RESUMEN
The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain-containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZFON) mice. NKT populations in PLZFON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet+ NKT1 and RORγt+ NKT17 subsets dramatically reduced and the emergence of a T-bet-RORγt- NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells' entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZFON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation.
Asunto(s)
Factores de Transcripción de Tipo Kruppel , Células T Asesinas Naturales , Acetilación , Animales , Diferenciación Celular , Inmunidad Innata , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Linfocitos/metabolismo , Ratones , Células T Asesinas Naturales/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de ZincRESUMEN
Human SETD2 is the unique histone methyltransferase that generates H3K36 trimethylation (H3K36me3), an epigenetic mark that plays a key role in normal hematopoiesis. Interestingly, recurrent inactivating mutations of SETD2 and aberrant H3K36me3 are increasingly reported to be involved in hematopoietic malignancies. Benzene (BZ) is a ubiquitous environmental pollutant and carcinogen that causes leukemia. The leukemogenic properties of BZ depend on its biotransformation in the bone marrow into oxidative metabolites, in particular 1,4-benzoquinone (BQ). This hematotoxic metabolite can form DNA and protein adducts that result in the damage and the alteration of cellular processes. Recent studies suggest that BZ-dependent leukemogenesis could depend on epigenetic perturbations, notably aberrant histone methylation. We investigated whether H3K36 trimethylation by SETD2 could be impacted by BZ and its hematotoxic metabolites. Herein, we show that BQ, the major leukemogenic metabolite of BZ, inhibits irreversibly the human histone methyltransferase SETD2, resulting in decreased H3K36me3. Our mechanistic studies further indicate that the BQ-dependent inactivation of SETD2 is due to covalent binding of BQ to reactive Zn-finger cysteines within the catalytic domain of the enzyme. The formation of these quinoprotein adducts results in loss of enzyme activity and protein crosslinks/oligomers. Experiments conducted in hematopoietic cells confirm that exposure to BQ results in the formation of SETD2 crosslinks/oligomers and concomitant loss of H3K36me3 in cells. Taken together, our data indicate that BQ, a major hematotoxic metabolite of BZ, could contribute to BZ-dependent leukemogenesis by perturbing the functions of SETD2, a histone lysine methyltransferase of hematopoietic relevance. SIGNIFICANCE STATEMENT: Benzoquinone is a major leukemogenic metabolite of benzene. Dysregulation of histone methyltransferase is involved in hematopoietic malignancies. This study found that benzoquinone irreversibly impairs SET domain containing 2, a histone H3K36 methyltransferase that plays a key role in hematopoiesis. Benzoquinone forms covalent adducts on Zn-finger cysteines within the catalytic site, leading to loss of activity, protein crosslinks/oligomers, and concomitant decrease of H3K36me3 histone mark. These data provide evidence that a leukemogenic metabolite of benzene can impair a key epigenetic enzyme.
Asunto(s)
Benceno/metabolismo , Benceno/toxicidad , Benzoquinonas/toxicidad , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Benceno/química , Benzoquinonas/química , Línea Celular , Cisteína/química , Cisteína/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Humanos , Leucemia/inducido químicamente , Leucemia/genética , Leucemia/metabolismo , Metilación , Cultivo Primario de Células , Dedos de Zinc/efectos de los fármacosRESUMEN
During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.
Asunto(s)
Compuestos de Bifenilo/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Unión al GTP Monoméricas/metabolismo , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/metabolismo , Nitrofenoles/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Células Madre/metabolismo , Sulfonamidas/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Estimación de Kaplan-Meier , Ratones , Ratones Transgénicos , Proteínas de Unión al GTP Monoméricas/genética , Síndromes Mielodisplásicos/mortalidad , Piperazinas/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Células Madre/efectos de los fármacos , Transcriptoma/efectos de los fármacosRESUMEN
Protein tyrosine phosphatase, nonreceptor type 2 (PTPN2) is mainly expressed in hematopoietic cells, where it negatively regulates growth factor and cytokine signaling. PTPN2 is an important regulator of hematopoiesis and immune/inflammatory responses, as evidenced by loss-of-function mutations of PTPN2 in leukemia and lymphoma and knockout mice studies. Benzene is an environmental chemical that causes hematological malignancies, and its hematotoxicity arises from its bioactivation in the bone marrow to electrophilic metabolites, notably 1,4-benzoquinone, a major hematotoxic benzene metabolite. Although the molecular bases for benzene-induced leukemia are not well-understood, it has been suggested that benzene metabolites alter topoisomerases II function and thereby significantly contribute to leukemogenesis. However, several studies indicate that benzene and its hematotoxic metabolites may also promote the leukemogenic process by reacting with other targets and pathways. Interestingly, alterations of cell-signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), have been proposed to contribute to benzene-induced malignant blood diseases. We show here that 1,4-benzoquinone directly impairs PTPN2 activity. Mechanistic and kinetic experiments with purified human PTPN2 indicated that this impairment results from the irreversible formation (kinact = 645 m-1·s-1) of a covalent 1,4-benzoquinone adduct at the catalytic cysteine residue of the enzyme. Accordingly, cell experiments revealed that 1,4-benzoquinone exposure irreversibly inhibits cellular PTPN2 and concomitantly increases tyrosine phosphorylation of STAT1 and expression of STAT1-regulated genes. Our results provide molecular and cellular evidence that 1,4-benzoquinone covalently modifies key signaling enzymes, implicating it in benzene-induced malignant blood diseases.
Asunto(s)
Benceno , Benzoquinonas/metabolismo , Leucemia , Proteínas de Neoplasias , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Factor de Transcripción STAT1 , Transducción de Señal/efectos de los fármacos , Benceno/farmacocinética , Benceno/farmacología , Células HEK293 , Humanos , Células Jurkat , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 2/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/genéticaRESUMEN
Etoposide is a widely prescribed anticancer drug that is, however, associated with an increased risk of secondary leukemia. Although the molecular basis underlying the development of these leukemias remains poorly understood, increasing evidence implicates the interaction of etoposide metabolites [i.e., etoposide quinone (EQ)] with topoisomerase II enzymes. However, effects of etoposide quinone on other cellular targets could also be at play. We investigated whether T-cell protein tyrosine phosphatase (TCPTP), a protein tyrosine phosphatase that plays a key role in normal and malignant hematopoiesis through regulation of Janus kinase/signal transducer and activator of transcription signaling, could be a target of EQ. We report here that EQ is an irreversible inhibitor of TCPTP phosphatase (IC50 = â¼7 µM, second-order rate inhibition constant of â¼810 M-1â min-1). No inhibition was observed with the parent drug. The inhibition by EQ was found to be due to the formation of a covalent adduct at the catalytic cysteine residue in the active site of TCPTP. Exposure of human hematopoietic cells (HL60 and Jurkat) to EQ led to inhibition of endogenous TCPTP and concomitant increase in STAT1 tyrosine phosphorylation. Our results suggest that in addition to alteration of topoisomerase II functions, EQ could also contribute to etoposide-dependent leukemogenesis through impairment of key hematopoietic signaling enzymes, such as TCPTP.
Asunto(s)
Etopósido/química , Proteína Tirosina Fosfatasa no Receptora Tipo 2/química , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Quinonas/farmacología , Sitios de Unión , Dominio Catalítico , Cisteína/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Células Jurkat , Fosforilación/efectos de los fármacos , Quinonas/química , Factor de Transcripción STAT1/metabolismoRESUMEN
Germ cells and adult stem cells maintain tissue homeostasis through a finely tuned program of responses to both physiological and stress-related signals. PLZF (Promyelocytic Leukemia Zinc Finger protein), a member of the POK family of transcription factors, acts as an epigenetic regulator of stem cell maintenance in germ cells and haematopoietic stem cells. We identified L1 retrotransposons as the primary targets of PLZF. PLZF-mediated DNA methylation induces silencing of the full-length L1 gene and inhibits L1 retrotransposition. Furthermore, PLZF causes the formation of barrier-type boundaries by acting on inserted truncated L1 sequences in protein coding genes. Cell stress releases PLZF-mediated repression, resulting in L1 activation/retrotransposition and impaired spermatogenesis and myelopoiesis. These results reveal a novel mechanism of action by which, PLZF represses retrotransposons, safeguarding normal progenitor homeostasis.
Asunto(s)
Epigenómica , Regulación de la Expresión Génica , Células Germinativas/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Elementos de Nucleótido Esparcido Largo/genética , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 5'/genética , Animales , Diferenciación Celular , Inmunoprecipitación de Cromatina , Metilación de ADN , Células Germinativas/citología , Ratones , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Células Madre/citología , Transcripción GenéticaRESUMEN
CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Pruebas de Enzimas/métodos , Colorantes Fluorescentes/metabolismo , Histonas/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Colorantes Fluorescentes/química , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/químicaRESUMEN
Transposable elements (TE) represent around 40% of the human genome. They are endogenous mobile DNA sequences able to jump and duplicate in the host genome. TE have long been considered as "junk" DNA but are now believed to be important regulators of gene expression by participating to the establishment of the DNA methylation profile. Recent advances in genome sequencing reveals a higher transposition frequency and TE driven gene expression in somatic cells than previously thought. As TE propagation is deleterious and may be involved in oncogenic mechanisms, host cells have developed silencing mechanisms mainly described in germinal and embryonic cells. However, somatic cells are also proned to TE transposition and use specific mechanisms involving tumor suppressor proteins including p53, Rb and PLZF. These transcription factors specifically target genomic retrotransposon sequences, histone deacetylase and DNA methylase activities, inducing epigenetic modifications related to gene silencing. Thus, these transcription factors negatively regulate TE expression by the formation of DNA methylation profil in somatic cells possibly associated with oncogenic mechanisms.
Asunto(s)
Epigénesis Genética/genética , Secuencias Repetitivas de Ácidos Nucleicos , Retroelementos/fisiología , Animales , Metilación de ADN , Regulación de la Expresión Génica , Humanos , Elementos de Nucleótido Esparcido Largo , Neoplasias/genéticaRESUMEN
BACKGROUND: The outcome of Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remain dismal despite the development of treatment. Targeted therapy is gaining more and more attention in improving prognosis. METHODS: Expression of BRAF was analyzed by RT-qPCR in AML and MDS patients. Cells viability treated by drugs was measured by CCK-8 assay. Network pharmacology and RNA-sequence were used to analyze the mechanism of drugs and verified in vitro and xenograft tumor model. RESULTS: Here we showed that BRAF was overexpressed in AML and MDS patients, and correlated with poor prognosis. The BRAF inhibitor-Vemurafenib (VEM) could significantly induce senescence, proliferation inhibition and apoptosis in AML cells, which can be enhanced by Bortezomib (BOR). This inhibitory effect was also verified in CD34 + cells derived from AML patients. Mechanistically, we showed that VEM combined with BOR could turn on HIPPO signaling pathway, thereby inducing cellular senescence in AML cells and xenograft mouse. CONCLUSIONS: Taken together, our findings demonstrate a significant upregulation of BRAF expression in AML and MDS patients, which is associated with unfavorable clinical outcomes. We also discovered that the BRAF inhibitor Vemurafenib induces cellular senescence through activation of the HIPPO signaling pathway. Analysis of BRAF expression holds promise as a prognostic indicator and potential therapeutic target for individuals with AML and MDS.
Asunto(s)
Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Humanos , Animales , Ratones , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Vía de Señalización Hippo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/complicaciones , Síndromes Mielodisplásicos/patologíaRESUMEN
Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.
Asunto(s)
Inflamación , Leucemia Mieloide Aguda , Ratones Noqueados , FN-kappa B , Proteína-Arginina N-Metiltransferasas , Transducción de Señal , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Inflamación/metabolismo , Inflamación/genética , Péptidos y Proteínas de Señalización Intracelular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
WHIM Syndrome is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report a decrease in bone mineral density in 25% of WHIM patients and bone defects leading to osteoporosis in a WHIM mouse model. Imbalanced bone tissue is observed in mutant mice combining reduced osteoprogenitor cells and increased osteoclast numbers. Mechanistically, impaired CXCR4 desensitization disrupts cell cycle progression and osteogenic commitment of skeletal stromal/stem cells, while increasing their pro-osteoclastogenic capacities. Impaired osteogenic differentiation is evidenced in primary bone marrow stromal cells from WHIM patients. In mice, chronic treatment with the CXCR4 antagonist AMD3100 normalizes in vitro osteogenic fate of mutant skeletal stromal/stem cells and reverses in vivo the loss of skeletal cells, demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of skeletal stromal/stem cells. Our study provides mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of skeletal cells and the balance between bone formation and resorption.
Asunto(s)
Síndromes de Inmunodeficiencia , Osteoporosis , Enfermedades de Inmunodeficiencia Primaria , Receptores CXCR4 , Animales , Ratones , Síndromes de Inmunodeficiencia/genética , Mutación , Osteogénesis/genética , Osteoporosis/genética , Enfermedades de Inmunodeficiencia Primaria/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , HumanosRESUMEN
Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolytic activity. Here we show that patients with MDS clones harbouring mutations in the TET2 gene are characterised by phenotypic defects in their circulating NK cells. Remarkably, NK cells and MDS clones from the same patient share the TET2 genotype, and the NK cells are characterised by increased methylation of genomic DNA and reduced expression of Killer Immunoglobulin-like receptors (KIR), perforin, and TNF-α. In vitro inhibition of TET2 in NK cells of healthy donors reduces their cytotoxicity, supporting its critical role in NK cell function. Conversely, NK cells from patients treated with azacytidine (#NCT02985190; https://clinicaltrials.gov/ ) show increased KIR and cytolytic protein expression, and IFN-γ production. Altogether, our findings show that, in addition to their oncogenic consequences in the myeloid cell subsets, TET2 mutations contribute to repressing NK-cell function in MDS patients.
Asunto(s)
Dioxigenasas , Síndromes Mielodisplásicos , Humanos , Metilación , Síndromes Mielodisplásicos/metabolismo , Células Asesinas Naturales , Azacitidina/farmacología , Receptores KIR/genética , Mutación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/metabolismoRESUMEN
Arginine methylation is a common post-translational modification affecting protein activity and the transcription of target genes when methylation occurs on histone tails. There are nine protein arginine methyltransferases (PRMTs) in mammals, divided into subgroups depending on the methylation they form on a molecule of arginine. During the formation and maturation of the different types of blood cells, PRMTs play a central role by controlling cell differentiation at the transcriptional level. PRMT enzymatic activity is necessary for many cellular processes in hematological malignancies, such as the activation of cell cycle and proliferation, inhibition of apoptosis, DNA repair processes, RNA splicing, and transcription by methylating histone tails' arginine. Chemical tools have been developed to inhibit the activity of PRMTs and have been tested in several models of hematological malignancies, including primary samples from patients, xenografts into immunodeficient mice, mouse models, and human cell lines. They show a significant effect by reducing cell viability and increasing the overall survival of mice. PRMT5 inhibitors have a strong therapeutic potential, as phase I clinical trials in hematological malignancies that use these molecules show promising results, thus, underlining PRMT inhibitors as useful therapeutic tools for cancer treatment in the future.
RESUMEN
The human genome is composed of unique DNA sequences that encode proteins and unique sequence noncoding RNAs that are essential for normal development and cellular differentiation. The human genome also contains over 50% of genome sequences that are repeat in nature (tandem and interspersed repeats) that are now known to contribute dynamically to genetic diversity in populations, to be transcriptionally active under certain physiological conditions, and to be aberrantly active in disease states including cancer, where consequences are pleiotropic with impact on cancer cell phenotypes and on the tumor immune microenvironment. Repeat element-derived RNAs play unique roles in exogenous and endogenous cell signaling under normal and disease conditions. A key component of repeat element-derived transcript-dependent signaling occurs via triggering of innate immune receptor signaling that then feeds forward to inflammatory responses through interferon and NFκB signaling. It has recently been shown that cancer cells display abnormal transcriptional activity of repeat elements and that this is linked to either aggressive disease and treatment failure or to improved prognosis/treatment response, depending on cell context and the amplitude of the so-called 'viral mimicry' response that is engaged. 'Viral mimicry' refers to a cellular state of active antiviral response triggered by endogenous nucleic acids often derived from aberrantly transcribed endogenous retrotransposons and other repeat elements. In this paper, the literature regarding transcriptional activation of repeat elements and engagement of inflammatory signaling in normal (focusing on hematopoiesis) and cancer is reviewed with an emphasis on the role of innate immune receptor signaling, in particular by dsRNA receptors of the RIG-1 like receptor family and interferons/NFκB. How repeat element-derived RNA reprograms cell identity through RNA-guided chromatin state modulation is also discussed.
RESUMEN
Etoposide is an extensively prescribed anticancer drug that, unfortunately, causes therapy-related leukemia. The mechanisms by which etoposide induces secondary hematopoietic malignancies are poorly documented. However, etoposide-related leukemogenesis is known to depend on oxidative metabolites of etoposide, notably etoposide quinone, that can react with protein cysteine residues such as in topoisomerases II. CREBBP is a major histone acetyltransferase that functions mainly as a transcriptional co-activator. This epigenetic enzyme is considered as a tumor suppressor that plays a major role in hematopoiesis. Genetic alterations affecting CREBBP activity are highly common in hematopoietic malignancies. We report here that CREBBP is impaired by etoposide quinone. Molecular and kinetic analyses show that this inhibition occurs through the rapid and covalent (kinhib = 16.102 M-1. s-1) adduction of etoposide quinone with redox sensitive cysteine residues within the RING and PHD Zn2+-fingers of CREBBP catalytic core leading to subsequent release of Zn2+. In agreement with these findings, experiments conducted in cells and in mice treated with etoposide showed irreversible inhibition of endogenous CREBBP activity and decreased H3K18 and H3K27 acetylation. As shown for topoisomerases II, our work thus suggests that the leukemogenic metabolite etoposide quinone can impair the epigenetic CREBBP acetyltransferase through reaction with redox sensitive cysteine residues.
Asunto(s)
Antineoplásicos , Cisteína , Animales , Proteína de Unión a CREB/metabolismo , Etopósido , Humanos , Ratones , Oxidación-Reducción , Estrés Oxidativo , Quinonas , ZincRESUMEN
Acute promyelocytic leukemia(APL) is characterized by the t(15;17) chromosomal translocation leading to the formation of the PML-RARalpha oncoprotein. This leukemia has attracted considerable interest in recent years, being the first in which therapies that specifically target the underlying molecular lesion, i.e., all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), leading to induction of differentiation and apoptosis have been successfully used in clinical practice. The advent of ATRA therapy has transformed APL from being a disease with a poor outlook to one of the most prognostically favorable subsets of acute myeloid leukemia. Further improvements in outcome may be achieved with the use of ATO, which achieves high rates of remission in the relatively small proportion of patients now relapsing following standard first-line therapy with ATRA and anthracycline-based chemotherapy. Moreover, recent studies have suggested that ATO and ATRA, or even ATO alone, used as front-line treatment of PML-RARA- associated APL can induce long-term remissions. This raises the possibility that some patients can be cured using differentiation therapies alone, without the need for chemotherapy, thereby potentially reducing treatment-related toxicity. It is clear that the success of such an approach is critically dependent upon molecular diagnostics and monitoring for minimal residual disease (MRD) to distinguish those patients who can potentially be cured with differentiation therapy from those requiring additional myelosuppressive agents. This represents an exciting new phase in the treatment of acute leukemia, highlighting the potential of molecularly targeted and MRD-directed therapies to achieve an individualized approach to patient management.
Asunto(s)
Antineoplásicos/uso terapéutico , Arsenicales/uso terapéutico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Óxidos/uso terapéutico , Tretinoina/uso terapéutico , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Trióxido de Arsénico , Arsenicales/efectos adversos , Arsenicales/farmacología , Diferenciación Celular/efectos de los fármacos , Disnea/inducido químicamente , Fiebre/inducido químicamente , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Leucocitosis/inducido químicamente , Modelos Biológicos , Neoplasia Residual , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Óxidos/efectos adversos , Óxidos/farmacología , Transcripción Genética , Resultado del Tratamiento , Tretinoina/efectos adversos , Tretinoina/farmacologíaRESUMEN
Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARalpha by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARalpha oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARalpha-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARalpha+RARalpha/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARalpha APL. RARalpha-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARalpha-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype.
Asunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 17/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Receptores de Ácido Retinoico/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , Cromatina/genética , Metilación de ADN , Progresión de la Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Promielocítica Aguda/patología , Datos de Secuencia Molecular , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Receptores de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Retinoides/farmacología , Regulación hacia ArribaRESUMEN
Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Acetilación , Acetiltransferasas/análisis , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente Directa , Colorantes Fluorescentes , Regulación Neoplásica de la Expresión Génica , Células HeLa , Histona Acetiltransferasas , Humanos , Factores de Transcripción de Tipo Kruppel , Leucemia Promielocítica Aguda/genética , Microscopía Confocal , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas Represoras/química , Proteínas Represoras/genética , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Dedos de ZincRESUMEN
Fanconi anemia (FA) causes bone marrow failure early during childhood, and recent studies indicate that a hematopoietic defect could begin in utero. We performed a unique kinetics study of hematopoiesis in Fancg-/- mouse embryos, between the early embryonic day 11.5 (E11.5) to E12.5 developmental window (when the highest level of hematopoietic stem cells [HSC] amplification takes place) and E14.5. This study reveals a deep HSC defect with exhaustion of proliferative and self-renewal capacities very early during development, together with severe FA clinical and biological manifestations, which are mitigated at E14.5 due to compensatory mechanisms that help to ensure survival of Fancg-/- embryos. It also reports that a deep HSC defect is also observed during human FA development, and that human FA fetal liver (FL) HSCs present a transcriptome profile similar to that of mouse E12.5 Fancg-/- FL HSCs. Altogether, our results highlight that early mouse FL could represent a good alternative model for studying Fanconi pathology.
Asunto(s)
Desarrollo Embrionario , Anemia de Fanconi/patología , Células Madre Hematopoyéticas/patología , Animales , Apoptosis , Ciclo Celular , Daño del ADN , Embrión de Mamíferos/patología , Eritrocitos/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Femenino , Ontología de Genes , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Hígado/embriología , Hígado/metabolismo , Ratones Endogámicos C57BL , Fenotipo , Placenta/metabolismo , Embarazo , Transcriptoma/genéticaRESUMEN
Differentiation induction is an effective therapy for acute promyelocytic leukemia (APL), which dramatically responds to all-trans-retinoic acid (ATRA). Recent studies have indicated that combinatorial use of retinoid and nonretinoid compounds, such as histone deacetylase inhibitors, arsenics, and PKA agonists, has higher therapeutic value in this disease and potentially in other malignancies. In a screen of 370 compounds, we identified benzodithiophene analogues as potent enhancers of ATRA-induced APL cell differentiation. These effects were not associated with changes in global histone acetylation and, for the most potent compounds, were exerted at very low nanomolar concentrations, and were paralleled by enhancement of some, but not all, ATRA-modulated gene expressions. Investigating the mechanism underlying the effects of these drugs on ATRA-induced APL cell differentiation, we have shown that benzodithiophenes enhance ATRA-mediated dissociation and association of corepressor N-CoR and coactivator p300 acetyltransferase, respectively, with retinoic acid receptor (RAR) alpha proteins. These data suggest that benzodithiophenes act at the level of receptor activation, possibly by affecting posttranslational modification of the receptor (and/or coregulators), thus leading to an enhancement in ATRA-mediated effects on gene expression and APL cell differentiation. Given the specificities of these low benzodithiophene concentrations for PML-RARalpha and RARalpha, these drugs may be useful for combinatorial differentiation therapy of APL and possibly other acute myelogenous leukemia subtypes in which the overall ATRA signaling is suppressed.