RESUMEN
Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.
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Células de la Médula Ósea/fisiología , Neutrófilos/fisiología , Animales , Células de la Médula Ósea/inmunología , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Ratones , Neoplasias Experimentales/inmunología , Neutrófilos/inmunologíaRESUMEN
Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS). Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. In order to further investigate the role of this splice factor in RNA splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or ≥1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in both competitive and non-competitive reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not in human cells contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to accurately model ZRSR2-mutant MDS in mice.
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Síndromes Mielodisplásicos , Empalme del ARN , Ribonucleoproteínas , Factor de Empalme U2AF , Animales , Intrones , Ratones , Mutación , Síndromes Mielodisplásicos/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Empalmosomas/genética , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib's efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management.
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Ácido Graso Desaturasas/genética , Carioferinas/genética , Liposarcoma/tratamiento farmacológico , Oligopéptidos/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bortezomib/farmacología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Ácido Graso Desaturasas/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hidrazinas/farmacología , Liposarcoma/genética , Liposarcoma/patología , Piperazinas/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Triazoles/farmacología , Proteína Exportina 1RESUMEN
Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12-THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients.
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Anilidas/farmacología , Biomarcadores de Tumor/genética , Reparación del ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Pirimidinas/farmacología , Mutaciones Letales Sintéticas , Animales , Apoptosis , Proteína BRCA1/genética , Proteína BRCA2/genética , Proliferación Celular , Quimioterapia Combinada , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.
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Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Diferenciación Celular/genética , Regulación de la Expresión Génica/genética , Granulocitos/metabolismo , Mielopoyesis/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Acute myeloid leukemia is characterized by arrested differentiation, and agents that overcome this block are therapeutically useful, as shown by the efficacy of all-trans retinoic acid in acute promyelocytic leukemia. However, the early promise of differentiation therapy did not translate into clinical benefit for other subtypes of acute myeloid leukemia, in which cytotoxic chemotherapeutic regimens remained the standard of care. Recent advances, including insights from sequencing of acute myeloid leukemia genomes, have led to the development of targeted therapies, comprising agents that induce differentiation of leukemic cells in preclinical models and clinical trials, thus rejuvenating interest in differentiation therapy. These agents act on various cellular processes including dysregulated metabolic programs, signaling pathways, epigenetic machinery and the cell cycle. In particular, inhibitors of mutant IDH1/2 and FLT3 have shown clinical benefit, leading to approval by regulatory bodies of their use. Besides the focus on recently approved differentiation therapies, this review also provides an overview of differentiation- inducing agents being tested in clinical trials or investigated in preclinical research. Combinatorial strategies are currently being tested for several agents (inhibitors of KDM1A, DOT1L, BET proteins, histone deacetylases), which were not effective in clinical studies as single agents, despite encouraging anti-leukemic activity observed in preclinical models. Overall, recently approved drugs and new investigational agents being developed highlight the merits of differentiation therapy; and ongoing studies promise further advances in the treatment of acute myeloid leukemia in the near future.
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Antineoplásicos , Leucemia Mieloide Aguda , Leucemia Promielocítica Aguda , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Diferenciación Celular , Histona Demetilasas , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Tretinoina/farmacologíaRESUMEN
Myelodysplastic syndromes (MDS) are characterized by recurrent somatic alterations often affecting components of RNA splicing machinery. Mutations of splice factors SF3B1, SRSF2, ZRSR2 and U2AF1 occur in >50% of MDS. To assess the impact of spliceosome mutations on splicing and to identify common pathways/genes affected by distinct mutations, we performed RNA-sequencing of MDS bone marrow samples harboring spliceosome mutations (including hotspot alterations of SF3B1, SRSF2 and U2AF1; small deletions of SRSF2 and truncating mutations of ZRSR2), and devoid of other common co-occurring mutations. We uncover the landscape of splicing alterations in each splice factor mutant MDS and demonstrate that small deletions in SRSF2 cause highest number of splicing alterations compared with other spliceosome mutations. Although the mis-spliced events observed in different splice factor mutations were largely non-overlapping, a subset of genes, including EZH2, were aberrantly spliced in multiple mutant groups. We also verified aberrant splicing of key genes USP9X, USP24 (deubiquitinating enzymes), LUC7L2 (splice factor) and EED (PRC2 component) in MDS harboring small deletions of SRSF2. Pathway analysis revealed that mis-spliced genes in different mutant groups were enriched in RNA splicing and transport as well as several signaling cascades, suggesting converging biological consequences downstream of distinct spliceosome mutations. Our study reveals splicing signatures of each splice factor mutation and identifies shared and distinct sets of mis-spliced genes and affected biological processes in different spliceosome mutant MDS.
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Mutación , Síndromes Mielodisplásicos , Factores de Empalme de ARN , Empalme del ARN , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismoRESUMEN
ZBTB transcription factors orchestrate gene transcription during tissue development. However, their roles in glioblastoma (GBM) remain unexplored. Here, through a functional screening of ZBTB genes, we identify that BCL6 is required for GBM cell viability and that BCL6 overexpression is associated with worse prognosis. In a somatic transgenic mouse model, depletion of Bcl6 inhibits the progression of KrasG12V-driven high-grade glioma. Transcriptome analysis demonstrates the involvement of BCL6 in tumor protein p53 (TP53), erythroblastic leukemia viral oncogene homolog (ErbB), and MAPK signaling pathways. Indeed, BCL6 represses the expression of wild-type p53 and its target genes in GBM cells. Knockdown of BCL6 augments the activation of TP53 pathway in response to radiation. Importantly, we discover that receptor tyrosine kinase AXL is a transcriptional target of BCL6 in GBM and mediates partially the regulatory effects of BCL6 on both MEK-ERK (mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase) and S6K-RPS6 (ribosomal protein S6 kinase-ribosomal protein S6) axes. Similar to BCL6 silencing, depletion of AXL profoundly attenuates GBM proliferation both in vitro and in vivo. Moreover, targeted inhibition of BCL6/nuclear receptor corepressor 1 (NCoR) complex by peptidomimetic inhibitor not only significantly decreases AXL expression and the activity of MEK-ERK and S6K-RPS6 cascades but also displays a potent antiproliferative effect against GBM cells. Together, these findings uncover a glioma-promoting role of BCL6 and provide the rationale of targeting BCL6 as a potential therapeutic approach.
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Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Gefitinib , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Mutantes , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-6/genética , Quinazolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
The cellular differentiation pathway originating from the bone marrow leading to early T lymphocytes remains poorly understood. The view that T cells branch off from a lymphoid-restricted pathway has recently been challenged by a model proposing a common progenitor for T cell and myeloid lineages. We generated interleukin-7 receptor alpha (Il7r) Cre recombinase knockin mice and traced lymphocyte development by visualizing the history of Il7r expression. Il7r fate mapping labeled all T cells but few myeloid cells. More than 85% of T cell progenitors were Il7r reporter(+) and, hence, had arisen from an Il7r-expressing pathway. In contrast, the overwhelming majority of myeloid cells in the thymus were derived from Il7r reporter(-) cells. Thus, lymphoid-restricted progenitors are the major route to T cells, and distinct origins of lymphoid and myeloid lineages represent a fundamental hallmark of hematopoiesis.
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Linaje de la Célula , Células Mieloides/citología , Células Mieloides/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Alelos , Animales , Diferenciación Celular , Ratones , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/inmunologíaRESUMEN
Maturation of granulocytes is dependent on controlled gene expression by myeloid lineage restricted transcription factors. CEBPE is one of the essential transcription factors required for granulocytic differentiation. Identification of downstream targets of CEBPE is vital to understand better its role in terminal granulopoiesis. In this study, we have identified Card10 as a novel target of CEBPE. We show that CEBPE binds to regulatory elements upstream of the murine Card10 locus, and expression of CARD10 is significantly reduced in Cebpe knock-out mice. Silencing Card10 in a human cell line and in murine primary cells impaired granulopoiesis, affecting expression of genes involved in myeloid cell development and function. Taken together, our data demonstrate for the first time that Card10 is expressed in granulocytes and is a direct target of CEBPE with functions extending to myeloid differentiation.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Diferenciación Celular , Granulocitos/citología , Animales , Sitios de Unión , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica , Granulocitos/metabolismo , Humanos , Ratones , Células Mieloides , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Chromosomal translocation t(8;21)(q22;q22) which leads to the generation of oncogenic RUNX1-RUNX1T1 (AML1-ETO) fusion is observed in approximately 10% of acute myelogenous leukemia (AML). To identify somatic mutations that co-operate with t(8;21)-driven leukemia, we performed whole and targeted exome sequencing of an Asian cohort at diagnosis and relapse. We identified high frequency of truncating alterations in ASXL2 along with recurrent mutations of KIT, TET2, MGA, FLT3, and DHX15 in this subtype of AML. To investigate in depth the role of ASXL2 in normal hematopoiesis, we utilized a mouse model of ASXL2 deficiency. Loss of ASXL2 caused progressive hematopoietic defects characterized by myeloid hyperplasia, splenomegaly, extramedullary hematopoiesis, and poor reconstitution ability in transplantation models. Parallel analyses of young and >1-year old Asxl2-deficient mice revealed age-dependent perturbations affecting, not only myeloid and erythroid differentiation, but also maturation of lymphoid cells. Overall, these findings establish a critical role for ASXL2 in maintaining steady state hematopoiesis, and provide insights into how its loss primes the expansion of myeloid cells.
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Diferenciación Celular/genética , Proliferación Celular/genética , Hematopoyesis/genética , Células Mieloides/metabolismo , Proteínas Represoras/genética , Enfermedad Aguda , Animales , Perfilación de la Expresión Génica/métodos , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Mielopoyesis/genéticaRESUMEN
Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.
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Exoma , Leucemia Mieloide Aguda , Mutación , Tirosina Quinasa 3 Similar a fms/genética , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN/genética , Femenino , Humanos , Quimioterapia de Inducción , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Masculino , Recurrencia , Estudios RetrospectivosRESUMEN
The transcription factor CCAAT enhancer binding protein α (C/EBPα) is a master regulator in granulopoiesis and is frequently disrupted in acute myeloid leukemia (AML). We have previously shown that C/EBPα exerts its effects by regulating microRNAs (miRs) such as miR-223 and miR-34a. Here, we confirm miR-30c as a novel important target of C/EBPα during granulopoiesis. Thus, wild-type C/EBPα-p42 directly upregulates miR-30c expression, whereas C/EBPα-p30, found in AML, does not. miR-30c is downregulated in AML, especially in normal karyotype AML patients with CEBPA mutations. An induced C/EBPα knockout in mice leads to a significant downregulation of miR-30c expression in bone marrow cells. We identified NOTCH1 as a direct target of miR-30c. Finally, a block of miR-30c prevents C/EBPα-induced downregulation of Notch1 protein and leads to a reduced CD11b expression in myeloid differentiation. Our study presents the first evidence that C/EBPα, miR-30c, and Notch1 together play a critical role in granulocytic differentiation and AML, and particularly in AML with CEBPA mutations. These data reveal the importance of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.
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Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Granulocitos/citología , Leucemia Mieloide Aguda/metabolismo , MicroARNs/metabolismo , Receptor Notch1/metabolismo , Animales , Diferenciación Celular/fisiología , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Granulocitos/metabolismo , Humanos , Immunoblotting , Leucemia Mieloide Aguda/genética , Leucopoyesis/fisiología , Ratones , Ratones Noqueados , MicroARNs/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
1,2,3-Triazole-based heterocycles have previously been shown to possess significant anticancer activity in various tumor models. In the present study, we attached a 1,2,3-triazole moiety to the third position of a 1,2-benzisoxazole heterocycle via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with various alkynes and established for the title compounds significant antiproliferative effect against human acute myeloid leukemia (AML) cells. Among the tested compounds, 3-(4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl)benzo[d]isoxazole (PTB) was found to be the most potent antiproliferative agent with an IC50 of 2 µM against MV4-11 cells using MTT assay. Notably, PTB induced cytotoxicity in MOLM13, MOLM14 and MV4-11 cells with selectivity over normal bone marrow cells (C57BL/6). Furthermore, PTB was found to induce cytotoxicity by increasing apoptosis of AML cells (MOLM13, MOLM14 and MV4-11) as well as sub-G1 cell population and apoptotic cells at submicromolar concentrations, as shown by flow cytometry and Annexin-V staining, respectively. On the protein level we suggested histone deacetylases (HDACs) as the potential protein target of those compounds in silico, and the predicted target was next experimentally validated by measuring the variations in the levels of p21, cyclin D and acetylation of histone H3 and tubulin. Molecular docking analysis of the title compounds with the second deacetylase domain of HDAC6 displayed high degree of shape complementarity to the binding site of the enzyme, forming multiple molecular interactions in the hydrophobic region as well as a hydrogen bond to the phenol side-chain of Tyr-782. Thus, 1,2,3-triazole derivatives appear to represent a class of novel, biologically active ligands against histone deacetylases which deserve to be further evaluated in their applications in the cancer field.
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Antineoplásicos/síntesis química , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidores de Histona Desacetilasas/síntesis química , Histona Desacetilasas/química , Triazoles/química , Tubulina (Proteína)/metabolismo , Acetilación , Animales , Antineoplásicos/química , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/toxicidad , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Isoxazoles/química , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Triazoles/síntesis química , Triazoles/toxicidadAsunto(s)
Linaje de la Célula , Leucemia/genética , Mutación , Proteínas de Neoplasias/genética , Recurrencia Local de Neoplasia/genética , Transcriptoma , Enfermedad Aguda , ADN de Neoplasias/genética , Humanos , Leucemia/clasificación , Leucemia/patología , Recurrencia Local de Neoplasia/patología , PronósticoRESUMEN
The adenosine triphosphate (ATP)-dependent chromatin remodeling complex, SWItch/Sucrose Non-Fermentable (SWI/SNF), has been implicated in normal hematopoiesis. The AT-rich interaction domain 1B (ARID1B) and its paralog, ARID1A, are mutually exclusive, DNA-interacting subunits of the BRG1/BRM-associated factor (BAF) subclass of SWI/SNF complex. Although the role of several SWI/SNF components in hematopoietic differentiation and stem cell maintenance has been reported, the function of ARID1B in hematopoietic development has not been defined. To this end, we generated a mouse model of Arid1b deficiency specifically in the hematopoietic compartment. Unlike the extensive phenotype observed in mice deficient in its paralog, ARID1A, Arid1b knockout (KO) mice exhibited a modest effect on steady-state hematopoiesis. Nonetheless, transplantation experiments showed that the reconstitution of myeloid cells in irradiated recipient mice was dependent on ARID1B. Furthermore, to assess the effect of the complete loss of ARID1 proteins in the BAF complex, we generated mice lacking both ARID1A and ARID1B in the hematopoietic compartment. The double-KO mice succumbed to acute bone marrow failure resulting from complete loss of BAF-mediated chromatin remodeling activity. Our Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed that >80% of loci regulated by ARID1B were distinct from those regulated by ARID1A; and ARID1B controlled expression of genes crucial in myelopoiesis. Overall, loss of ARID1B affected chromatin dynamics in murine hematopoietic stem and progenitor cells, albeit to a lesser extent than cells lacking ARID1A.
Asunto(s)
Hematopoyesis , Proteínas Nucleares , Animales , Ratones , Diferenciación Celular/genética , Cromatina , Hematopoyesis/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Alternative lengthening of telomeres (ALT) supports telomere maintenance in 10-15% of cancers, thus representing a compelling target for therapy. By performing anti-cancer compound library screen on isogenic cell lines and using extrachromosomal telomeric C-circles, as a bona fide marker of ALT activity, we identify a receptor tyrosine kinase inhibitor ponatinib that deregulates ALT mechanisms, induces telomeric dysfunction, reduced ALT-associated telomere synthesis, and targets, in vivo, ALT-positive cells. Using RNA-sequencing and quantitative phosphoproteomic analyses, combined with C-circle level assessment, we find an ABL1-JNK-JUN signalling circuit to be inhibited by ponatinib and to have a role in suppressing telomeric C-circles. Furthermore, transcriptome and interactome analyses suggest a role of JUN in DNA damage repair. These results are corroborated by synergistic drug interactions between ponatinib and either DNA synthesis or repair inhibitors, such as triciribine. Taken together, we describe here a signalling pathway impacting ALT which can be targeted by a clinically approved drug.
Asunto(s)
Transducción de Señal , Telómero , Supervivencia Celular , Transducción de Señal/efectos de los fármacos , Regulación de la Expresión Génica , Reparación del ADN , Replicación del ADN , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Humanos , Animales , Ratones , Línea Celular TumoralRESUMEN
Despite the remarkable clinical responses achieved with immune checkpoint blockade therapy, the response rate is relatively low and only a subset of patients can benefit from the treatment. Aberrant RNA accumulation can mediate IFN signaling and stimulate an immune response, suggesting that targeting RNA decay machinery might sensitize tumor cells to immunotherapy. With this in mind, we identified an RNA exoribonuclease, XRN1, as a potential therapeutic target to suppress RNA decay and stimulate antitumor immunity. Silencing of XRN1 suppressed tumor growth in syngeneic immunocompetent mice and potentiated immunotherapy efficacy, while silencing of XRN1 alone did not affect tumor growth in immunodeficient mice. Mechanistically, XRN1 depletion activated IFN signaling and the viral defense pathway; both pathways play determinant roles in regulating immune evasion. Aberrant RNA-sensing signaling proteins (RIG-I/MAVS) mediated the expression of IFN genes, as depletion of each of them blunted the elevation of antiviral/IFN signaling in XRN1-silenced cells. Analysis of pan-cancer CRISPR-screening data indicated that IFN signaling triggered by XRN1 silencing is a common phenomenon, suggesting that the effect of XRN1 silencing may be extended to multiple types of cancers. Overall, XRN1 depletion triggers aberrant RNA-mediated IFN signaling, highlighting the importance of the aberrant RNA-sensing pathway in regulating immune responses. These findings provide the molecular rationale for developing XRN1 inhibitors and exploring their potential clinical application in combination with cancer immunotherapy. SIGNIFICANCE: Targeting XRN1 activates an intracellular innate immune response mediated by RNA-sensing signaling and potentiates cancer immunotherapy efficacy, suggesting inhibition of RNA decay machinery as a novel strategy for cancer treatment.
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Neoplasias , ARN , Animales , Ratones , Exonucleasas/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Inmunoterapia , Neoplasias/genética , Neoplasias/terapia , Estabilidad del ARN , Transducción de SeñalRESUMEN
Hematopoietic stem cell (HSC) transplantation is limited by histocompatibility barriers and by lack of space in bone marrow niches. These obstacles prevent in vivo analysis of histoincompatible mutant stem cells and of HSC functions in non-irradiated mice. By genetically combining immunodeficiency with impaired function of the growth factor receptor Kit in mice, we generated a 'universal' HSC recipient that efficiently accepts long-term histocompatible and histoincompatible HSCs without prior irradiation.
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Proteínas de Unión al ADN/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Inmunidad Innata/inmunología , Proteínas Proto-Oncogénicas c-kit/inmunología , Animales , Supervivencia Celular/inmunología , Células Cultivadas , Proteínas de Unión al ADN/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/genética , Irradiación Corporal TotalRESUMEN
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. SIGNIFICANCE: Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.This article is highlighted in the In This Issue feature, p. 275.