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1.
Blood ; 136(24): 2764-2773, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33301029

RESUMO

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.


Assuntos
Regulação Leucêmica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Transplante de Neoplasias
2.
Haematologica ; 107(1): 86-99, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33375773

RESUMO

Chromosomal translocations involving KMT2A gene are one of the most common genetic alterations found in pediatric acute myeloid leukemias (AML) although the molecular mechanisms that initiate the disease remain incompletely defined. To elucidate these initiating events we have used a human model system of AML driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observe the development of a profound hypomethylation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem cell associated genes along with skewed expression in other genes such as S100A8/9 implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-seq data showed that there were relatively few leukemiaspecific changes and the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed the adenylate cyclase gene ADCY9 as an essential gene in KM3-AML, and suggest the potential for autocrine signalling through the chemokine receptor CCR1 and CCL23 ligand. Together, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.


Assuntos
Epigênese Genética , Leucemia Mieloide Aguda , Leucemia Mieloide , Adenilil Ciclases , Criança , Metilação de DNA , Histona-Lisina N-Metiltransferase , Humanos , Histona Desmetilases com o Domínio Jumonji , Leucemia Mieloide Aguda/genética , Mutação , Proteína de Leucina Linfoide-Mieloide , Translocação Genética
3.
Nature ; 532(7600): 508-511, 2016 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-27121842

RESUMO

Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Autorrenovação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Contagem de Células , Autorrenovação Celular/genética , Regulação para Baixo/genética , Feminino , Sangue Fetal/citologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais/genética
4.
Genes Chromosomes Cancer ; 59(2): 125-130, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31515871

RESUMO

Infant acute lymphoblastic leukemias (ALL) are rare hematological malignancies occurring in children younger than 1 year of age, most frequently associated with KMT2A rearrangements (KMT2A-r). The smaller subset without KMT2A-r, which represents 20% of infant ALL cases, is poorly characterized. Here we report two cases of chemotherapy-sensitive non-KMT2A-r infant ALL. Transcriptome analyses revealed identical ACIN1-NUTM1 gene fusions in both cases, derived from cryptic chromosomal rearrangements undetected by standard cytogenetic approaches. Two isoforms of the gene fusion, joining exons 3 or 4 of ACIN1 to exon 3 of NUTM1, were identified. Both fusion transcripts contained the functional DNA-binding SAP (SAF-A/B, Acinus, and PIAS) domain of ACIN1 and most of NUTM1. The detection of the ACIN1-NUTM1 fusion by RT-PCR allowed the molecular monitoring of minimal residual disease in a clinical setting. Based on publicly available genomic datasets and literature review, we predict that NUTM1 gene fusions are recurrent events in infant ALL. As such, we propose two clinically relevant assays to screen for NUTM1 rearrangements in bone marrow cells, independent of the fusion partner: NUMT1 immunohistochemistry and NUTM1 RNA expression. In sum, our study identifies ACIN1-NUTM1 as a recurrent and possibly cryptic fusion in non-KMT2A-r infant ALL, provides clinical tools to screen for NUTM1-rearranged leukemia and contributes to the refinement of this new subgroup.


Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aberrações Cromossômicas , Citogenética , Fusão Gênica , Rearranjo Gênico/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imuno-Histoquímica , Recém-Nascido , Leucemia Mieloide Aguda/genética , Masculino , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo
5.
Genes Chromosomes Cancer ; 57(6): 311-319, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29427526

RESUMO

The advent of large scale genomic sequencing technologies significantly improved the molecular classification of acute megakaryoblastic leukaemia (AMKL). AMKL represents a subset (∼10%) of high fatality pediatric acute myeloid leukemia (AML). Recurrent and mutually exclusive chimeric gene fusions associated with pediatric AMKL are found in 60%-70% of cases and include RBM15-MKL1, CBFA2T3-GLIS2, NUP98-KDM5A and MLL rearrangements. In addition, another 4% of AMKL harbor NUP98 rearrangements (NUP98r), with yet undetermined fusion partners. We report a novel NUP98-BPTF fusion in an infant presenting with primary refractory AMKL. In this NUP98r, the C-terminal chromatin recognition modules of BPTF, a core subunit of the NURF (nucleosome remodeling factor) ATP-dependent chromatin-remodeling complex, are fused to the N-terminal moiety of NUP98, creating an in frame NUP98-BPTF fusion, with structural homology to NUP98-KDM5A. The leukemic blasts expressed two NUP98-BPTF splicing variants, containing one or two tandemly spaced PHD chromatin reader domains. Our study also identified an unreported wild type BPTF splicing variant encoding for 2 PHD domains, detected both in normal cord blood CD34+ cells and in leukemic blasts, as with the fly BPTF homolog, Nurf301. Disease course was marked by rapid progression and primary chemoresistance, with ultimately significant tumor burden reduction following treatment with a clofarabine containing regimen. In sum, we report 2 novel NUP98-BPTF fusion isoforms that contribute to refine the NUP98r subgroup of pediatric AMKL. Multicenter clinical trials are critically required to determine the frequency of this fusion in AMKL patients and explore innovative treatment strategies for a disease still plagued with poor outcomes.


Assuntos
Antígenos Nucleares/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas do Tecido Nervoso/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fatores de Transcrição/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Humanos , Lactente , Cariotipagem , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Masculino , Splicing de RNA
6.
BMC Genomics ; 17: 75, 2016 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-26810393

RESUMO

BACKGROUND: Cancer genomics projects are producing ever-increasing amounts of rich and diverse data from patient samples. The ability to easily visualize this data in an integrated an intuitive way is currently limited by the current software available. As a result, users typically must use several different tools to view the different data types for their cohort, making it difficult to have a simple unified view of their data. RESULTS: Here we present Cascade, a novel web based tool for the intuitive 3D visualization of RNA-seq data from cancer genomics experiments. The Cascade viewer allows multiple data types (e.g. mutation, gene expression, alternative splicing frequency) to be simultaneously displayed, allowing a simplified view of the data in a way that is tuneable based on user specified parameters. The main webpage of Cascade provides a primary view of user data which is overlaid onto known biological pathways that are either predefined or added by users. A space-saving menu for data selection and parameter adjustment allows users to access an underlying MySQL database and customize the features presented in the main view. CONCLUSIONS: There is currently a pressing need for new software tools to allow researchers to easily explore large cancer genomics datasets and generate hypotheses. Cascade represents a simple yet intuitive interface for data visualization that is both scalable and customizable.


Assuntos
Genômica/métodos , Neoplasias/genética , Software , Humanos
7.
Blood ; 122(9): 1545-55, 2013 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-23777767

RESUMO

Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Histona Desmetilases com o Domínio Jumonji/fisiologia , Interferência de RNA/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/fisiologia , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Estudos de Validação como Assunto
8.
Bioinformatics ; 29(17): 2075-83, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23786767

RESUMO

MOTIVATION: The growth of next-generation sequencing (NGS) has not only dramatically accelerated the pace of research in the field of genomics, but it has also opened the door to personalized medicine and diagnostics. The resulting flood of data has led to the rapid development of large numbers of bioinformatic tools for data analysis, creating a challenging situation for researchers when choosing and configuring a variety of software for their analysis, and for other researchers trying to replicate their analysis. As NGS technology continues to expand from the research environment into clinical laboratories, the challenges associated with data analysis have the potential to slow the adoption of this technology. RESULTS: Here we discuss the potential of virtual machines (VMs) to be used as a method for sharing entire installations of NGS software (bioinformatic 'pipelines'). VMs are created by programs designed to allow multiple operating systems to co-exist on a single physical machine, and they can be made following the object-oriented paradigm of encapsulating data and methods together. This allows NGS data to be distributed within a VM, along with the pre-configured software for its analysis. Although VMs have historically suffered from poor performance relative to native operating systems, we present benchmarking results demonstrating that this reduced performance can now be minimized. We further discuss the many potential benefits of VMs as a solution for NGS analysis and describe several published examples. Lastly, we consider the benefits of VMs in facilitating the introduction of NGS technology into the clinical environment. CONTACT: brian.wilhelm@umontreal.ca.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Software , Genômica/métodos
9.
Blood ; 120(3): 592-602, 2012 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-22661698

RESUMO

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.


Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Inativação Gênica/imunologia , Células Matadoras Naturais/citologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neoplasias/genética , Neoplasias/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transexualidade/genética
10.
Nature ; 453(7199): 1239-43, 2008 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-18488015

RESUMO

Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and that many functional properties of transcripts are based not on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or few experimental conditions. Here we applied direct high-throughput sequencing of complementary DNAs (RNA-Seq), supplemented with data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including rapid proliferation, meiotic differentiation and environmental stress, as well as in RNA processing mutants to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to sample transcriptomes deeply at maximal resolution. In contrast to hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not robustly transcribed or rapidly degraded. The combined sequencing and strand-specific array data provide rich condition-specific information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus improving the existing genome annotation. Sequence reads spanning exon-exon or exon-intron junctions give unique insight into a surprising variability in splicing efficiency across introns, genes and conditions. Splicing efficiency was largely coordinated with transcript levels, and increased transcription led to increased splicing in test genes. Hundreds of introns showed such regulated splicing during cellular proliferation or differentiation.


Assuntos
Células Eucarióticas/metabolismo , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Schizosaccharomyces/genética , Processamento Alternativo/genética , Imunoprecipitação da Cromatina , Éxons/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Íntrons/genética , RNA Polimerase II/metabolismo , RNA Fúngico/análise , RNA Fúngico/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas de Schizosaccharomyces pombe/genética , Sensibilidade e Especificidade , Transcrição Gênica/genética
11.
Blood Adv ; 8(1): 112-129, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-37729615

RESUMO

ABSTRACT: Acute megakaryoblastic leukemia (AMKL) is a rare, developmentally restricted, and highly lethal cancer of early childhood. The paucity and hypocellularity (due to myelofibrosis) of primary patient samples hamper the discovery of cell- and genotype-specific treatments. AMKL is driven by mutually exclusive chimeric fusion oncogenes in two-thirds of the cases, with CBFA2T3::GLIS2 (CG2) and NUP98 fusions (NUP98r) representing the highest-fatality subgroups. We established CD34+ cord blood-derived CG2 models (n = 6) that sustain serial transplantation and recapitulate human leukemia regarding immunophenotype, leukemia-initiating cell frequencies, comutational landscape, and gene expression signature, with distinct upregulation of the prosurvival factor B-cell lymphoma 2 (BCL2). Cell membrane proteomic analyses highlighted CG2 surface markers preferentially expressed on leukemic cells compared with CD34+ cells (eg, NCAM1 and CD151). AMKL differentiation block in the mega-erythroid progenitor space was confirmed by single-cell profiling. Although CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with venetoclax, they were vulnerable to strategies that target the megakaryocytic prosurvival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor navitoclax and DT2216, a selective BCL-XL proteolysis-targeting chimera degrader developed to limit thrombocytopenia in patients. NUP98r AMKL were also sensitive to BCL-XL inhibition but not the NUP98r monocytic leukemia, pointing to a lineage-specific dependency. Navitoclax or DT2216 treatment in combination with low-dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.


Assuntos
Antineoplásicos , Leucemia Megacarioblástica Aguda , Humanos , Criança , Pré-Escolar , Animais , Camundongos , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Proteômica , Fatores de Transcrição , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras
12.
Blood ; 118(17): 4682-9, 2011 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-21900201

RESUMO

The three-amino-acid loop extension (TALE) class homeodomain proteins MEIS1 and PKNOX1 (PREP1) share the ability to interact with PBX and HOX family members and bind similar DNA sequences but appear to play opposing roles in tumor development. Elevated levels of MEIS1 accelerate development of HOX- and MLL-induced leukemias, and this pro-tumorigenic property has been associated with transcriptional activity of MEIS1. In contrast, reduction of PKNOX1 levels has been linked with cancer development despite the absence of an identifiable transactivating domain. In this report, we show that a chimeric protein generated by fusion of the MEIS1 C-terminal region encompassing the transactivating domain with the full-length PKNOX1 (PKNOX1-MC) acquired the ability to accelerate the onset of Hoxa9-induced leukemia in the mouse bone marrow transduction/transplantation model. Gene expression profiling of primary bone marrow cells transduced with Hoxa9 plus Meis1, or Hoxa9 plus Pknox1-MC revealed perturbations in overlapping functional gene subsets implicated in DNA packaging, chromosome organization, and in cell cycle regulation. Together, results presented in this report suggest that the C-terminal domain of MEIS1 confers to PKNOX1 an ectopic transactivating function that promotes leukemogenesis by regulating expression of genes involved in chromatin accessibility and cell cycle progression.


Assuntos
Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/química , Domínios e Motivos de Interação entre Proteínas/fisiologia , Animais , Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Cromatina/metabolismo , Proteínas de Homeodomínio/genética , Leucemia/genética , Leucemia/metabolismo , Leucemia/mortalidade , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Transfecção
13.
Blood ; 117(2): e27-38, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-20980679

RESUMO

The molecular mechanisms regulating self-renewal of leukemia stem cells remain poorly understood. Here we report the generation of 2 closely related leukemias created through the retroviral overexpression of Meis1 and Hoxa9. Despite their apparent common origin, these clonal leukemias exhibit enormous differences in stem cell frequency (from 1 in 1.4, FLA2; to 1 in 347, FLB1), suggesting that one of these leukemias undergoes nearly unlimited self-renewal divisions. Using next-generation RNA-sequencing, we characterized the transcriptomes of these phenotypically similar, but biologically distinct, leukemias, identifying hundreds of differentially expressed genes and a large number of structural differences (eg, alternative splicing and promoter usage). Focusing on ligand-receptor pairs, we observed high expression levels of Sdf1-Cxcr4; Jagged2-Notch2/1; Osm-Gp130; Scf-cKit; and Bmp15-Tgfb1/2. Interestingly, the integrin beta 2-like gene (Itgb2l) is both highly expressed and differentially expressed between our 2 leukemias (∼ 14-fold higher in FLA2 than FLB1). In addition, gene ontology analysis indicated G-protein-coupled receptor had a much higher proportion of differential expression (22%) compared with other classes (∼ 5%), suggesting a potential role regulating subtle changes in cellular behavior. These results provide the first comprehensive transcriptome analysis of a leukemia stem cell and document an unexpected level of transcriptome variation between phenotypically similar leukemic cells.


Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas , Animais , Células Clonais , Citometria de Fluxo , Vetores Genéticos , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Proteína Meis1 , Proteínas de Neoplasias/genética , Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
14.
Front Oncol ; 13: 1211540, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37456227

RESUMO

Methyltransferases are enzymes fundamental to a wide range of normal biological activities that can become dysregulated during oncogenesis. For instance, the recent description of the methyltransferase-like (METTL) family of enzymes, has demonstrated the importance of the N6-adenosine-methyltransferase (m6A) modification in transcripts in the context of malignant transformation. Because of their importance, numerous METTL family members have been biochemically characterized to identify their cellular substrates, however some members such as METTL7B, recently renamed TMT1B and which is the subject of this review, remain enigmatic. First identified in the stacked Golgi, TMT1B is also localized to the endoplasmic reticulum as well as lipid droplets and has been reported as being upregulated in a wide range of cancer types including lung cancer, gliomas, and leukemia. Interestingly, despite evidence that TMT1B might act on protein substrates, it has also been shown to act on small molecule alkyl thiol substrates such as hydrogen sulfide, and its loss has been found to affect cellular proliferation and migration. Here we review the current evidence for TMT1B's activity, localization, and potential biological role in the context of both normal and cancerous cell types.

15.
Blood ; 116(10): 1678-84, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-20522713

RESUMO

It is believed that hemopoietic stem cells (HSC), which colonize the fetal liver (FL) rapidly, expand to establish a supply of HSCs adequate for maintenance of hemopoiesis throughout life. Accordingly, FL HSCs are actively cycling as opposed to their predominantly quiescent bone marrow counterparts, suggesting that the FL microenvironment provides unique signals that support HSC proliferation and self-renewal. We now report the generation and characterization of mice with a mutant allele of Baf250a lacking exons 2 and 3. Baf250a(E2E3/E2E3) mice are viable until E19.5, but do not survive beyond birth. Most interestingly, FL HSC numbers are markedly higher in these mice than in control littermates, thus raising the possibility that Baf250a determines the HSC pool size in vivo. Limit dilution experiments indicate that the activity of Baf250a(E2E3/E2E3) HSC is equivalent to that of the wild-type counterparts. The Baf250a(E2E3/E2E3) FL-derived stroma, in contrast, exhibits a hemopoiesis-supporting potential superior to the developmentally matched controls. To our knowledge, this demonstration is the first that a mechanism operating in a cell nonautonomous manner canexpand the pool size of the fetal HSC populations.


Assuntos
Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Fígado/metabolismo , Mutação , Proteínas Nucleares/genética , Alelos , Animais , Western Blotting , Contagem de Células , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Fígado/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Tempo , Fatores de Transcrição
16.
PLoS Genet ; 5(8): e1000626, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19714215

RESUMO

The regulation of the G1- to S-phase transition is critical for cell-cycle progression. This transition is driven by a transient transcriptional wave regulated by transcription factor complexes termed MBF/SBF in yeast and E2F-DP in mammals. Here we apply genomic, genetic, and biochemical approaches to show that the Yox1p homeodomain protein of fission yeast plays a critical role in confining MBF-dependent transcription to the G1/S transition of the cell cycle. The yox1 gene is an MBF target, and Yox1p accumulates and preferentially binds to MBF-regulated promoters, via the MBF components Res2p and Nrm1p, when they are transcriptionally repressed during the cell cycle. Deletion of yox1 results in constitutively high transcription of MBF target genes and loss of their cell cycle-regulated expression, similar to deletion of nrm1. Genome-wide location analyses of Yox1p and the MBF component Cdc10p reveal dozens of genes whose promoters are bound by both factors, including their own genes and histone genes. In addition, Cdc10p shows promiscuous binding to other sites, most notably close to replication origins. This study establishes Yox1p as a new regulatory MBF component in fission yeast, which is transcriptionally induced by MBF and in turn inhibits MBF-dependent transcription. Yox1p may function together with Nrm1p to confine MBF-dependent transcription to the G1/S transition of the cell cycle via negative feedback. Compared to the orthologous budding yeast Yox1p, which indirectly functions in a negative feedback loop for cell-cycle transcription, similarities but also notable differences in the wiring of the regulatory circuits are evident.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Retroalimentação Fisiológica , Proteínas de Homeodomínio/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Regulação para Baixo , Regulação Fúngica da Expressão Gênica , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Ligação Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Transcrição Gênica
17.
Mol Cell Biol ; 27(11): 4058-69, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17371846

RESUMO

In this study, we characterize a four-protein nucleosome-binding complex from Schizosaccharomyces pombe, termed SAPHIRE, that includes two orthologs of human Lsd1, a histone demethylase. The SAPHIRE complex is essential for cell viability, whereas saphire mutants lacking key conserved catalytic residues are viable but thermosensitive, suggesting that SAPHIRE has both an important enzymatic function and an essential nonenzymatic function. SAPHIRE is present in (or adjacent to) particular heterochromatic loci and also in the transcription start site regions of many highly active polymerase II genes. However, ribosomal protein genes are notably SAPHIRE deficient. SAPHIRE promotes activation, as target genes are selectively attenuated in saphire mutants. Interestingly, saphire mutants display increased histone H3 lysine 4 dimethylation, a modification typically associated with euchromatin. SAPHIRE localization is dynamic, as activated genes rapidly acquire SAPHIRE. Furthermore, saphire mutants dramatically shift a heterochromatin-euchromatin boundary in Chr1, suggesting a novel role in boundary regulation.


Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Genoma Fúngico , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequência de Aminoácidos , Animais , Sobrevivência Celular , Cromatina/química , Resposta ao Choque Térmico , Histona Desmetilases , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Alinhamento de Sequência , Telômero/metabolismo , Ativação Transcricional
18.
Methods ; 48(3): 249-57, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19336255

RESUMO

The ability to quantitatively survey the global behavior of transcriptomes has been a key milestone in the field of systems biology, enabled by the advent of DNA microarrays. While this approach has literally transformed our vision and approach to cellular physiology, microarray technology has always been limited by the requirement to decide, a priori, what regions of the genome to examine. While very high density tiling arrays have reduced this limitation for simpler organisms, it remains an obstacle for larger, more complex, eukaryotic genomes. The recent development of "next-generation" massively parallel sequencing (MPS) technologies by companies such as Roche (454 GS FLX), Illumina (Genome Analyzer II), and ABI (AB SOLiD) has completely transformed the way in which quantitative transcriptomics can be done. These new technologies have reduced both the cost-per-reaction and time required by orders of magnitude, making the use of sequencing a cost-effective option for many experimental approaches. One such method that has recently been developed uses MPS technology to directly survey the RNA content of cells, without requiring any of the traditional cloning associated with EST sequencing. This approach, called "RNA-seq", can generate quantitative expression scores that are comparable to microarrays, with the added benefit that the entire transcriptome is surveyed without the requirement of a priori knowledge of transcribed regions. The important advantage of this technique is that not only can quantitative expression measures be made, but transcript structures including alternatively spliced transcript isoforms, can also be identified. This article discusses the experimental approach for both sample preparation and data analysis for the technique of RNA-seq.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Animais , Sequência de Bases , Humanos
19.
Exp Hematol ; 74: 1-12, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31154068

RESUMO

Leukemia is a complex genetic disease caused by errors in differentiation, growth, and apoptosis of hematopoietic cells in either lymphoid or myeloid lineages. Large-scale genomic characterization of thousands of leukemia patients has produced a tremendous amount of data that have enabled a better understanding of the differences between adult and pediatric patients. For instance, although phenotypically similar, pediatric and adult myeloid leukemia patients differ in their mutational profiles, typically involving either chromosomal translocations or recurrent single-base-pair mutations, respectively. To elucidate the molecular mechanisms underlying the biology of this cancer, continual efforts have been made to develop more contextually and biologically relevant experimental models. Leukemic cell lines, for example, provide an inexpensive and tractable model but often fail to recapitulate critical aspects of tumor biology. Likewise, murine leukemia models of leukemia have been highly informative but also do not entirely reproduce the human disease. More recent advances in the development of patient-derived xenografts (PDXs) or human models of leukemias are poised to provide a more comprehensive, and biologically relevant, approach to directly assess the impact of the in vivo environment on human samples. In this review, the advantages and limitations of the various current models used to functionally define the genetic requirements of leukemogenesis are discussed.


Assuntos
Diferenciação Celular , Leucemia Mieloide , Neoplasias Experimentais , Translocação Genética , Adolescente , Animais , Criança , Pré-Escolar , Feminino , Xenoenxertos , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Leucemia Mieloide/terapia , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia
20.
Blood Adv ; 3(21): 3307-3321, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31698461

RESUMO

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.


Assuntos
Biomarcadores , Modelos Animais de Doenças , Leucemia Megacarioblástica Aguda/etiologia , Leucemia Megacarioblástica Aguda/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biologia Computacional/métodos , Suscetibilidade a Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Leucemia Megacarioblástica Aguda/terapia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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