RESUMO
The BloodChIP Xtra database (http://bloodchipXtra.vafaeelab.com/) facilitates genome-wide exploration and visualization of transcription factor (TF) occupancy and chromatin configuration in rare primary human hematopoietic stem (HSC-MPP) and progenitor (CMP, GMP, MEP) cells and acute myeloid leukemia (AML) cell lines (KG-1, ME-1, Kasumi1, TSU-1621-MT), along with chromatin accessibility and gene expression data from these and primary patient AMLs. BloodChIP Xtra features significantly more datasets than our earlier database BloodChIP (two primary cell types and two cell lines). Improved methodologies for determining TF occupancy and chromatin accessibility have led to increased availability of data for rare primary cell types across the spectrum of healthy and AML hematopoiesis. However, there is a continuing need for these data to be integrated in an easily accessible manner for gene-based queries and use in downstream applications. Here, we provide a user-friendly database based around genome-wide binding profiles of key hematopoietic TFs and histone marks in healthy stem/progenitor cell types. These are compared with binding profiles and chromatin accessibility derived from primary and cell line AML and integrated with expression data from corresponding cell types. All queries can be exported to construct TF-gene and protein-protein networks and evaluate the association of genes with specific cellular processes.
Assuntos
Sítios de Ligação , Perfilação da Expressão Gênica , Leucemia Mieloide Aguda , Humanos , Cromatina/genética , Regulação da Expressão Gênica , Leucemia Mieloide Aguda/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).
Assuntos
Antineoplásicos , Desmetilação , Síndromes Mielodisplásicas , Oncogenes , Regulação para Cima , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desmetilação/efeitos dos fármacos , Humanos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oncogenes/efeitos dos fármacos , Oncogenes/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Células-Tronco Hematopoéticas , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Regulação da Expressão Gênica , Hematopoese/genética , Cromatina/metabolismoRESUMO
Myelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.
Assuntos
Leucemia Mielomonocítica Crônica , Síndromes Mielodisplásicas , Humanos , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Células-Tronco Hematopoéticas/metabolismo , Monócitos , Células ClonaisRESUMO
Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation, but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesized that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, and RUNX1) bind key hematopoietic genes in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other's, regulatory elements. However, their mutual regulation during normal hematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. In this study, we integrated bulk and single-cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists, with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and leukemic cells. The heptad factors GATA2, TAL1, and ERG formed an integrated subcircuit that regulates stem cell-to-erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits can be harnessed to promote specific cell-type transitions and overcome dysregulated hematopoiesis.
Assuntos
Fator de Transcrição GATA2/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Células Eritroides/metabolismo , Células Eritroides/patologia , Redes Reguladoras de Genes , Hematopoese , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Regulador Transcricional ERG/genéticaRESUMO
Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
Assuntos
Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/genética , Histona Desmetilases/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição da Família Snail/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células HEK293 , Células HL-60 , Histona Desmetilases/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Telomerase is a ribonucleoprotein complex that maintains the length and integrity of telomeres, and thereby enables cellular proliferation. Understanding the regulation of telomerase in hematopoietic cells is relevant to the pathogenesis of leukemia, in which telomerase is constitutively activated, as well as bone marrow failure syndromes that feature telomerase insufficiency. Past studies showing high levels of telomerase in human erythroblasts and a prevalence of anemia in disorders of telomerase insufficiency provide the rationale for investigating telomerase regulation in erythroid cells. Here it is shown for the first time that the telomerase RNA-binding protein dyskerin (encoded by DKC1) is dramatically upregulated as human hematopoietic stem and progenitor cells commit to the erythroid lineage, driving an increase in telomerase activity in the presence of limiting amounts of TERT mRNA. It is also shown that upregulation of DKC1 was necessary for expansion of glycophorin A+ erythroblasts and sufficient to extend telomeres in erythroleukemia cells. Chromatin immunoprecipitation and reporter assays implicated GATA1-mediated transcriptional regulation of DKC1 in the modulation of telomerase in erythroid lineage cells. Together these results describe a novel mechanism of telomerase regulation in erythroid cells which contrasts with mechanisms centered on transcriptional regulation of TERT that are known to operate in other cell types. This is the first study to reveal a biological context in which telomerase is upregulated by DKC1 and to implicate GATA1 in telomerase regulation. The results from this study are relevant to hematopoietic disorders involving DKC1 mutations, GATA1 deregulation and/or telomerase insufficiency.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Eritroblastos/metabolismo , Fator de Transcrição GATA1/metabolismo , Proteínas Nucleares/metabolismo , Telomerase , Proteínas de Ciclo Celular/genética , Fator de Transcrição GATA1/genética , Humanos , Proteínas Nucleares/genética , Telomerase/genética , Telomerase/metabolismo , Regulação para CimaRESUMO
Acute myeloid leukemia (AML) is a complex disease characterized by a diverse range of recurrent molecular aberrations that occur in many different combinations. Components of transcriptional networks are a common target of these aberrations, leading to network-wide changes and deployment of novel or developmentally inappropriate transcriptional programs. Genome-wide techniques are beginning to reveal the full complexity of normal hematopoietic stem cell transcriptional networks and the extent to which they are deregulated in AML, and new understandings of the mechanisms by which AML cells maintain self-renewal and block differentiation are starting to emerge. The hope is that increased understanding of the network architecture in AML will lead to identification of key oncogenic dependencies that are downstream of multiple network aberrations, and that this knowledge will be translated into new therapies that target these dependencies. Here, we review the current state of knowledge of network perturbation in AML with a focus on major mechanisms of transcription factor dysregulation, including mutation, translocation, and transcriptional dysregulation, and discuss how these perturbations propagate across transcriptional networks. We will also review emerging mechanisms of network disruption, and briefly discuss how increased knowledge of network disruption is already being used to develop new therapies.
Assuntos
Redes Reguladoras de Genes/genética , Leucemia Mieloide Aguda/genética , Transcrição Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Mutação/genética , Fatores de Transcrição/genética , Translocação Genética/genéticaRESUMO
MOTIVATION: Long non-coding RNAs (lncRNAs) have been implicated in the regulation of diverse biological functions. The number of newly identified lncRNAs has increased dramatically in recent years but their expression and function have not yet been described from most diseases. To elucidate lncRNA function in human disease, we have developed a novel network based method (NLCFA) integrating correlations between lncRNA, protein coding genes and noncoding miRNAs. We have also integrated target gene associations and protein-protein interactions and designed our model to provide information on the combined influence of mRNAs, lncRNAs and miRNAs on cellular signal transduction networks. RESULTS: We have generated lncRNA expression profiles from the CD34+ haematopoietic stem and progenitor cells (HSPCs) from patients with Myelodysplastic syndromes (MDS) and healthy donors. We report, for the first time, aberrantly expressed lncRNAs in MDS and further prioritize biologically relevant lncRNAs using the NLCFA. Taken together, our data suggests that aberrant levels of specific lncRNAs are intimately involved in network modules that control multiple cancer-associated signalling pathways and cellular processes. Importantly, our method can be applied to prioritize aberrantly expressed lncRNAs for functional validation in other diseases and biological contexts. AVAILABILITY AND IMPLEMENTATION: The method is implemented in R language and Matlab. CONTACT: xizhou@wakehealth.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Anotação de Sequência Molecular/métodos , Síndromes Mielodisplásicas/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Software , Idoso , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Neoplasias/genética , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodosRESUMO
Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.
Assuntos
Embrião de Mamíferos/metabolismo , Endoglina/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Hematopoese/fisiologia , Sondas Moleculares/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Endoglina/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Sondas Moleculares/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-ALL and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remains poorly understood. In order to discover transcriptional regulators of ERG, we employed a quantitative mass spectrometry-based method to identify factors binding the 321 bp ERG +85 stem cell enhancer region in MOLT-4 T-ALL and KG-1 AML cells. Using this approach, we identified a number of known binders of the +85 enhancer in leukaemic cells along with previously unknown binders, including ETV6 and IKZF1. We confirmed that ETV6 and IKZF1 were also bound at the +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells. Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators not just of ERG, but also of a number of genes regulated by a densely interconnected network of seven transcription factors. At last, we show that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis.
Assuntos
Fator de Transcrição Ikaros/fisiologia , Proteínas Proto-Oncogênicas c-ets/fisiologia , Proteínas Repressoras/fisiologia , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Sequência Consenso , Elementos Facilitadores Genéticos , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Ligação Proteica , Proteoma , Proteômica , Regulador Transcricional ERG/fisiologia , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Glucocorticoids are critical components of combination chemotherapy regimens in pediatric acute lymphoblastic leukemia (ALL). The proapoptotic BIM protein is an important mediator of glucocorticoid-induced apoptosis in normal and malignant lymphocytes, whereas the antiapoptotic BCL2 confers resistance. The signaling pathways regulating BIM and BCL2 expression in glucocorticoid-treated lymphoid cells remain unclear. In this study, pediatric ALL patient-derived xenografts (PDXs) inherently sensitive or resistant to glucocorticoids were exposed to dexamethasone in vivo. Microarray analysis showed that KLF13 and MYB gene expression changes were significantly greater in dexamethasone-sensitive than -resistant PDXs. Chromatin immunoprecipitation (ChIP) analysis detected glucocorticoid receptor (GR) binding at the KLF13 promoter to trigger KLF13 expression only in sensitive PDXs. Next, KLF13 bound to the MYB promoter, deactivating MYB expression only in sensitive PDXs. Sustained MYB expression in resistant PDXs resulted in maintenance of BCL2 expression and inhibition of apoptosis. ChIP sequencing analysis revealed a novel GR binding site in a BIM intronic region (IGR) that was engaged only in dexamethasone-sensitive PDXs. The absence of GR binding at the BIM IGR was associated with BIM silencing and dexamethasone resistance. This study has identified novel mechanisms of opposing BCL2 and BIM gene regulation that control glucocorticoid-induced apoptosis in pediatric ALL cells in vivo.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Proteínas de Membrana/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Glucocorticoides/genética , Animais , Antineoplásicos Hormonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Imunoprecipitação da Cromatina , Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genome-wide combinatorial binding patterns for key transcription factors (TFs) have not been reported for primary human hematopoietic stem and progenitor cells (HSPCs), and have constrained analysis of the global architecture of molecular circuits controlling these cells. Here we provide high-resolution genome-wide binding maps for a heptad of key TFs (FLI1, ERG, GATA2, RUNX1, SCL, LYL1, and LMO2) in human CD34(+) HSPCs, together with quantitative RNA and microRNA expression profiles. We catalog binding of TFs at coding genes and microRNA promoters, and report that combinatorial binding of all 7 TFs is favored and associated with differential expression of genes and microRNA in HSPCs. We also uncover a previously unrecognized association between FLI1 and RUNX1 pairing in HSPCs, we establish a correlation between the density of histone modifications that mark active enhancers and the number of overlapping TFs at a peak, we demonstrate bivalent histone marks at promoters of heptad target genes in CD34(+) cells that are poised for later expression, and we identify complex relationships between specific microRNAs and coding genes regulated by the heptad. Taken together, these data reveal the power of integrating multifactor sequencing of chromatin immunoprecipitates with coding and noncoding gene expression to identify regulatory circuits controlling cell identity.
Assuntos
Estudo de Associação Genômica Ampla , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Fatores de Transcrição/genética , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Análise por Conglomerados , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Humanos , RNA não Traduzido , Transcrição GênicaRESUMO
Aberrant transcriptional programs in combination with abnormal proliferative signaling drive leukemic transformation. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. Ets Related Gene (ERG) is a component of normal and leukemic stem cell signatures and high ERG expression is a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG promoters and +85 stem cell enhancer and a heptad of transcription factors that combinatorially regulate genes in HSCs. Gene expression signatures derived from ERG promoter-stem cell enhancer and heptad activity are associated with clinical outcome when ERG expression alone fails. We also show that the heptad signature is associated with AMLs that lack somatic mutations in NPM1 and confers an adverse prognosis when associated with FLT3 mutations. Taken together, these results suggest that transcriptional regulators cooperate to establish or maintain primitive stem cell-like signatures in leukemic cells and that the underlying pattern of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Elementos Facilitadores Genéticos/genética , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA2/fisiologia , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células K562 , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/fisiologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/fisiologia , Nucleofosmina , Prognóstico , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína Proto-Oncogênica c-fli-1/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Regulador Transcricional ERGRESUMO
Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.
Assuntos
Sistemas CRISPR-Cas , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/metabolismo , Sumoilação/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , FemininoRESUMO
The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Linfócitos T/metabolismo , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas com Domínio LIM , Metaloproteínas/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sobrevida , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Linfócitos T/patologia , Transativadores/antagonistas & inibidores , Transativadores/química , Transativadores/genética , Regulador Transcricional ERGRESUMO
The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.
Assuntos
Leucemia , Fatores de Transcrição , Animais , Masculino , Camundongos , Proteínas Correpressoras , Regulação da Expressão Gênica , Genes ReguladoresRESUMO
Oncofetal protein SALL4 is critical for cancer cell survival. Targeting SALL4, however, is only applicable in a fraction of cancer patients who are positive for this gene. To overcome this limitation, we propose to induce a cancer vulnerability by engineering a partial dependency upon SALL4. Following exogenous expression of SALL4, SALL4-negative cancer cells became partially dependent on SALL4. Treatment of SALL4-negative cells with the FDA-approved hypomethylating agent 5-aza-2'-deoxycytidine (DAC) resulted in transient upregulation of SALL4. DAC pretreatment sensitized SALL4-negative cancer cells to entinostat, which negatively affected SALL4 expression through a microRNA, miRNA-205, both in culture and in vivo. Moreover, SALL4 was essential for the efficiency of sequential treatment of DAC and entinostat. Overall, this proof-of-concept study provides a framework whereby the targeting pathways such as SALL4-centered therapy can be expanded, sensitizing cancer cells to treatment by transient target induction and engineering a dependency. SIGNIFICANCE: These findings provide a therapeutic approach for patients harboring no suitable target by induction of a SALL4-mediated vulnerability.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Animais , Apoptose , Benzamidas/administração & dosagem , Proliferação de Células , Decitabina/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Piridinas/administração & dosagem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissue-derived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocyte-derived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth.