RESUMO
Homeobox genes encode developmental transcription factors regulating tissue-specific differentiation processes and drive cancerogenesis when deregulated. Dendritic cells (DCs) are myeloid immune cells occurring as two types, either conventional or plasmacytoid DCs. Recently, we showed that the expression of NKL-subclass homeobox gene VENTX is restricted to conventional DCs, regulating developmental genes. Here, we identified and investigated homeobox genes specifically expressed in plasmacytoid DCs (pDCs) and derived blastic plasmacytoid dendritic cell neoplasm (BPDCN). We analyzed gene expression data, performed RQ-PCR, protein analyses by Western blot and immuno-cytology, siRNA-mediated knockdown assays and subsequent RNA-sequencing and live-cell imaging. Screening of public gene expression data revealed restricted activity of the CUT-class homeobox gene CUX2 in pDCs. An extended analysis of this homeobox gene class in myelopoiesis showed that additional CUX2 activity was restricted to myeloid progenitors, while BPDCN patients aberrantly expressed ONECUT2, which remained silent in the complete myeloid compartment. ONECUT2 expressing BPDCN cell line CAL-1 served as a model to investigate its regulation and oncogenic activity. The ONECUT2 locus at 18q21 was duplicated and activated by IRF4, AUTS2 and TNF-signaling and repressed by BMP4-, TGFb- and IL13-signalling. Functional analyses of ONECUT2 revealed the inhibition of pDC differentiation and of CDKN1C and CASP1 expression, while SMAD3 and EPAS1 were activated. EPAS1 in turn enhanced survival under hypoxic conditions which thus may support dendritic tumor cells residing in hypoxic skin lesions. Collectively, we revealed physiological and aberrant activities of CUT-class homeobox genes in myelopoiesis including pDCs and in BPDCN, respectively. Our data may aid in the diagnosis of BPDCN patients and reveal novel therapeutic targets for this fatal malignancy.
Assuntos
Genes Homeobox , Neoplasias Hematológicas , Humanos , Diferenciação Celular , Linhagem Celular , Células Mieloides/metabolismo , Células Dendríticas/metabolismo , Neoplasias Hematológicas/patologia , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genéticaRESUMO
T-box genes encode transcription factors, which control developmental processes and promote cancer if deregulated. Recently, we described the lymphoid TBX-code, which collates T-box gene activities in normal lymphopoiesis, enabling identification of members deregulated in lymphoid malignancies. Here, we have extended this analysis to cover myelopoiesis, compiling the myeloid TBX-code and, thus, highlighting which of these genes might be deregulated in myeloid tumor types. We analyzed public T-box gene expression datasets bioinformatically for normal and malignant cells. Candidate T-box-gene-expressing model cell lines were identified and examined by RQ-PCR, Western Blotting, genomic profiling, and siRNA-mediated knockdown combined with RNA-seq analysis and live-cell imaging. The established myeloid TBX-code comprised 10 T-box genes, including progenitor-cell-restricted TBX1. Accordingly, we detected aberrant expression of TBX1 in 10% of stem/progenitor-cell-derived chronic myeloid leukemia (CML) patients. The classic CML cell line K-562 expressed TBX1 at high levels and served as a model to identify TBX1 activators, including transcription factor GATA1 and genomic amplification of the TBX1 locus at 22q11; inhibitors, including BCR::ABL1 fusion and downregulated GNAI2, as well as BMP, FGF2, and WNT signaling; and the target genes CDKN1A, MIR17HG, NAV1, and TMEM38A. The establishment of the myeloid TBX-code permitted identification of aberrant TBX1 expression in subsets of CML patients and cell lines. TBX1 forms an integral part of an oncogenic regulatory network impacting proliferation, survival, and differentiation. Thus, the data spotlight novel diagnostic markers and potential therapeutic targets for this malignancy.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Humanos , Genes cdc , Western Blotting , Linhagem Celular Tumoral , Proteínas com Domínio T/genéticaRESUMO
Homeobox genes encode transcription factors regulating basic developmental processes. They are arranged according to sequence similarities of their conserved homeobox in 11 classes, including TALE. Recently, we have reported the so-called TALE-code. This gene signature describes physiological expression patterns of all active TALE-class homeobox genes in the course of hematopoiesis. The TALE-code allows the evaluation of deregulated TALE homeobox genes in leukemia/lymphoma. Here, we extended the TALE-code to include the stages of pro-B-cells and pre-B-cells in early B-cell development. Detailed analysis of the complete lineage of B-cell differentiation revealed expression of TALE homeobox genes IRX1 and MEIS1 exclusively in pro-B-cells. Furthermore, we identified aberrant expression of IRX2, IRX3 and MEIS1 in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) which originates from early B-cell progenitors. The data showed correlated activities of deregulated TALE-class members with particular BCP-ALL subtype markers, namely IRX2 with TCF3/E2A-fusions, IRX3 with ETV6/TEL-fusions, and MEIS1 with KMT2A/MLL-fusions. These correlations were also detected in BCP-ALL cell lines which served as experimental models. We performed siRNA-mediated knockdown experiments and reporter gene assays to analyze regulatory connections. The results showed mutual activation of IRX1 and TCF3. In contrast, IRX2 directly repressed wild-type TCF3 while the fusion gene TCF3::PBX1 lost the binding site for IRX2 and remained unaltered. IRX3 mutually activated fusion gene ETV6::RUNX1 while activating itself by aberrantly expressed transcription factor KLF15. Finally, KMT2A activated MEIS1 which in turn supported the expression of IRX3. In summary, we revealed normal TALE homeobox gene expression in early B-cell development and identified aberrant activities of IRX2, IRX3 and MEIS1 in particular subtypes of BCP-ALL. Thus, these TALE homeobox genes may serve as novel diagnostic markers and therapeutic targets.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Meis1/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Precursoras de Linfócitos B/patologia , RNA Interferente PequenoRESUMO
Homeobox genes encode transcription factors that control basic developmental decisions. Knowledge of their hematopoietic activities casts light on normal and malignant immune cell development. Recently, we constructed the so-called lymphoid TALE-code that codifies expression patterns of all active TALE class homeobox genes in early hematopoiesis and lymphopoiesis. Here, we present the corresponding myeloid TALE-code to extend this gene signature, covering the entire hematopoietic system. The collective data showed expression patterns for eleven TALE homeobox genes and highlighted the exclusive expression of IRX1 in megakaryocyte-erythroid progenitors (MEPs), implicating this TALE class member in a specific myeloid differentiation process. Analysis of public profiling data from acute myeloid leukemia (AML) patients revealed aberrant activity of IRX1 in addition to IRX3 and IRX5, indicating an oncogenic role for these TALE homeobox genes when deregulated. Screening of RNA-seq data from 100 leukemia/lymphoma cell lines showed overexpression of IRX1, IRX3, and IRX5 in megakaryoblastic and myelomonocytic AML cell lines, chosen as suitable models for studying the regulation and function of these homeo-oncogenes. Genomic copy number analysis of IRX-positive cell lines demonstrated chromosomal amplification of the neighboring IRX3 and IRX5 genes at position 16q12 in MEGAL, underlying their overexpression in this cell line model. Comparative gene expression analysis of these cell lines revealed candidate upstream factors and target genes, namely the co-expression of GATA1 and GATA2 together with IRX1, and of BMP2 and HOXA10 with IRX3/IRX5. Subsequent knockdown and stimulation experiments in AML cell lines confirmed their activating impact in the corresponding IRX gene expression. Furthermore, we demonstrated that IRX1 activated KLF1 and TAL1, while IRX3 inhibited GATA1, GATA2, and FST. Accordingly, we propose that these regulatory relationships may represent major physiological and oncogenic activities of IRX factors in normal and malignant myeloid differentiation, respectively. Finally, the established myeloid TALE-code is a useful tool for evaluating TALE homeobox gene activities in AML.
Assuntos
Sistema Hematopoético , Leucemia Mieloide Aguda , Expressão Ectópica do Gene , Genes Homeobox , Sistema Hematopoético/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Células Progenitoras Mieloides/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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 , TranscriptomaRESUMO
NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte-erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.
Assuntos
Células Eritroides/citologia , Proteínas de Homeodomínio/genética , Leucemia Eritroblástica Aguda/genética , Megacariócitos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Endotélio/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Humanos , Fatores Reguladores de Interferon/genética , Leucemia Eritroblástica Aguda/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição SOXF/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Hereditary Hemorrhagic Telangiectasia (HHT) is a rare and systemic disorder which is characterized by recurrent epistaxis, mucocutaneous telangiectases, and visceral arteriovenous malformations (AVM). An interdisciplinary concept is recommended. MATERIAL AND METHODS: We performed a retrospective review of consecutive patients who were referred to our newly established HHT Center of Excellence (HHT COE) for evaluation and treatment between April 2014 and August 2019. RESULTS: A network of over 20 departments was established at the University Hospital Essen. In 261 of the 282 patients (93â%), who were referred to the hospital's COE, the HHT diagnosis was at least possible. Most patients suffered from several symptoms (epistaxis and / or telangiectasia: >â80â%, visceral involvement: 65â%) and received a variety of treatments, often in a multidisciplinary setting. Alongside this direct treatment, the COE leader manages the coordination of the center and its public relations, which involves more than 900 e-mails per year. International collaboration and exchanges of expertise within the European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN) can improve the treatment of patients with HHT particularly where these cases are complex. CONCLUSIONS: An HHT COE provides an interdisciplinary network where highly specialized diagnostic and therapeutic processes can be updated and optimized continuously.
Assuntos
Telangiectasia Hemorrágica Hereditária , Epistaxe/etiologia , Epistaxe/terapia , Humanos , Doenças Raras , Estudos Retrospectivos , Telangiectasia Hemorrágica Hereditária/diagnóstico , Telangiectasia Hemorrágica Hereditária/terapiaRESUMO
The malaria parasite P. falciparum exports a large number of proteins to its host cell, the mature human erythrocyte. Although the function of the majority of these proteins is not well understood, many exported proteins appear to play a role in modification of the erythrocyte following invasion. Protein export to the erythrocyte is a secretory process that begins with entry to the endoplasmic reticulum. For most exported proteins, this step is mediated by hydrophobic signal peptides found towards the N-terminal end of proteins. The signal peptides present on P. falciparum exported proteins often differ in length from those found in other systems, and generally contain a highly extended N-terminal region. Here we have investigated the function of these extended N-terminal regions, using the exported parasite protein GBP130 as a model. Surprisingly, several deletions of the extended N-terminal regions of the GBP130 signal peptide have no effect on the ability of the signal peptide to direct a fluorescent reporter to the secretory pathway. Addition of the same N-terminal extension to a canonical signal peptide does not affect transport of either soluble or membrane proteins to their correct respective subcellular localisations. Finally, we show that extended signal peptides are able to complement canonical signal peptides in driving protein traffic to the apicoplast of the parasite, and are also functional in a mammalian cell system. Our study is the first detailed analysis of an extended P. falciparum signal peptide and suggests that N-terminal extensions of exported Plasmodium falciparum proteins are not required for entry to the secretory system, and are likely to be involved in other, so far unknown, processes.
Assuntos
Plasmodium falciparum/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Protozoários/metabolismo , Apicoplastos/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Mutação , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Proteínas de Protozoários/química , Via Secretória , SolubilidadeRESUMO
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/genéticaRESUMO
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 WntRESUMO
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 FluorescenteRESUMO
Chromosomal or mutational activation of BCL6 (at 3q27) typifies diffuse large B-cell lymphoma (DLBCL) which in the germinal center subtype may be accompanied by focal amplification of chromosome band 13q31 effecting upregulation of miR-17~92. Using long distance inverse-polymerase chain reaction, we mapped and sequenced six breakpoints of a complex BCL6 rearrangement t(3;13)(q27;q31)t(12;13)(p11;q31) in DLBCL cells, which places miR-17~92 antisense within the resulting ITPR2-BCL6 chimeric fusion gene rearrangement. MiR-17~92 members were upregulated ~15-fold over controls in a copy number independent manner consistent with structural deregulation. MIR17HG and ITPR2-BCL6 were, despite their close configuration, independently expressed, discounting antisense regulation. MIR17HG in t(3;13)t(12;13) cells proved highly responsive to treatment with histone deacetylase inhibitors implicating epigenetic deregulation, consistent with which increased histone-H3 acetylation was detected by chromatin immunoprecipitation near the upstream MIR17HG breakpoint. Remarkably, 5/6 DNA breaks in the t(3;13)t(12;13) precisely cut at stress-induced DNA duplex destabilization (SIDD) peaks reminiscent of chromosomal fragile sites, while the sixth lay 150 bp distant. Extended SIDD profiling showed that additional oncomiRs also map to SIDD peaks. Fluorescence in situ hybridization analysis showed that 11 of 52 (21%) leukemia-lymphoma (L-L) cell lines with 13q31 involvement bore structural rearrangements at/near MIR17HG associated with upregulation. As well as fueling genome instability, SIDD peaks mark regulatory nuclear-scaffold matrix attachment regions open to nucleosomal acetylation. Collectively, our data indict a specific DNA instability motif (SIDD) in chromosome rearrangement, specifically alterations activating miR-17~92 epigenetically via promoter hyperacetylation, and supply a model for the clustering of oncomiRs near cancer breakpoints.
Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Acetilação , Imunoprecipitação da Cromatina , Bandeamento Cromossômico , Pontos de Quebra do Cromossomo , Sítios Frágeis do Cromossomo/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Receptores de Inositol 1,4,5-Trifosfato/genética , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Longo não Codificante , Sequências Reguladoras de Ácido Nucleico/genética , Translocação GenéticaRESUMO
The human family of ETS transcription factors numbers 28 genes which control multiple aspects of development, notably the differentiation of blood and immune cells. Otherwise, aberrant expression of ETS genes is reportedly involved in forming leukemia and lymphoma. Here, we comprehensively mapped ETS gene activities in early hematopoiesis, lymphopoiesis and all mature types of lymphocytes using public datasets. We have termed the generated gene expression pattern lymphoid ETS-code. This code enabled identification of deregulated ETS genes in patients with lymphoid malignancies, revealing 12 aberrantly expressed members in Hodgkin lymphoma (HL). For one of these, ETS gene ETV3, expression in stem and progenitor cells in addition to that in developing and mature T-cells was mapped together with downregulation in B-cell differentiation. In contrast, subsets of HL patients aberrantly overexpressed ETV3, indicating oncogenic activity in this B-cell malignancy. Analysis of ETV3-overexpressing HL cell line SUP-HD1 demonstrated genomic duplication of the ETV3 locus at 1q23, GATA3 as mutual activator, and suppressed BMP-signalling as mutual downstream effect. Additional examination of the neighboring ETS genes ETS1 and FLI1 revealed physiological activities in B-cell development and aberrant downregulation in HL patient subsets. SUP-HD1 showed genomic loss on chromosome 11, del(11)(q22q25), targeting both ETS1 and FLI1, underlying their downregulation. Furthermore, in the same cell line we identified PBX1-mediated overexpression of RIOK2 which inhibited ETS1 and activated JAK2 expression. Collectively, we codified normal ETS gene activities in lymphopoiesis and identified oncogenic ETS members in HL.
Assuntos
Doença de Hodgkin , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Doença de Hodgkin/patologia , Diferenciação Celular/genética , Proteínas Proto-Oncogênicas c-ets , Linhagem Celular , Proteína Proto-Oncogênica c-ets-1/genéticaRESUMO
BCL2, BCL6 and MYC are major oncogenes in B-cell lymphoma. Their aberrant activation frequently occurs via chromosomal translocations which juxtapose light or heavy chain immunoglobulin (IG) genes to BCL2 and MYC or fuse diverse partner genes with BCL6. So-called double-hit lymphomas usually carry BCL2 and MYC rearrangements, while triple-hit lymphomas additionally bear BCL6-fusions. All these translocations are of diagnostic relevance and usually denote poor prognosis. Here, we genomically characterized classic follicular lymphoma (FL) cell line SC-1, thereby identifying t(14;18)(q32;q21) juxtaposing IGH and BCL2, t(8;14)(q24;q32) juxtaposing IGH and MYC, and t(3;3)(q25;q27) fusing MBNL1 to BCL6. In addition, we found that SC-1 carries a novel chromosomal rearrangement, t(14;17)(q32;q21), which, though present at establishment, has remained unreported until now. We further show that t(14;17)(q32;q21) juxtaposes IGH with the HOXB gene cluster at 17q21 and affect the oncogenic activation of both homeobox gene HOXB5 and neighboring micro-RNA gene miR10a. Moreover, we detected aberrant overexpression of HOXB5 in subsets of Burkitt lymphoma, FL, and multiple myeloma patients, confirming the clinical relevance of its deregulation. In SC-1, HOXB5 activation was additionally supported by co-expression of hematopoietic stem cell factor ZNF521, indicating an aberrant impact in cell differentiation. Functional investigations showed that HOXB5 represses the apoptotic driver BCL2L11 and promotes survival in the presence of etoposide, and that miR10a inhibits BCL6 and may thus play an oncogenic role in later stages of lymphomagenesis. Collectively, we characterize triple-hit B-cell line SC-1 and identify the aberrant expression of HOXB5 and miR10a, both novel oncogenes in B-cell lymphoma.
RESUMO
Hodgkin/Reed-Sternberg (HRS) cells represent the malignant fraction of infiltrated lymph nodes in Hodgkin lymphoma (HL). Although HRS cells display multiple chromosomal aberrations, few are recurrent and the targeted genes unknown. However, understanding the pathology of HL and developing rational therapies may well require identifying putative deregulated genes. Here, we analyzed the karyotype of the well-defined HL cell line L-1236 by spectral karyotyping and identified multiple abnormalities, therein, notably t(4;8)(q27;q24) which includes two breakpoint regions previously highlighted in HL. Target genes at 4q27 and 8q24 were shortlisted by high density genomic arrays and fluorescence in situ hybridization. Expression analysis of candidate target genes revealed conspicuous activation of phosphodiesterase PDE5A at 4q27 and inhibition of homeobox gene ZHX2 at 8q24. Treatment of L-1236 with PDE5A-inhibitor sildenafil or with siRNA directed against PDE5A and concomitant stimulation with cyclic guanosine monophosphate (cGMP) resulted in enhanced apoptosis, indicating PDE5A as an oncogene. Expression profiling of L-1236 cells following siRNA-mediated knockdown of ZHX2 showed inhibition of genes regulating differentiation and apoptosis, suggesting tumor suppressor activity of ZHX2. Downstream genes included STAT1 and several STAT1-target genes, indicating activation of STAT1-signaling by ZHX2 as analyzed by RQ-PCR and western blot. Taken together, we have identified a novel aberration with recurrent breakpoints in HL, t(4;8)(q27;q24), which activate PDE5A and repress ZHX2, deregulating apoptosis, differentiation, and STAT1-signaling in HL cells.
Assuntos
Cromossomos Humanos Par 4 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Genes Homeobox , Doença de Hodgkin/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Translocação Genética , Adolescente , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Criança , Pré-Escolar , Cromossomos Humanos Par 8 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Doença de Hodgkin/enzimologia , Humanos , Hibridização in Situ Fluorescente/métodos , Lactente , Cariótipo , Masculino , Análise em Microsséries/métodos , Fator de Transcrição STAT1/genéticaRESUMO
STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL.
RESUMO
Cutaneous T-cell lymphoma (CTCL) is a severe lymphoid malignancy with a worse prognosis lacking curative treatment regimens. Several gene mutations and deregulated pathways, including NFkB signaling, have been implicated in its pathogenesis. Accordingly, CTCL cell line HUT-78 reportedly contains mutated NFKB2, which is constitutively activated via partial gene deletion, also demonstrating that genomic rearrangements cause driving mutations in this malignancy. Here, along with HUT-78, we analyzed CTCL cell line HH to identify additional aberrations underlying gene deregulation. Karyotyping and genomic profiling of HH showed several rearrangements worthy of detailed investigation. Corresponding to the established karyotype, RNA-seq data and PCR analysis confirmed the presence of t(3;17)(q28;q25), generating a novel fusion gene, FOXK2::TP63. Furthermore, chromosomal rearrangement t(1;4)(p32;q25) was connected to amplification at 4q24-26, affecting aberrant NFKB1 overexpression thereat. Transcription factor binding-site analysis and knockdown experiments demonstrated that IRF4 contributed to NFKB1 expression. Within the same amplicon, we identified amplification and overexpression of NFkB signaling activator CAMK2D (4q26) and p53-inhibitor UBE2D3 (4q24). Genomic profiling data for HUT-78 detailed a deletion at 10q25 underlying reported NFKB2 activation. Moreover, amplifications of ID1 (20q11) and IKZF2 (2q34) in this cell line drove overexpression of these NK cell differentiation factors and possibly thus formed corresponding lineage characteristics. Target gene analysis for NFKB1 via siRNA-mediated knockdown in HH revealed activation of TP63, MIR155, and NOTCH pathway component RBPJ. Finally, treatment of HH with NFkB inhibitor demonstrated a role for NFkB in supporting proliferation, while usage of inhibitor DAPT showed significant survival effects via the NOTCH pathway. Collectively, our data suggest that NFkB and/or NOTCH inhibitors may represent reasonable treatment options for subsets of CTCL patients.
RESUMO
T-box genes encode transcription factors which control basic processes in development of several tissues including cell differentiation in the hematopoietic system. Here, we analyzed the physiological activities of all 17 human T-box genes in early hematopoiesis and in lymphopoiesis including developing and mature B-cells, T-cells, natural killer (NK)-cells and innate lymphoid cells. The resultant expression pattern comprised six genes, namely EOMES, MGA, TBX1, TBX10, TBX19 and TBX21. We termed this gene signature TBX-code which enables discrimination of normal and aberrant activities of T-box genes in lymphoid malignancies. Accordingly, expression analysis of T-box genes in Hodgkin lymphoma (HL) patients using a public profiling dataset revealed overexpression of EOMES, TBX1, TBX2, TBX3, TBX10, TBX19, TBX21 and TBXT while MGA showed aberrant downregulation. Analysis of T-cell acute lymphoid leukemia patients indicated aberrant overexpression of six T-box genes while no deregulated T-box genes were detected in anaplastic large cell lymphoma patients. As a paradigm we focused on TBX3 which was ectopically activated in about 6% of HL patients analyzed. Normally, TBX3 is expressed in tissues like lung, adrenal gland and retina but not in hematopoiesis. HL cell line KM-H2 expressed enhanced TBX3 levels and was used as an in vitro model to identify upstream regulators and downstream targets in this malignancy. Genomic studies of this cell line showed focal amplification of the TBX3 locus at 12q24 which may underlie its aberrant expression. In addition, promoter analysis and comparative expression profiling of HL cell lines followed by knockdown experiments revealed overexpressed transcription factors E2F4 and FOXC1 and chromatin modulator KDM2B as functional activators. Furthermore, we identified repressed target genes of TBX3 in HL including CDKN2A, NFKBIB and CD19, indicating its respective oncogenic function in proliferation, NFkB-signaling and B-cell differentiation. Taken together, we have revealed a lymphoid TBX-code and used it to identify an aberrant network around deregulated T-box gene TBX3 in HL which promotes hallmark aberrations of this disease. These findings provide a framework for future studies to evaluate deregulated T-box genes in lymphoid malignancies.
Assuntos
Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Proteínas com Domínio T/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Linfopoese/fisiologia , Proteínas com Domínio T/genética , Linfócitos T/metabolismoRESUMO
Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation and are grouped into classes and subclasses according to sequence similarities. Here, we analyzed the activities of the 20 members strong TALE homeobox gene class in early hematopoiesis and in lymphopoiesis including developing and mature B-cells, T-cells, natural killer (NK)-cells and innate lymphoid cells (ILC). The resultant expression pattern comprised eleven genes and which we termed TALE-code enables discrimination of normal and aberrant activities of TALE homeobox genes in lymphoid malignancies. Subsequent expression analysis of TALE homeobox genes in public datasets of Hodgkin lymphoma (HL) patients revealed overexpression of IRX3, IRX4, MEIS1, MEIS3, PBX1, PBX4 and TGIF1. As paradigm we focused on PBX1 which was deregulated in about 17% HL patients. Normal PBX1 expression was restricted to hematopoietic stem cells and progenitors of T-cells and ILCs but absent in B-cells, reflecting its roles in stemness and early differentiation. HL cell line SUP-HD1 expressed enhanced PBX1 levels and served as an in vitro model to identify upstream regulators and downstream targets in this malignancy. Genomic studies of this cell line therein showed a gain of the PBX1 locus at 1q23 which may underlie its aberrant expression. Comparative expression profiling analyses of HL patients and cell lines followed by knockdown experiments revealed NFIB and TLX2 as target genes activated by PBX1. HOX proteins operate as cofactors of PBX1. Accordingly, our data showed that HOXB9 overexpressed in HL coactivated TLX2 but not NFIB while activating TNFRSF9 without PBX1. Further downstream analyses showed that TLX2 activated TBX15 which operated anti-apoptotically. Taken together, we discovered a lymphoid TALE-code and identified an aberrant network around deregulated TALE homeobox gene PBX1 which may disturb B-cell differentiation in HL by reactivation of progenitor-specific genes. These findings may provide the framework for future studies to exploit possible vulnerabilities of malignant cells in therapeutic scenarios.