RESUMO
Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.
Assuntos
Aptâmeros de Nucleotídeos , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Inativação de Genes , Linfócitos T , Humanos , Edição de Genes/métodos , Aptâmeros de Nucleotídeos/genética , Linfócitos T/metabolismo , Linfócitos T/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Técnicas de Introdução de Genes/métodos , TransgenesRESUMO
The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.
Assuntos
Citidina/análogos & derivados , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , tRNA Metiltransferases/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular Tumoral , Uso do Códon , Sequência Consenso , Citidina/metabolismo , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Metilação , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Transcriptoma , tRNA Metiltransferases/deficiênciaRESUMO
In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-7R genes.
Assuntos
Interleucina-10/metabolismo , Ativação de Macrófagos , Fator de Transcrição MafB/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sequência de Bases , Linhagem Celular , Sondas de DNA , Inativação Gênica , Humanos , Fator de Transcrição MafB/genética , Metaloproteinase 7 da Matriz/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Receptores de Interleucina-7/genéticaRESUMO
BACKGROUND: ZFP36 is an mRNA binding protein that exerts anti-tumor activity in glioblastoma by triggering cell death, associated to an increase in the stability of the kinase RIP1. METHODS: We used cell death assays, size exclusion chromatography, Co-Immunoprecipitation, shRNA lentivectors and glioma neural stem cells to determine the effects of ZFP36 on the assembly of a death complex containing RIP1 and on the induction of necroptosis. RESULTS: Here we demonstrate that ZFP36 promotes the assembly of the death complex called Ripoptosome and induces RIP1-dependent death. This involves the depletion of the ubiquitine ligases cIAP2 and XIAP and leads to the association of RIP1 to caspase-8 and FADD. Moreover, we show that ZFP36 controls RIP1 levels in glioma neural stem cell lines. CONCLUSIONS: We provide a molecular mechanism for the tumor suppressor role of ZFP36, and the first evidence for Ripoptosome assembly following ZFP36 expression. These findings suggest that ZFP36 plays an important role in RIP1-dependent cell death in conditions where IAPs are depleted.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Tristetraprolina/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose , Proteína 3 com Repetições IAP de Baculovírus , Linhagem Celular Tumoral , Estabilidade Enzimática , Glioma/patologia , Células HEK293 , Humanos , Células-Tronco Neoplásicas/metabolismo , Multimerização Proteica , ProteóliseRESUMO
Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.
Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Orosomucoide/metabolismo , Vitamina D/farmacologia , Perfilação da Expressão Gênica , Células HL-60 , Humanos , Macrófagos/metabolismo , Orosomucoide/genética , Orosomucoide/isolamento & purificação , Células U937RESUMO
In recent years, there has been an increase in research efforts surrounding RNA modification thanks to key breakthroughs in NGS-based whole transcriptome mapping methods. More than 100 modifications have been reported in RNAs, and some have been mapped at single-nucleotide resolution in the mammalian transcriptome. This has opened new research avenues in fields such as neurobiology, developmental biology, and oncology, among others. To date, we know that the RNA modification machinery finely tunes many diverse mechanisms involved in RNA processing and translation to regulate gene expression. However, it appears obvious to the research community that we have only just begun the process of understanding the several functions of the dynamic web of RNA modification, or the "epitranscriptome". To expand the data generated so far, recently published studies revealed a dual role for N6-methyladenosine (m6A), the most abundant mRNA modification, in driving both chromatin dynamics and transcriptional output. These studies showed that the m6A-modified, chromatin-associated RNAs could act as molecular docks, recruiting histone modification proteins and thus contributing to the regulation of local chromatin structure. Here, we review these latest exciting findings and outline outstanding research questions whose answers will help to elucidate the biological relevance of the m6A modification of chromatin-associated RNAs in mammalian cells.
Assuntos
Adenosina , RNA , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Animais , Cromatina/genética , Mamíferos/genética , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNARESUMO
Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Despite available therapeutic interventions, it is very difficult to treat, and a cure is not yet available. The intra-tumoral GBM heterogeneity is a crucial factor contributing to poor clinical outcomes. GBM derives from a small heterogeneous population of cancer stem cells (CSCs). In cancer tissue, CSCs are concentrated within the so-called niches, where they progress from a slowly proliferating phase. CSCs, as most tumor cells, release extracellular vesicles (EVs) into the surrounding microenvironment. To explore the role of EVs in CSCs and GBM tumor cells, we investigated the miRNA and protein content of the small EVs (sEVs) secreted by two GBM-established cell lines and by GBM primary CSCs using omics analysis. Our data indicate that GBM-sEVs are selectively enriched for miRNAs that are known to display tumor suppressor activity, while their protein cargo is enriched for oncoproteins and tumor-associated proteins. Conversely, among the most up-regulated miRNAs in CSC-sEVs, we also found pro-tumor miRNAs and proteins related to stemness, cell proliferation, and apoptosis. Collectively, our findings support the hypothesis that sEVs selectively incorporate different miRNAs and proteins belonging both to fundamental processes (e.g., cell proliferation, cell death, stemness) as well as to more specialized ones (e.g., EMT, membrane docking, cell junction organization, ncRNA processing).
RESUMO
Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-pointTM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.
Assuntos
Aptâmeros de Nucleotídeos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Edição de RNA , Animais , Bactérias/genética , Bactérias/metabolismo , Sistemas CRISPR-Cas , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Mutação INDEL , RNA Guia de Cinetoplastídeos/genética , Reparo de DNA por Recombinação , Sequenciamento do ExomaRESUMO
Transcription Factor for Immunoglobulin Heavy-Chain Enhancer 3 (Tfe3) is a transactivator of metabolic genes that are regulated through an EBox located in their promoters. It is involved in physiological processes such as osteoclast and macrophage differentiation, as well as in pathological processes such as translocations underlying different cancer diseases. MAFB is a basic region/leucine zipper transcription factor that affects transcription by binding specific DNA regions known as MARE. It plays a pivotal role in regulating lineage-specific hematopoiesis by repressing transcription of erythroid specific genes in myeloid cells and enhancing expression of macrophage and megakaryocytic genes. Here we have shown MAFB to be highly induced in human hematopoietic cells undergoing macrophage differentiation following Tfe3 ectopic expression, and to be down regulated, compared to the controls, in the same cell population following Phorbol Esters (PMA) dependent differentiation coupled to Tfe3 gene silencing. Electrophoretic mobility shift assays identified a Tfe3-binding site (EBox) in the MAFB promoter region that is conserved in different mammalian species. MAFB promoter was transactivated by co-expression of Tfe3 in reporter gene assays while deletion or mutation of the MAFB EBox prevented transactivation by Tfe3. Both of these genes were previously included in the group of transcription factors able to drive macrophage differentiation. The observation that MAFB belongs to the Tfe3 regulon suggests the existence of a pathway where these two gene families act synergistically to determine differentiation.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Fator de Transcrição MafB/genética , Fator de Transcrição MafB/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Sítios de Ligação/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Primers do DNA/genética , Expressão Gênica , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Regiões Promotoras Genéticas , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional , Células U937RESUMO
BACKGROUND: The uneven use of synonymous codons in the transcriptome regulates the efficiency and fidelity of protein translation rates. Yet, the importance of this codon bias in regulating cell state-specific expression programmes is currently debated. Here, we ask whether different codon usage controls gene expression programmes in self-renewing and differentiating embryonic stem cells. RESULTS: Using ribosome and transcriptome profiling, we identify distinct codon signatures during human embryonic stem cell differentiation. We find that cell state-specific codon bias is determined by the guanine-cytosine (GC) content of differentially expressed genes. By measuring the codon frequencies at the ribosome active sites interacting with transfer RNAs (tRNA), we further discover that self-renewing cells optimize translation of codons that depend on the inosine tRNA modification in the anticodon wobble position. Accordingly, inosine levels are highest in human pluripotent embryonic stem cells. This effect is conserved in mice and is independent of the differentiation stimulus. CONCLUSIONS: We show that GC content influences cell state-specific mRNA levels, and we reveal how translational mechanisms based on tRNA modifications change codon usage in embryonic stem cells.
Assuntos
Códon , Células-Tronco Embrionárias/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Animais , Composição de Bases , Autorrenovação Celular , HumanosRESUMO
The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.
Assuntos
5-Metilcitosina/metabolismo , Metilação de DNA , Células Epidérmicas/citologia , Metiltransferases/metabolismo , RNA/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Diferenciação Celular , Linhagem Celular , Citosina/metabolismo , Células Epidérmicas/metabolismo , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Humanos , Metiltransferases/genética , RNA/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
Loss-of-function mutations in the cytosine-5 RNA methylase NSUN2 cause neurodevelopmental disorders in humans, yet the underlying cellular processes leading to the symptoms that include microcephaly remain unclear. Here, we show that NSUN2 is expressed in early neuroepithelial progenitors of the developing human brain, and its expression is gradually reduced during differentiation of human neuroepithelial stem (NES) cells in vitro. In the developing Nsun2-/- mouse cerebral cortex, intermediate progenitors accumulate and upper-layer neurons decrease. Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5' tRNA fragments accumulate in Nsun2-/- brains. Neural differentiation of NES cells is impaired by both NSUN2 depletion and the presence of angiogenin. Since repression of NSUN2 also inhibited neural cell migration toward the chemoattractant fibroblast growth factor 2, we conclude that the impaired differentiation capacity in the absence of NSUN2 may be driven by the inability to efficiently respond to growth factors.
Assuntos
Diferenciação Celular/genética , Citosina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , RNA/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Movimento Celular , Feminino , Técnicas de Inativação de Genes , Humanos , Metilação , Metiltransferases/genética , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Organogênese/genética , Ribonuclease Pancreático/farmacologiaRESUMO
BACKGROUND: Overexpression of microRNA-31 (miR-31) is implicated in the pathogenesis of esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary zinc deficiency. Using a rat model that recapitulates features of human ESCC, the mechanism whereby Zn regulates miR-31 expression to promote ESCC is examined. METHODS: To inhibit in vivo esophageal miR-31 overexpression in Zn-deficient rats (n = 12-20 per group), locked nucleic acid-modified anti-miR-31 oligonucleotides were administered over five weeks. miR-31 expression was determined by northern blotting, quantitative polymerase chain reaction, and in situ hybridization. Physiological miR-31 targets were identified by microarray analysis and verified by luciferase reporter assay. Cellular proliferation, apoptosis, and expression of inflammation genes were determined by immunoblotting, caspase assays, and immunohistochemistry. The miR-31 promoter in Zn-deficient esophagus was identified by ChIP-seq using an antibody for histone mark H3K4me3. Data were analyzed with t test and analysis of variance. All statistical tests were two-sided. RESULTS: In vivo, anti-miR-31 reduced miR-31 overexpression (P = .002) and suppressed the esophageal preneoplasia in Zn-deficient rats. At the same time, the miR-31 target Stk40 was derepressed, thereby inhibiting the STK40-NF-κΒ-controlled inflammatory pathway, with resultant decreased cellular proliferation and activated apoptosis (caspase 3/7 activities, fold change = 10.7, P = .005). This same connection between miR-31 overexpression and STK40/NF-κΒ expression was also documented in human ESCC cell lines. In Zn-deficient esophagus, the miR-31 promoter region and NF-κΒ binding site were activated. Zn replenishment restored the regulation of this genomic region and a normal esophageal phenotype. CONCLUSIONS: The data define the in vivo signaling pathway underlying interaction of Zn deficiency and miR-31 overexpression in esophageal neoplasia and provide a mechanistic rationale for miR-31 as a therapeutic target for ESCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , MicroRNAs/antagonistas & inibidores , Oligonucleotídeos/farmacologia , Transdução de Sinais , Animais , Apoptose , Northern Blotting , Carcinoma de Células Escamosas/genética , Proliferação de Células , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Imuno-Histoquímica , Inflamação , Masculino , Análise em Microsséries , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase/métodos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Regulação para Cima , Zinco/deficiênciaRESUMO
RNA binding proteins belonging to the TIS11/TTP gene family regulate the stability of multiple targets. Their inactivation or deregulated expression has recently been related to cancer, and it has been suggested that they are capable of displaying tumor suppressor activities. Here we describe three new targets of ZFP36 (PIM-1, PIM-3 and XIAP) and show by different approaches that its ectopic expression is capable of impairing glioblastoma cell lines viability and invasiveness by interfering with different transduction pathways. Moreover, we provide evidence that compounds capable of inducing the expression of TIS11/TTP genes determine a comparable biological effect on the same cell contexts.
Assuntos
Transdução de Sinais , Tristetraprolina/metabolismo , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Proteínas Fúngicas , Glioblastoma/metabolismo , Glioblastoma/patologia , Células HEK293 , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Polifenóis/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Tristetraprolina/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3'-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by posttranscriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3' untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, because cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.
Assuntos
Antígenos CD34/metabolismo , Fator 1 de Resposta a Butirato/metabolismo , Diferenciação Celular , Células Eritroides/citologia , Células-Tronco Hematopoéticas/citologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas/genética , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Cinnamomum zeylanicum/química , Regulação para Baixo/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Sangue Fetal/citologia , Flavonoides/farmacologia , Inativação Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Polifenóis , Ligação Proteica/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tristetraprolina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.