism behind BCL6 diffuse large B-cell lymphoma. Enforced modulation of BCL6 affected 48/221 signature genes. Although BCL6 is known as a transcriptional repressor, 28 genes were up-regulated, including LMO2 and MYBL1 which, like BCL6, signify germinal center diffuse large B-cell lymphoma. Supporting the notion that BCL6 can induce gene expression, BCL6 and the majority of potential targets were co-regulated in a series of B-cell lines. In conclusion, genomic copy number aberrations, activation of AhR/ARNT, and overexpression of BCL6 are collectively responsible for differential expression of more than 100 genes in subclones of the U-2932 cell line. It is particularly interesting that BCL6 - regulated by AhR/ARNT and wild-type MEF2B - may drive expression of germinal center markers in diffuse large B-cell lymphoma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas com Domínio LIM/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Hidrocarboneto Arílico/fisiologia , Transativadores/genética , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/biossíntese , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/fisiologia , Humanos , Proteínas com Domínio LIM/biossíntese , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Fatores de Transcrição MEF2/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-6 , Transativadores/biossíntese
17.
Genes Chromosomes Cancer ; 53(11): 917-33, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25043849

RESUMO

FOX genes encode transcription factors which regulate basic developmental processes during embryogenesis and in the adult. Several FOX genes show deregulated expression in particular malignancies, representing oncogenes or tumor suppressors. Here, we screened six Hodgkin lymphoma (HL) cell lines for FOX gene activity by comparative microarray profiling, revealing overexpression of FOXC1 and FOXD1, and reduced transcription of FOXN3, FOXO1, and FOXP1. In silico expression analyses of these FOX gene candidates in HL patient samples supported the cell line data. Chromosomal analyses demonstrated an amplification of the FOXC1 locus at 6p25 and a gain of the FOXR2 locus at Xp11, indicting genomic aberrations for their upregulation. Comparative expression profiling and ensuing stimulation experiments revealed implementation of the TGFß- and WNT-signaling pathways in deregulation of FOXD1 and FOXN3. Functional analysis of FOXP1 implicated miR9 and miR34a as upstream regulators and PAX5, TCF3, and RAG2 as downstream targets. A similar exercise for FOXC1 revealed repression of MSX1 and activation of IPO7, both mediating inhibition of the B-cell specific homeobox gene ZHX2. Taken together, our data show that aberrantly expressed FOX genes and their downstream targets are involved in the pathogenesis of HL via deregulation of B-cell differentiation and may represent useful diagnostic markers and/or therapeutic targets.


Assuntos
Fatores de Transcrição Forkhead/genética , Doença de Hodgkin/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Loci Gênicos , Doença de Hodgkin/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , MicroRNAs/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt
18.
Int J Mol Sci ; 15(8): 13932-7, 2014 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-25116689

RESUMO

Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLß, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.


Assuntos
Proteínas de Transporte/metabolismo , Centrossomo/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Células HEK293 , Humanos , Microscopia Confocal , Proteínas MutL
19.
Genes Chromosomes Cancer ; 52(8): 741-7, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23630094

RESUMO

Chromosomal rearrangements are common features of most cancers, where they contribute to deregulated gene expression. Chromothripsis is a recently described oncogenic mechanism whereby small genomic pieces originating from one chromosomal region undergo massive rearrangements in a single step. Here, we document chromothripsis in Hodgkin lymphoma (HL) cell lines by genomic profiling, showing alternating amplicons of defined chromosomal regions. In L-1236 cells, fluorescent in situ hybridization analyses identified aberrations affecting amplified chromosomal segments that derived from the long arm regions of chromosomes 3 and 9 and that colocalized to a derivative chromosome 6, indicating the cataclysmic origin of this mutation. The ABL1 gene at 9q34 was targeted by these rearrangements leading to its overexpression in L-1236 cells, correlating with pharmacological resistance to treatment with the kinase inhibitor dasatinib. Collectively, we identified and characterized chromothriptic rearrangements in HL cell lines to serve as models for analyzing this novel oncogenomic mechanism.


Assuntos
Aberrações Cromossômicas , Rearranjo Gênico , Doença de Hodgkin/genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 9/genética , Genes abl/genética , Doença de Hodgkin/patologia , Humanos , Hibridização in Situ Fluorescente
20.
J Cell Biochem ; 114(10): 2405-14, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23696135

RESUMO

The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLß (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.


Assuntos
Reparo de Erro de Pareamento de DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , DNA/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Ensaio Cometa , DNA/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo de Erro de Pareamento de DNA/genética , Enzimas Reparadoras do DNA/genética , Humanos , Imunoprecipitação
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