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1.
Mol Cell ; 81(16): 3323-3338.e14, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352207

RESUMO

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.


Assuntos
Carcinogênese/genética , Metiltransferases/genética , Neoplasias/genética , tRNA Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Metilação , Neoplasias/patologia , Oncogenes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
2.
Sci Adv ; 7(27)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34193418

RESUMO

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates the rapid development of new therapies against coronavirus disease 2019 (COVID-19) infection. Here, we present the identification of 200 approved drugs, appropriate for repurposing against COVID-19. We constructed a SARS-CoV-2-induced protein network, based on disease signatures defined by COVID-19 multiomics datasets, and cross-examined these pathways against approved drugs. This analysis identified 200 drugs predicted to target SARS-CoV-2-induced pathways, 40 of which are already in COVID-19 clinical trials, testifying to the validity of the approach. Using artificial neural network analysis, we classified these 200 drugs into nine distinct pathways, within two overarching mechanisms of action (MoAs): viral replication (126) and immune response (74). Two drugs (proguanil and sulfasalazine) implicated in viral replication were shown to inhibit replication in cell assays. This unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials.


Assuntos
Reposicionamento de Medicamentos , SARS-CoV-2/fisiologia , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Humanos , Redes Neurais de Computação , Proguanil/farmacologia , Proguanil/uso terapêutico , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Sulfassalazina/farmacologia , Replicação Viral/efeitos dos fármacos
3.
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
4.
Nature ; 593(7860): 597-601, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902106

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

5.
Leukemia ; 35(4): 1012-1022, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32764680

RESUMO

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.


Assuntos
Rearranjo Gênico , Predisposição Genética para Doença , Histona Acetiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Apoptose/genética , Biomarcadores Tumorais , Diferenciação Celular , Linhagem Celular Tumoral , Gerenciamento Clínico , Epigênese Genética , Técnicas de Inativação de Genes , Estudos de Associação Genética , Histona Acetiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Células Mieloides/metabolismo , Células Mieloides/patologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
6.
Curr Opin Hematol ; 28(2): 80-85, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33337619

RESUMO

PURPOSE OF REVIEW: In recent years, the N6-methyladenosine (m6A) modification of RNA has been shown to play an important role in the development of acute myeloid leukemia (AML) and the maintenance of leukemic stem cells (LSCs). In this review we summarise the recent findings in the field of epitranscriptomics related to m6A and its relevance in AML. RECENT FINDINGS: Recent studies have focused on the role of m6A regulators in the development of AML and their potential as translational targets. The writer Methyltransferase Like 3 and its binding partner Methyltransferase Like 14, as well as the reader YTH domain-containing family protein 2, were shown to be vital for LSC survival, and their loss has detrimental effects on AML cells. Similar observations were made with the demethylases fat mass and obesity-associated protein and AlkB homologue 5 RNA demethylase. Of importance, loss of any of these genes has little to no effect on normal hemopoietic stem cells, suggesting therapeutic potential. SUMMARY: The field of epitranscriptomics is still in its infancy and the importance of m6A and other RNA-modifications in AML will only come into sharper focus. The development of therapeutics targeting RNA-modifying enzymes may open up new avenues for treatment of such malignancies.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , RNA/metabolismo , Adenosina/metabolismo , Animais , Biomarcadores , Gerenciamento Clínico , Suscetibilidade a Doenças , Epigenômica/métodos , Humanos , Leucemia Mieloide Aguda/patologia , Metilação , RNA/genética
7.
ACS Cent Sci ; 6(12): 2196-2208, 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33376781

RESUMO

The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2'-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner. Here we describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, we have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N6-methyladenosine (m6A), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we show that METTL16 deposits m6A to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation.

8.
Nat Commun ; 11(1): 1407, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179751

RESUMO

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets.


Assuntos
Transformação Celular Neoplásica , Histona-Lisina N-Metiltransferase/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Oncogene ; 39(9): 2030, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31754212

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

10.
Nat Struct Mol Biol ; 26(10): 858-862, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31582848

RESUMO

Exciting research has connected specific RNA modifications to chromatin, providing evidence for co-transcriptional deposition and function in gene regulation. Here we review insights gained from studying the co-transcriptional roles of RNA modifications, and their influence in normal and disease contexts. We also discuss how the availability of novel technical approaches could raise the translational potential of targeting RNA-modifying enzymes for the treatment of disease.


Assuntos
Cromatina/metabolismo , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Animais , Cromatina/genética , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Humanos , RNA/genética , Transcrição Genética
11.
Oncogene ; 38(7): 998-1018, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30190546

RESUMO

Cell cycle regulation, especially faithful DNA replication and mitosis, are crucial to maintain genome stability. Cyclin-dependent kinase (CDK)/cyclin complexes drive most processes in cellular proliferation. In response to DNA damage, cell cycle surveillance mechanisms enable normal cells to arrest and undergo repair processes. Perturbations in genomic stability can lead to tumor development and suggest that cell cycle regulators could be effective targets in anticancer therapy. However, many clinical trials ended in failure due to off-target effects of the inhibitors used. Here, we investigate in vivo the importance of WEE1- and MYT1-dependent inhibitory phosphorylation of mammalian CDK1. We generated Cdk1AF knockin mice, in which two inhibitory phosphorylation sites are replaced by the non-phosphorylatable amino acids T14A/Y15F. We uncovered that monoallelic expression of CDK1AF is early embryonic lethal in mice and induces S phase arrest accompanied by γH2AX and DNA damage checkpoint activation in mouse embryonic fibroblasts (MEFs). The chromosomal fragmentation in Cdk1AF MEFs does not rely on CDK2 and is partly caused by premature activation of MUS81-SLX4 structure-specific endonuclease complexes, as well as untimely onset of chromosome condensation followed by nuclear lamina disassembly. We provide evidence that tumor development in liver expressing CDK1AF is inhibited. Interestingly, the regulatory mechanisms that impede cell proliferation in CDK1AF expressing cells differ partially from the actions of the WEE1 inhibitor, MK-1775, with p53 expression determining the sensitivity of cells to the drug response. Thus, our work highlights the importance of improved therapeutic strategies for patients with various cancer types and may explain why some patients respond better to WEE1 inhibitors.


Assuntos
Proteína Quinase CDC2/metabolismo , Perda do Embrião/enzimologia , Embrião de Mamíferos/enzimologia , Mitose , Fase S , Substituição de Aminoácidos , Animais , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perda do Embrião/genética , Perda do Embrião/patologia , Embrião de Mamíferos/patologia , Ativação Enzimática , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 9(1): 5378, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30568163

RESUMO

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Epigênese Genética , Células HL-60 , Hematopoese , Humanos , Células K562 , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA
13.
Nat Genet ; 50(6): 883-894, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29736013

RESUMO

The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição GATA/genética , Histona Desmetilases/genética , Leucemia Mieloide/genética , Proteínas Proto-Oncogênicas c-ets/genética , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Histonas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteômica/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Ativação Transcricional
14.
Blood ; 131(15): 1639-1653, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29463564

RESUMO

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.


Assuntos
Glutamina/metabolismo , Leucemia Mieloide Aguda , Mutação , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms , Sistemas CRISPR-Cas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Estudo de Associação Genômica Ampla , Glutamina/genética , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Células THP-1 , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
15.
Nature ; 552(7683): 126-131, 2017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29186125

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia.


Assuntos
Adenosina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Metiltransferases/metabolismo , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Adenosina/genética , Adenosina/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Cromatina/metabolismo , Feminino , Genes Neoplásicos/genética , Humanos , Leucemia Mieloide Aguda/patologia , Metiltransferases/química , Metiltransferases/deficiência , Metiltransferases/genética , Camundongos , Biossíntese de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Sítio de Iniciação de Transcrição
16.
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 , Penetrância , Fenótipo , Fatores de Transcrição/genética , Transcriptoma/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
17.
Sci Rep ; 7(1): 2244, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28533524

RESUMO

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Estudo de Associação Genômica Ampla , Animais , Proteínas Ligadas por GPI/metabolismo , Edição de Genes/métodos , Biblioteca Gênica , Vetores Genéticos , Estudo de Associação Genômica Ampla/métodos , Humanos , Lentivirus/genética , Camundongos , Fenótipo , RNA Guia , Transdução de Sinais
18.
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
19.
Pathol Res Pract ; 210(8): 533-40, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24947414

RESUMO

Our purpose was to investigate the expression pattern of BRCA1 protein in sporadic breast carcinomas, as well as the clinicopathological and prognostic value of its subcellular localizations. Immunohistochemistry was performed on paraffin embedded tissue specimens from 111 sporadic, invasive breast carcinomas to detect the expression of the proteins BRCA1, ER, PR, erbB2, p53 and Ki67. BRCA1 protein was detected in the nuclei and the cytoplasm of the tumor cells. Nuclear BRCA1 immunoreactivity showed no relation with the classic clinicopathological markers and the expression of cerbB2, p53 and Ki67. Reduced expression of nuclear BRCA1 protein was found to exert an independent favorable impact on both the overall and relapse-free (RF) survival of the patients (p=0.019 and p=0.043, respectively). Cytoplasmic BRCA1 was associated with none of the classic histomorphological indices, except from the lymph node metastasis, with which its relation was found to be inverse (p=0.05), prolonging the RF survival of the patients (p=0.05). Our findings suggest that BRCA1 protein depicts different prognostic significance, according to its subcellular distribution. Nuclear detection of the protein was associated with a worse prognosis, while the cytoplasmic one was related to fewer recurrences as a result of fewer lymph node metastases.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo
20.
Histopathology ; 62(3): 472-80, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23163571

RESUMO

AIMS: To study the clinicopathological and prognostic value of cyclin D1 overexpression in patients with breast carcinoma. METHODS AND RESULTS: Immunohistochemistry was performed on paraffin-embedded tissue specimens from 290 invasive breast carcinomas to detect the proteins cyclin D1, oestrogen receptor (ER), progesterone receptor (PR), p53, c-erbB2, and topoisomerase IIα (topoIIα). Cyclin D1 staining was quantified using a computerized image analysis method. Cyclin D1 overexpression characterized smaller, ER-positive and PR-positive tumours (P = 0.017, P < 0.0001, and P < 0.0001, respectively), of a lower histological and nuclear grade (P = 0.011 and P < 0.0001, respectively), and with reduced expression of topoIIα (P = 0.001) and p53 (P < 0.001). Cyclin D1 was found to have an independent favourable impact on the overall survival of both the unselected cohort of patients (P = 0.011) and of patients with ER-negative and lymph node-positive tumours (P = 0.034 and P = 0.015, respectively). In triple-negative tumours, cyclin D1 overexpression was found to have independent favourable impacts on both overall and relapse-free survival (P = 0.002 for both). CONCLUSIONS: This is the first immunohistochemical study to dissociate the advantageous prognostic effect of cyclin D1 overexpression from its association with ER expression, and to provide evidence that cyclin D1 overexpression may be a marker of prolonged survival in patient subgroups with aggressive phenotypes.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Ciclina D1/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/patologia , Ciclina D1/análise , Intervalo Livre de Doença , Feminino , Humanos , Interpretação de Imagem Assistida por Computador , Imuno-Histoquímica , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Fenótipo , Prognóstico , Modelos de Riscos Proporcionais , Receptores de Estrogênio/biossíntese
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