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1.
Nature ; 554(7690): 62-68, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29364867

RESUMO

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Evolução Molecular , Dosagem de Genes , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Animais , Carcinogênese/genética , Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Progressão da Doença , Feminino , Genes myc , Genes p53 , Humanos , Masculino , Camundongos , Mutação , Subunidade p52 de NF-kappa B/genética , Metástase Neoplásica/genética , Proteínas Nucleares/genética , Fenótipo , Fosfoproteínas/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Fator de Crescimento Transformador beta1/genética , Proteínas de Sinalização YAP
2.
Blood ; 131(6): 649-661, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29282219

RESUMO

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.


Assuntos
Calreticulina/genética , Autorrenovação Celular/genética , Células-Tronco Hematopoéticas/fisiologia , Mielofibrose Primária/genética , Trombocitose/genética , Animais , Células Cultivadas , Homozigoto , Leucocitose/genética , Leucocitose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Esplenomegalia/genética , Esplenomegalia/patologia , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologia
3.
Blood ; 130(17): 1911-1922, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-28835438

RESUMO

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD , but not Npm1cA/+;NrasG12D/+ , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+ During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.


Assuntos
Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Alelos , Animais , Diferenciação Celular , Autorrenovação Celular , Sobrevivência Celular/genética , Progressão da Doença , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Células-Tronco Multipotentes/metabolismo , Mielopoese , Proteínas Nucleares/metabolismo , Nucleofosmina , Penetrância , Fenótipo , Fatores de Transcrição/genética , Transcriptoma/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
4.
Proc Natl Acad Sci U S A ; 111(27): 9840-5, 2014 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-24958871

RESUMO

Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2-deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.


Assuntos
Divisão Celular/fisiologia , Células-Tronco Embrionárias/enzimologia , Histona Desacetilase 1/fisiologia , Histona Desacetilase 2/fisiologia , Células-Tronco Pluripotentes/enzimologia , Acetilação , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo
5.
Blood ; 121(8): 1335-44, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23287868

RESUMO

Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 misregulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.


Assuntos
Transformação Celular Neoplásica/genética , Instabilidade Genômica/genética , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Linfócitos T/enzimologia , Animais , Animais Recém-Nascidos , Transformação Celular Neoplásica/imunologia , Cromatina/genética , Aberrações Cromossômicas , Dano ao DNA/genética , Dano ao DNA/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Feminino , Instabilidade Genômica/imunologia , Haploinsuficiência/genética , Haploinsuficiência/imunologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/citologia , Timo/citologia , Transcriptoma/imunologia
6.
Proc Natl Acad Sci U S A ; 107(18): 8242-7, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20404188

RESUMO

Histone deacetylases (HDAC) 1 and 2 are highly similar enzymes that help regulate chromatin structure as the core catalytic components of corepressor complexes. Although tissue-specific deletion of HDAC1 and HDAC2 has demonstrated functional redundancy, germ-line deletion of HDAC1 in the mouse causes early embryonic lethality, whereas HDAC2 does not. To address the unique requirement for HDAC1 in early embryogenesis we have generated conditional knockout embryonic stem (ES) cells in which HDAC1 or HDAC2 genes can be inactivated. Deletion of HDAC1, but not HDAC2, causes a significant reduction in the HDAC activity of Sin3A, NuRD, and CoREST corepressor complexes. This reduced corepressor activity results in a specific 1.6-fold increase in histone H3 K56 acetylation (H3K56Ac), thus providing genetic evidence that H3K56Ac is a substrate of HDAC1. In culture, ES cell proliferation was unaffected by loss of either HDAC1 or HDAC2. Rather, we find that loss of HDAC1 affects ES cell differentiation. ES cells lacking either HDAC1 or HDAC2 were capable of forming embryoid bodies (EBs), which stimulates differentiation into the three primary germ layers. However, HDAC1-deficient EBs were significantly smaller, showed spontaneous rhythmic contraction, and increased expression of both cardiomyocyte and neuronal markers. In summary, our genetic study of HDAC1 and HDAC2 in ES cells, which mimic the embryonic epiblast, has identified a unique requirement for HDAC1 in the optimal activity of HDAC1/2 corepressor complexes and cell fate determination during differentiation.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Animais , Biomarcadores , Linhagem Celular , Proliferação de Células , Regulação Enzimológica da Expressão Gênica , Histona Desacetilase 1/deficiência , Histona Desacetilase 2/deficiência , Camundongos , Ligação Proteica
7.
Stem Cell Reports ; 18(5): 1061-1074, 2023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-37028423

RESUMO

Perturbing expression is a powerful way to understand the role of individual genes, but can be challenging in important models. CRISPR-Cas screens in human induced pluripotent stem cells (iPSCs) are of limited efficiency due to DNA break-induced stress, while the less stressful silencing with an inactive Cas9 has been considered less effective so far. Here, we developed the dCas9-KRAB-MeCP2 fusion protein for screening in iPSCs from multiple donors. We found silencing in a 200 bp window around the transcription start site in polyclonal pools to be as effective as using wild-type Cas9 for identifying essential genes, but with much reduced cell numbers. Whole-genome screens to identify ARID1A-dependent dosage sensitivity revealed the PSMB2 gene, and enrichment of proteasome genes among the hits. This selective dependency was replicated with a proteasome inhibitor, indicating a targetable drug-gene interaction. Many more plausible targets in challenging cell models can be efficiently identified with our approach.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Sistemas CRISPR-Cas/genética , Genoma , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Sci Adv ; 8(31): eabn4886, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35921412

RESUMO

Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of premalignancy to cancer. To investigate this, we initiated preleukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA sequencing of preleukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of preleukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. That is, transcriptional variability confers forward momentum to cell fate systems, with differential multistage impact throughout cancer evolution.


Assuntos
Leucemia , Pré-Leucemia , Animais , Diferenciação Celular , Leucemia/genética , Camundongos , Pré-Leucemia/genética , Pré-Leucemia/patologia , Biossíntese de Proteínas
9.
Blood Adv ; 6(2): 386-398, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34638130

RESUMO

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis leading to peripheral cytopenias and in a substantial proportion of cases to acute myeloid leukemia. The deletion of the long arm of chromosome 11, del(11q), is a rare but recurrent clonal event in MDS. Here, we detail the largest series of 113 cases of MDS and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) harboring a del(11q) analyzed at clinical, cytological, cytogenetic, and molecular levels. Female predominance, a survival prognosis similar to other MDS, a low monocyte count, and dysmegakaryopoiesis were the specific clinical and cytological features of del(11q) MDS. In most cases, del(11q) was isolated, primary and interstitial encompassing the 11q22-23 region containing ATM, KMT2A, and CBL genes. The common deleted region at 11q23.2 is centered on an intergenic region between CADM1 (also known as Tumor Suppressor in Lung Cancer 1) and NXPE2. CADM1 was expressed in all myeloid cells analyzed in contrast to NXPE2. At the functional level, the deletion of Cadm1 in murine Lineage-Sca1+Kit+ cells modifies the lymphoid-to-myeloid ratio in bone marrow, although not altering their multilineage hematopoietic reconstitution potential after syngenic transplantation. Together with the frequent simultaneous deletions of KMT2A, ATM, and CBL and mutations of ASXL1, SF3B1, and CBL, we show that CADM1 may be important in the physiopathology of the del(11q) MDS, extending its role as tumor-suppressor gene from solid tumors to hematopoietic malignancies.


Assuntos
Molécula 1 de Adesão Celular/metabolismo , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Animais , Molécula 1 de Adesão Celular/genética , Deleção Cromossômica , Cromossomos Humanos Par 11 , Feminino , Genes Supressores de Tumor , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia
11.
Blood Adv ; 5(9): 2412-2425, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33956058

RESUMO

Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD-positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.


Assuntos
Leucemia Mieloide Aguda , Doença Aguda , Animais , Proteínas de Ligação a DNA , Histona-Lisina N-Metiltransferase , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , Proteínas Nucleares/genética , Nucleofosmina
12.
Nat Genet ; 53(10): 1443-1455, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34556857

RESUMO

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.


Assuntos
Montagem e Desmontagem da Cromatina/genética , DNA de Neoplasias/química , Regulação Leucêmica da Expressão Gênica , Histonas/metabolismo , Leucemia Mieloide Aguda/genética , Mutação/genética , Processamento de Proteína Pós-Traducional , Animais , Sequência de Bases , Modelos Animais de Doenças , Elementos Facilitadores Genéticos/genética , Redes Reguladoras de Genes , Loci Gênicos , Humanos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Nucleofosmina , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Tirosina Quinase 3 Semelhante a fms/metabolismo
15.
Nat Commun ; 10(1): 5351, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767858

RESUMO

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.


Assuntos
Leucemia Mieloide/genética , Mutação , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , RNA Ribossômico/genética , Transcrição Gênica , Doença Aguda , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células HEK293 , Humanos , Células K562 , Leucemia Mieloide/patologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Nucleofosmina , Biossíntese de Proteínas/genética , Células THP-1 , Transplante Heterólogo
16.
Sci Transl Med ; 9(402)2017 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-28794285

RESUMO

We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.


Assuntos
Benzotiazóis/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Fluoruracila/uso terapêutico , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Qualidade de Vida , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores
17.
Cell Rep ; 17(4): 1193-1205, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760321

RESUMO

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L, BCL2, and MEN1, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose KAT2A as a candidate for downstream study. KAT2A inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Terapia de Alvo Molecular , Adulto , Apoptose , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Humanos , Reprodutibilidade dos Testes
18.
Mol Cell Biol ; 30(20): 4851-63, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20713442

RESUMO

Lysine-specific demethylase 1 (LSD1), which demethylates mono- and dimethylated histone H3-Lys4 as part of a complex including CoREST and histone deacetylases (HDACs), is essential for embryonic development in the mouse beyond embryonic day 6.5 (e6.5). To determine the role of LSD1 during this early period of embryogenesis, we have generated loss-of-function gene trap mice and conditional knockout embryonic stem (ES) cells. Analysis of postimplantation gene trap embryos revealed that LSD1 expression, and therefore function, is restricted to the epiblast. Conditional deletion of LSD1 in mouse ES cells, the in vitro counterpart of the epiblast, revealed a reduction in CoREST protein and associated HDAC activity, resulting in a global increase in histone H3-Lys56 acetylation, but not H3-Lys4 methylation. Despite this biochemical perturbation, ES cells with LSD1 deleted proliferate normally and retain stem cell characteristics. Loss of LSD1 causes the aberrant expression of 588 genes, including those coding for transcription factors with roles in anterior/posterior patterning and limb development, such as brachyury, Hoxb7, Hoxd8, and retinoic acid receptor γ (RARγ). The gene coding for brachyury, a key regulator of mesodermal differentiation, is a direct target gene of LSD1 and is overexpressed in e6.5 Lsd1 gene trap embryos. Thus, LSD1 regulates the expression and appropriate timing of key developmental regulators, as part of the LSD1/CoREST/HDAC complex, during early embryonic development.


Assuntos
Perfilação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Proteínas Correpressoras , Primers do DNA/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/metabolismo , Histona Desmetilases , Camundongos , Camundongos Knockout , Oxirredutases N-Desmetilantes/deficiência , Oxirredutases N-Desmetilantes/genética , Gravidez , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
19.
Genome Res ; 17(6): 708-19, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17567991

RESUMO

Butyrate is a histone deacetylase inhibitor (HDACi) with anti-neoplastic properties, which theoretically reactivates epigenetically silenced genes by increasing global histone acetylation. However, recent studies indicate that a similar number or even more genes are down-regulated than up-regulated by this drug. We treated hepatocarcinoma HepG2 cells with butyrate and characterized the levels of acetylation at DNA-bound histones H3 and H4 by ChIP-chip along the ENCODE regions. In contrast to the global increases of histone acetylation, many genomic regions close to transcription start sites were deacetylated after butyrate exposure. In order to validate these findings, we found that both butyrate and trichostatin A treatment resulted in histone deacetylation at selected regions, while nucleosome loss or changes in histone H3 lysine 4 trimethylation (H3K4me3) did not occur in such locations. Furthermore, similar histone deacetylation events were observed when colon adenocarcinoma HT-29 cells were treated with butyrate. In addition, genes with deacetylated promoters were down-regulated by butyrate, and this was mediated at the transcriptional level by affecting RNA polymerase II (POLR2A) initiation/elongation. Finally, the global increase in acetylated histones was preferentially localized to the nuclear periphery, indicating that it might not be associated to euchromatin. Our results are significant for the evaluation of HDACi as anti-tumourogenic drugs, suggesting that previous models of action might need to be revised, and provides an explanation for the frequently observed repression of many genes during HDACi treatment.


Assuntos
Adenocarcinoma/metabolismo , Butiratos/farmacologia , Neoplasias do Colo/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
20.
Genome Res ; 17(6): 691-707, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17567990

RESUMO

We generated high-resolution maps of histone H3 lysine 9/14 acetylation (H3ac), histone H4 lysine 5/8/12/16 acetylation (H4ac), and histone H3 at lysine 4 mono-, di-, and trimethylation (H3K4me1, H3K4me2, H3K4me3, respectively) across the ENCODE regions. Studying each modification in five human cell lines including the ENCODE Consortium common cell lines GM06990 (lymphoblastoid) and HeLa-S3, as well as K562, HFL-1, and MOLT4, we identified clear patterns of histone modification profiles with respect to genomic features. H3K4me3, H3K4me2, and H3ac modifications are tightly associated with the transcriptional start sites (TSSs) of genes, while H3K4me1 and H4ac have more widespread distributions. TSSs reveal characteristic patterns of both types of modification present and the position relative to TSSs. These patterns differ between active and inactive genes and in particular the state of H3K4me3 and H3ac modifications is highly predictive of gene activity. Away from TSSs, modification sites are enriched in H3K4me1 and relatively depleted in H3K4me3 and H3ac. Comparison between cell lines identified differences in the histone modification profiles associated with transcriptional differences between the cell lines. These results provide an overview of the functional relationship among histone modifications and gene expression in human cells.


Assuntos
Genoma Humano/fisiologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transcrição Gênica/fisiologia , Células HeLa , Humanos , Células K562
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