RESUMO
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
Assuntos
Histona Acetiltransferases/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Linhagem Celular Tumoral , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
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
Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.
Assuntos
Azacitidina/farmacologia , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Células Cultivadas , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/fisiologiaRESUMO
Although many genes are known to be critical for early hematopoiesis in the embryo, it remains unclear whether distinct regulatory pathways exist to control hematopoietic specification versus hematopoietic stem cell (HSC) emergence and function. Due to their interaction with key regulators of hematopoietic commitment, particular interest has focused on the role of the ETS family of transcription factors; of these, ERG is predicted to play an important role in the initiation of hematopoiesis, yet we do not know if or when ERG is required. Using in vitro and in vivo models of hematopoiesis and HSC development, we provide strong evidence that ERG is at the center of a distinct regulatory program that is not required for hematopoietic specification or differentiation but is critical for HSC maintenance during embryonic development. We show that, from the fetal period, ERG acts as a direct upstream regulator of Gata2 and Runx1 gene activity. Without ERG, physiological HSC maintenance fails, leading to the rapid exhaustion of definitive hematopoiesis.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas Oncogênicas/metabolismo , Animais , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/genética , Fatores de Transcrição , Regulador Transcricional ERGRESUMO
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
The ETS transcription factor ERG plays a central role in definitive hematopoiesis, and its overexpression in acute myeloid leukemia (AML) is associated with a stem cell signature and poor prognosis. Yet how ERG causes leukemia is unclear. Here we show that pan-hematopoietic ERG expression induces an early progenitor myeloid leukemia in transgenic mice. Integrated genome-scale analysis of gene expression and ERG binding profiles revealed that ERG activates a transcriptional program similar to human AML stem/progenitor cells and to human AML with high ERG expression. This transcriptional program was associated with activation of RAS that was required for leukemia cells growth in vitro and in vivo. We further show that ERG induces expression of the Pim1 kinase oncogene through a novel hematopoietic enhancer validated in transgenic mice and human CD34(+) normal and leukemic cells. Pim1 inhibition disrupts growth and induces apoptosis of ERG-expressing leukemic cells. The importance of the ERG/PIM1 axis is further underscored by the poorer prognosis of AML highly expressing ERG and PIM1. Thus, integrative genomic analysis demonstrates that ERG causes myeloid progenitor leukemia characterized by an induction of leukemia stem cell transcriptional programs. Pim1 and the RAS pathway are potential therapeutic targets of these high-risk leukemias.
Assuntos
Regulação Leucêmica da Expressão Gênica/fisiologia , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos , Elementos Facilitadores Genéticos/genética , Genômica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células Progenitoras Mieloides/fisiologia , Transplante de Neoplasias , Transcrição Gênica/fisiologia , Regulador Transcricional ERGRESUMO
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
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
Chromatin regulation involves the selective recruitment of chromatin factors to facilitate DNA repair, replication and transcription. Here we demonstrate the utility of coupling unbiased functional genomics with chromatin immunoprecipitation (CRISPR-ChIP) to identify the factors associated with active chromatin modifications in mammalian cells. Specifically, an integrated reporter containing a cis-regulatory element of interest and a single guide RNA provide a chromatinized template for a direct readout for regulators of histone modifications associated with actively transcribed genes such as H3K4me3 and H3K79me2. With CRISPR-ChIP, we identify all the nonredundant COMPASS complex members required for H3K4me3 and demonstrate that RNA polymerase II is dispensable for the maintenance of H3K4me3. As H3K79me2 has a putative oncogenic function in leukemia cells driven by MLL translocations, using CRISPR-ChIP we reveal a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex directed by the MLL fusion protein in a Menin-dependent manner and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10. Overall, CRISPR-ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Leucemia , Animais , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Fatores de Transcrição/genética , Proteína de Leucina Linfoide-Mieloide/genética , Cromatina , Leucemia/genética , Imunoprecipitação da Cromatina , Mamíferos/genéticaRESUMO
Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.
Assuntos
Interferons , Metiltransferases , Animais , Camundongos , Interferons/genética , Metiltransferases/genética , Metiltransferases/metabolismo , RNA de Cadeia DuplaRESUMO
Mouse haematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5), on the ventral surface of the dorsal aorta, by endothelial-to-haematopoietic transition. We investigated whether mesenchymal stem cells, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta-gonad-mesonephros and contribute to the development of the dorsal aorta and endothelial-to-haematopoietic transition. Here we show that mesoderm-derived PDGFRA+ stromal cells (Mesp1der PSCs) contribute to the haemogenic endothelium of the dorsal aorta and populate the E10.5-E11.5 aorta-gonad-mesonephros but by E13.5 were replaced by neural-crest-derived PSCs (Wnt1der PSCs). Co-aggregating non-haemogenic endothelial cells with Mesp1der PSCs but not Wnt1der PSCs resulted in activation of a haematopoietic transcriptional programme in endothelial cells and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA or BMP, WNT and NOTCH signalling interrupted this reprogramming event. Together, aorta-gonad-mesonephros Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.
Assuntos
Hemangioblastos , Mesonefro , Animais , Aorta , Hematopoese/genética , Células-Tronco Hematopoéticas , Mesoderma , CamundongosRESUMO
The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição GATA/metabolismo , Leucemia/metabolismo , Metaloproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Telomerase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Fatores de Transcrição GATA/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Proteínas com Domínio LIM , Leucemia/genética , Metaloproteínas/genética , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Telomerase/genética , Análise Serial de Tecidos , Transativadores/genéticaRESUMO
Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.
Assuntos
Antígenos CD/genética , Sangue/metabolismo , Endotélio/metabolismo , Fatores de Transcrição GATA/fisiologia , Hemangioblastos/fisiologia , Proteína Proto-Oncogênica c-ets-1/fisiologia , Receptores de Superfície Celular/genética , Animais , Antígenos CD/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Endoglina , Fatores de Transcrição GATA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hemangioblastos/metabolismo , Sistema Hematopoético/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Fatores Imunológicos/farmacologia , Terapia de Alvo Molecular , Fatores de Transcrição/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Descoberta de Drogas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Fatores Imunológicos/química , Fatores Imunológicos/uso terapêutico , Inflamação/tratamento farmacológico , Neoplasias/tratamento farmacológico , Domínios Proteicos/efeitos dos fármacos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Ibrutinib plus venetoclax is a highly effective combination in mantle cell lymphoma. However, strategies to enable the evaluation of therapeutic response are required. Our prospective analyses of patients within the AIM study revealed genomic profiles that clearly dichotomized responders and nonresponders. Mutations in ATM were present in most patients who achieved a complete response, while chromosome 9p21.1-p24.3 loss and/or mutations in components of the SWI-SNF chromatin-remodeling complex were present in all patients with primary resistance and two-thirds of patients with relapsed disease. Circulating tumor DNA analysis revealed that these alterations could be dynamically monitored, providing concurrent information on treatment response and tumor evolution. Functional modeling demonstrated that compromise of the SWI-SNF complex facilitated transcriptional upregulation of BCL2L1 (Bcl-xL) providing a selective advantage against ibrutinib plus venetoclax. Together these data highlight important insights into the molecular basis of therapeutic response and provide a model for real-time assessment of innovative targeted therapies.