RESUMEN
Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.
Asunto(s)
Genoma Humano , Leucemia Mieloide Aguda , Humanos , Cromatina/genética , Metilación de ADN , Leucemia Mieloide Aguda/genética , Genoma Humano/genética , Regiones Promotoras Genéticas , Elementos de Facilitación Genéticos , Silenciador del Gen , Reproducibilidad de los Resultados , Sistemas CRISPR-Cas , Análisis de Secuencia , ADN (Citosina-5-)-Metiltransferasas , Regulación Leucémica de la Expresión GénicaRESUMEN
Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the prognosis of AML patients remains poor and there is a need for the evaluation of promising therapeutic candidates to treat the disease. The objective of this study was to evaluate the efficacy of duocarmycin Stable A (DSA) in AML cells in vitro. We hypothesized that DSA would induce DNA damage in the form of DNA double-strand breaks (DSBs) and exert cytotoxic effects on AML cells within the picomolar range. Human AML cell lines Molm-14 and HL-60 were used to perform 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), DNA DSBs, cell cycle, 5-ethynyl-2-deoxyuridine (EdU), colony formation unit (CFU), Annexin V, RNA sequencing and other assays described in this study. Our results showed that DSA induced DNA DSBs, induced cell cycle arrest at the G2M phase, reduced proliferation and increased apoptosis in AML cells. Additionally, RNA sequencing results showed that DSA regulates genes that are associated with cellular processes such as DNA repair, G2M checkpoint and apoptosis. These results suggest that DSA is efficacious in AML cells and is therefore a promising potential therapeutic candidate that can be further evaluated for the treatment of AML.
Asunto(s)
Apoptosis , Proliferación Celular , Duocarmicinas , Leucemia Mieloide Aguda , Humanos , Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Proliferación Celular/efectos de los fármacos , Duocarmicinas/farmacología , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Células HL-60 , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacosRESUMEN
High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.
Asunto(s)
Quinasa de la Caseína II/genética , Resistencia a Antineoplásicos , Regulación Leucémica de la Expresión Génica , Factor de Transcripción Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína bcl-X/genética , Animales , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
Aberrant activation of the TAL1 is associated with up to 60% of T-ALL cases and is involved in CTCF-mediated genome organization within the TAL1 locus, suggesting that CTCF boundary plays a pathogenic role in T-ALL. Here, we show that -31-Kb CTCF binding site (-31CBS) serves as chromatin boundary that defines topologically associating domain (TAD) and enhancer/promoter interaction required for TAL1 activation. Deleted or inverted -31CBS impairs TAL1 expression in a context-dependent manner. Deletion of -31CBS reduces chromatin accessibility and blocks long-range interaction between the +51 erythroid enhancer and TAL1 promoter-1 leading to inhibition of TAL1 expression in erythroid cells, but not T-ALL cells. However, in TAL1-expressing T-ALL cells, the leukemia-prone TAL1 promoter-IV specifically interacts with the +19 stem cell enhancer located 19 Kb downstream of TAL1 and this interaction is disrupted by the -31CBS inversion in T-ALL cells. Inversion of -31CBS in Jurkat cells alters chromatin accessibility, histone modifications and CTCF-mediated TAD leading to inhibition of TAL1 expression and TAL1-driven leukemogenesis. Thus, our data reveal that -31CBS acts as critical regulator to define +19-enhancer and the leukemic prone promoter IV interaction for TAL1 activation in T-ALL. Manipulation of CTCF boundary can alter TAL1 TAD and oncogenic transcription networks in leukemogenesis.
Asunto(s)
Factor de Unión a CCCTC/genética , Carcinogénesis/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Sitios de Unión/genética , Cromatina/genética , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica , Genoma Humano/genética , Código de Histonas/genética , Humanos , Células Jurkat , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Unión Proteica/genética , Transcripción Genética/genéticaRESUMEN
Supplementing chemotherapy and radiotherapy with selenium has been shown to have benefits against various cancers. This approach has also been shown to alleviate the side effects associated with standard cancer therapies and improve the quality of life in patients. In addition, selenium levels in patients have been correlated with various cancers and have served as a diagnostic marker to track the efficiency of treatments or to determine whether these selenium levels cause or are a result of the disease. This concise review presents a survey of the selenium-based literature, with a focus on hematological malignancies, to demonstrate the significant impact of selenium in different cancers. The anti-cancer mechanisms and signaling pathways regulated by selenium, which impart its efficacious properties, are discussed. An outlook into the relationship between selenium and cancer is highlighted to guide future cancer therapy development.
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Neoplasias Hematológicas , Neoplasias , Selenio , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Neoplasias/tratamiento farmacológico , Calidad de Vida , Selenio/metabolismoRESUMEN
Cytokine receptor-like factor 2 B-cell acute lymphoblastic leukemia (CRLF2 B-ALL) is a high-risk subtype characterized by CRLF2 overexpression with poor survival rates in children and adults. CRLF2 and interleukin-7 receptor alpha (IL-7Rα) form a receptor for the cytokine thymic stromal lymphopoietin (TSLP), which induces JAK/STAT and PI3K/AKT/mTOR pathway signals. Previous studies from our group showed that low TSLP doses increased STAT5, AKT, and S6 phosphorylation and contributed to CRLF2 B-ALL cell survival. Here we investigated the role of TSLP in the survival and proliferation of CRLF2 B-ALL cells in vitro and in vivo. We hypothesized that high doses of TSLP increase CRLF2 signals and contribute to increased proliferation of CRLF2 B-ALL cells in vitro and in vivo. Interestingly, we observed the opposite effect. Specifically, high doses of TSLP induced apoptosis in human CRLF2 B-ALL cell lines in vitro, prevented engraftment of CRLF2 B-ALL cells, and prolonged the survival of +TSLP patient-derived-xenograft mice. Mechanistically, we showed that high doses of TSLP induced loss of its receptor and loss of CRLF2 signals in vitro. These results suggest that high doses of TSLP could be further investigated as a potential therapy for the treatment of CRLF2 B-ALL.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Linfopoyetina del Estroma Tímico , Animales , Humanos , Ratones , Citocinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Transducción de SeñalRESUMEN
Leukemic stem cells are multipotent, self-renewing, highly proliferative cells that can withstand drug treatments. Although currently available treatments potentially destroy blast cells, they fail to eradicate leukemic progenitor cells completely. Aldehyde dehydrogenase and STAT3 are frequently up-regulated in pre-leukemic stem cells as well as in acute myeloid leukemia (AML) expressing the CD34+CD38- phenotype. The Isatin analog, KS99 has shown anticancer activity against multiple myeloma which may, in part, be mediated by inhibition of Bruton's tyrosine kinase activation. Here we demonstrate that KS99 selectively targets leukemic stem cells with high aldehyde dehydrogenase activity and inhibits phosphorylation of STAT3. KS99 targeted cells co-expressing CD34, CD38, CD123, TIM-3, or CD96 immunophenotypes in AML, alone or in combination with the standard therapeutic agent cytarabine. AML with myelodysplastic-related changes was more sensitive than de novo AML with or without NPM1 mutation. KS99 treatment reduced the clonogenicity of primary human AML cells as compared to normal cord blood mononuclear cells. Downregulation of phosphorylated Bruton's tyrosine kinase, STAT3, and aldehyde dehydrogenase was observed, suggesting interaction with KS99 as predicted through docking. KS99 with or without cytarabine showed in vivo preclinical efficacy in human and mouse AML animal models and prolonged survival. KS99 was well tolerated with overall negligible adverse effects. In conclusion, KS99 inhibits aldehyde dehydrogenase and STAT3 activities and causes cell death of leukemic stem cells, but not normal hematopoietic stem and progenitor cells.
Asunto(s)
Isatina , Leucemia Mieloide Aguda , Animales , Antígenos CD34 , Citarabina , Subunidad alfa del Receptor de Interleucina-3 , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Células Madre Neoplásicas , NucleofosminaRESUMEN
Among brain tumors, Medulloblastoma (MB) is one of the most common, malignant, pediatric tumors of the cerebellum. It accounts for ~20% of all childhood central nervous system (CNS) tumors. Despite, tremendous advances in drug development processes, as well as novel drugs for MB the morbidity and mortality rates, remain high. Craniospinal radiation, high-dose chemotherapy, and surgical resection are the primary therapeutic strategies. Tremendous progress in the field of "genomics" with vast amounts of data has led to the identification of four distinct molecular subgroups in medulloblastoma: WNT group, SHH group, group-III, and group-IV. The identification of these subgroups has led to individualized treatment strategies for each subgroup. Here, we discuss the various molecular subgroups of medulloblastoma as well as the differences between them. We also highlight the latest treatment strategies available for medulloblastoma.
Asunto(s)
Neoplasias Cerebelosas/clasificación , Meduloblastoma/clasificación , HumanosRESUMEN
Ikaros is a DNA-binding protein that regulates gene expression and functions as a tumor suppressor in B-cell acute lymphoblastic leukemia (B-ALL). The full cohort of Ikaros target genes have yet to be identified. Here, we demonstrate that Ikaros directly regulates expression of the small GTPase, Rab20. Using ChIP-seq and qChIP we assessed Ikaros binding and the epigenetic signature at the RAB20 promoter. Expression of Ikaros, CK2, and RAB20 was determined by qRT-PCR. Overexpression of Ikaros was achieved by retroviral transduction, whereas shRNA was used to knockdown Ikaros and CK2. Regulation of transcription from the RAB20 promoter was analyzed by luciferase reporter assay. The results showed that Ikaros binds the RAB20 promoter in B-ALL. Gain-of-function and loss-of-function experiments demonstrated that Ikaros represses RAB20 transcription via chromatin remodeling. Phosphorylation by CK2 kinase reduces Ikaros' affinity toward the RAB20 promoter and abolishes its ability to repress RAB20 transcription. Dephosphorylation by PP1 phosphatase enhances both Ikaros' DNA-binding affinity toward the RAB20 promoter and RAB20 repression. In conclusion, the results demonstrated opposing effects of CK2 and PP1 on expression of Rab20 via control of Ikaros' activity as a transcriptional regulator. A novel regulatory signaling network in B-cell leukemia that involves CK2, PP1, Ikaros, and Rab20 is identified.
Asunto(s)
Factor de Transcripción Ikaros/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Enfermedad Aguda , Línea Celular Tumoral , Humanos , Factor de Transcripción Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas de Unión al GTP rab/genéticaRESUMEN
Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Epigénesis Genética , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Histona Desacetilasa 1/metabolismo , Factor de Transcripción Ikaros/metabolismo , Histona Demetilasas con Dominio de Jumonji/biosíntesis , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Represoras/biosíntesis , Transcripción Genética , Quinasa de la Caseína II/genética , Histona Desacetilasa 1/genética , Humanos , Factor de Transcripción Ikaros/genética , Histona Demetilasas con Dominio de Jumonji/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Proteínas Represoras/genética , Células U937RESUMEN
Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.
Asunto(s)
Señalización del Calcio , Glucólisis , Mitocondrias/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canales Catiónicos TRPM/metabolismo , Sustitución de Aminoácidos , Calcio , Línea Celular Tumoral , Supervivencia Celular , Eliminación de Gen , Humanos , Mitocondrias/genética , Mitocondrias/patología , Mutación Missense , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Neuroblastoma/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Canales Catiónicos TRPM/genéticaRESUMEN
Thymic stromal lymphopoietin (TSLP) and IL-7 are cytokines that signal via the IL-7 receptor alpha (IL-7Rα) to exert both overlapping and unique functions during early stages of mouse B-cell development. In human B lymphopoiesis, the requirement for IL-7Rα signaling is controversial and the roles of IL-7 and TSLP are less clear. Here, we evaluated human B-cell production using novel in vitro and xenograft models of human B-cell development that provide selective IL-7 and human TSLP (hTSLP) stimulation. We show that in vitro human B-cell production is almost completely blocked in the absence of IL-7Rα stimulation, and that either TSLP or IL-7 can provide a signal critical for the production and proliferation of human CD19(+) PAX5(+) pro-B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL-7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro-B cells under the influence of mouse IL-7 in a xenograft scenario is reduced by anti-IL-7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL-7Rα mediated signals for normal human B-cell production.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Citocinas/metabolismo , Interleucina-7/metabolismo , Linfopoyesis , Receptores de Interleucina-7/metabolismo , Transducción de Señal , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Células Cultivadas , Citocinas/farmacología , Expresión Génica , Humanos , Interleucina-7/farmacología , Linfopoyesis/efectos de los fármacos , Linfopoyesis/inmunología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacos , Linfopoyetina del Estroma TímicoRESUMEN
Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.
Asunto(s)
Quinasa de la Caseína II/antagonistas & inhibidores , Genes Supresores de Tumor , Factor de Transcripción Ikaros/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Animales , Apoptosis/efectos de los fármacos , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Factor de Transcripción Ikaros/genética , Ratones , Ratones Endogámicos NOD , Fosfatidilinositol 3-Quinasas , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Thymic stromal lymphopoietin (TSLP) stimulates in-vitro proliferation of human fetal B-cell precursors. However, its in-vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in-vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (-T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in -T mice. Patient-derived xenografts generated from +T as compared to -T mice showed a 3-6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from -T mice. +T/-T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.
Asunto(s)
Xenoinjertos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Receptores de Citocinas/metabolismo , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Xenoinjertos/inmunología , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Recuento de Linfocitos , Linfopoyesis/genética , Linfopoyesis/inmunología , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Citocinas/genética , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Transgenes , Trasplante HeterólogoRESUMEN
UNLABELLED: Ikaros is a zinc finger DNA-binding protein that regulates chromatin remodeling and the expression of genes involved in the cell cycle, apoptosis, and Notch signaling. It is a master regulator of lymphocyte differentiation and functions as a tumor suppressor in acute lymphoblastic leukemia. Nevertheless, no previous reports described effects of Ikaros on the life cycle of any human lymphotropic virus. Here, we demonstrate that full-length Ikaros (IK-1) functions as a major factor in the maintenance of viral latency in Epstein-Barr virus (EBV)-positive Burkitt's lymphoma Sal and MutuI cell lines. Either silencing of Ikaros expression by small hairpin RNA (shRNA) knockdown or ectopic expression of a non-DNA-binding isoform induced lytic gene expression. These effects synergized with other lytic inducers of EBV, including transforming growth factor ß (TGF-ß) and the hypoxia mimic desferrioxamine. Data from chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) and ChIP-sequencing (ChIP-seq) analyses indicated that Ikaros did not bind to either of the EBV immediate early genes BZLF1 and BRLF1. Rather, Ikaros affected the expression of Oct-2 and Bcl-6, other transcription factors that directly inhibit EBV reactivation and plasma cell differentiation, respectively. IK-1 also complexed with the EBV immediate early R protein in coimmunoprecipitation assays and partially colocalized with R within cells. The presence of R alleviated IK-1-mediated transcriptional repression, with IK-1 then cooperating with Z and R to enhance lytic gene expression. Thus, we conclude that Ikaros plays distinct roles at different stages of EBV's life cycle: it contributes to maintaining latency via indirect mechanisms, and it may also synergize with Z and R to enhance lytic replication through direct association with R and/or R-induced alterations in Ikaros' functional activities via cellular signaling pathways. IMPORTANCE: This is the first report showing that the cellular protein Ikaros, a known master regulator of hematopoiesis and critical tumor suppressor in acute lymphoblastic leukemia, also plays important roles in the life cycle of Epstein-Barr virus in B cells.
Asunto(s)
Linfocitos B/virología , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Factor de Transcripción Ikaros/metabolismo , Latencia del Virus , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Técnicas de Silenciamiento del Gen , Humanos , Unión ProteicaRESUMEN
Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function.
Asunto(s)
Núcleo Celular/metabolismo , Factor de Transcripción Ikaros/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Serina/metabolismo , Núcleo Celular/enzimología , Humanos , Factor de Transcripción Ikaros/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Quinasa SykRESUMEN
BACKGROUND: Ikaros is a DNA-binding protein that acts as master-regulator of hematopoiesis and a tumor suppressor. In thymocytes and T-cell leukemia, Ikaros negatively regulates transcription of terminal deoxynucleotide transferase (TdT), a key protein in lymphocyte differentiation. The signaling pathways that regulate Ikaros-mediated repression of TdT are unknown. Our previous work identified Casein Kinase II (CK2) and Protein Phosphatase 1 (PP1) as regulators of Ikaros DNA binding activity. Here, we investigated the role of PP1 and CK2 in regulating Ikaros-mediated control of TdT expression. PROCEDURES: Ikaros phosphomimetic and phosphoresistant mutants and specific CK2 and PP1 inhibitors were used in combination with quantitative chromatin immunoprecipitation (qChIP) and quantitative reverse transcriptase-PCR (q RT-PCR) assays to evaluate the role of CK2 and PP1 in regulating the ability of Ikaros to bind the TdT promoter and to regulate TdT expression. RESULTS: We demonstrate that phosphorylation of Ikaros by pro-oncogenic CK2 decreases Ikaros binding to the promoter of the TdT gene and reduces the ability of Ikaros to repress TdT expression during thymocyte differentiation. CK2 inhibition and PP1 activity restore Ikaros DNA-binding affinity toward the TdT promoter, as well as Ikaros-mediated transcriptional repression of TdT in primary thymocytes and in leukemia. CONCLUSION: These data establish that PP1 and CK2 signal transduction pathways regulate Ikaros-mediated repression of TdT in thymocytes and leukemia. These findings reveal that PP1 and CK2 have opposing effects on Ikaros-mediated repression of TdT and establish novel roles for PP1 and CK2 signaling in thymocyte differentiation and leukemia.
Asunto(s)
Quinasa de la Caseína II/metabolismo , ADN Nucleotidilexotransferasa/genética , Factor de Transcripción Ikaros/metabolismo , Leucemia de Células T/genética , Proteína Fosfatasa 1/metabolismo , Timocitos/metabolismo , Animales , Quinasa de la Caseína II/genética , Diferenciación Celular , Células Cultivadas , ADN Nucleotidilexotransferasa/metabolismo , Humanos , Factor de Transcripción Ikaros/genética , Leucemia de Células T/metabolismo , Leucemia de Células T/patología , Ratones , Fosforilación , Regiones Promotoras Genéticas , Proteína Fosfatasa 1/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Elementos Reguladores de la Transcripción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Timocitos/citologíaRESUMEN
Acute lymphoblastic leukemia (ALL) is the most common type of leukemia of childhood. Over the last 50 years there have been tremendous scientific advances in understanding the pathogenesis and the mechanisms that control cellular proliferation in ALL. These discoveries led to the development of efficient therapeutic regimens that greatly improved survival of children with ALL. Recently, several genes have been demonstrated to play a key role in tumor suppression and that their deregulation leads to malignant transformation and can affect overall survival. This review summarizes the role of Ikaros (IKZF1) in tumor suppression and regulation of gene expression in leukemia. Deletions and/or mutations of Ikaros have been detected in a large percentage of pediatric and adult ALL and reduced Ikaros function has been associated with poor outcome in ALL. Ikaros function in chromatin remodeling and epigenetic regulation of gene transcription emphasizes the important role of this protein in controlling cellular proliferation. In this review, we particularly focus on the role of signaling pathways in the regulation of Ikaros activity and its transcriptional control in leukemia.