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1.
J Exp Clin Cancer Res ; 41(1): 340, 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36482393

RESUMO

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). METHODS: We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). RESULTS: eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. CONCLUSIONS: We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.


Assuntos
Antineoplásicos , Citarabina , Fator de Iniciação 4A em Eucariotos , Leucemia Mieloide Aguda , Humanos , Citarabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2 , Linhagem Celular Tumoral , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Antineoplásicos/farmacologia
2.
Proc Natl Acad Sci U S A ; 116(8): 2925-2934, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718434

RESUMO

Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5'UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in the mRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics.


Assuntos
Reprogramação Celular/genética , Metabolismo Energético/genética , Neoplasias/genética , Proteínas Tirosina Fosfatases/genética , Quinases Proteína-Quinases Ativadas por AMP , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions , Proteínas de Ciclo Celular/genética , Ciclinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Regeneração Hepática/genética , Células MCF-7 , Magnésio/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias/patologia , Proteínas Quinases/genética
3.
Crit Rev Oncol Hematol ; 134: 65-70, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30771875

RESUMO

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the western world. CLL consists of the accumulation of malignant B-cells in the blood stream and homing tissues. Although treatable, this disease is not curable, and resistance or relapse is often present. In many cancers, the study of metabolic reprograming has uncovered novel targets that are already being exploited in the clinic. However, CLL metabolism is still poorly understood. The ability of CLL lymphocytes to adapt to diverse microenvironments is accompanied by modifications in cell metabolism, revealing the challenge of targeting the CLL lymphocytes present in all different compartments. Despite this, the study of CLL metabolism led to an ongoing clinical trial using glucose uptake and mitochondrial respiration inhibitors. In contrast, glutamine and fatty acid metabolism remain to be further exploited in CLL. Here, we summarize the present knowledge of CLL metabolism, as well as the metabolic influence of Myc, ATM and p53 on CLL lymphocytes.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Respiração Celular , Humanos
4.
Front Oncol ; 8: 411, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319974

RESUMO

Chronic Lymphocytic Leukemia (CLL) is an incurable disease, characterized by the accumulation of malignant B-lymphocytes in the blood stream (quiescent state) and homing tissues (where they can proliferate). In CLL, the targeting of B-cell receptor signaling through a Burton's tyrosine kinase inhibitor (ibrutinib) has rendered outstanding clinical results. However, complete remission is not guaranteed due to drug resistance or relapse, revealing the need for novel approaches for CLL treatment. The characterization of metabolic rewiring in proliferative cancer cells is already being applied for diagnostic and therapeutic purposes, but our knowledge of quiescent cell metabolism-relevant for CLL cells-is still fragmentary. Recently, we reported that glutamine metabolism in primary CLL cells bearing the del11q deletion is different from their del11q negative counterparts, making del11q cells especially sensitive to glutaminase and glycolysis inhibitors. In this work, we used our primary CLL lymphocyte bank and compounds interfering with central carbon metabolism to define metabolic traits associated with ibrutinib resistance. We observe a differential basal metabolite uptake linked to ibrutinib resistance, favoring glutamine uptake and catabolism. Upon ibrutinib treatment, the redox balance in ibrutinib resistant cells is shifted toward NADPH accumulation, without an increase in glutamine uptake, suggesting alternative metabolic rewiring such as the activation of fatty acid oxidation. In accordance to this idea, the curtailing of fatty acid oxidation by CPT1 inhibition (etomoxir) re-sensitized resistant cells to ibrutinib. Our results suggest that fatty acid oxidation could be explored as a target to overcome ibrutinib resistance.

6.
Cell Rep ; 15(11): 2340-7, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27239032

RESUMO

Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition.


Assuntos
Benzofuranos/farmacologia , Sistemas CRISPR-Cas/genética , Sistemas de Liberação de Medicamentos , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Alelos , Animais , Sequência de Bases , Resistência a Medicamentos/efeitos dos fármacos , Fator de Iniciação 4A em Eucariotos/metabolismo , Feminino , Loci Gênicos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação/genética , Células NIH 3T3 , Reprodutibilidade dos Testes , Triterpenos/farmacologia
7.
RNA ; 21(10): 1826-33, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26286746

RESUMO

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through partial complementary base-pairing to the 3' untranslated region (UTR) of target mRNAs. Inhibition of translation initiation has been identified as an early event of miRNA-mediated gene repression, but the underlying mechanistic details of this process are not well understood. Recently, eukaryotic initiation factor (eIF) 4AII was identified as a critical modulator of miRNA activity with depletion of this factor alleviating miRNA-mediated gene repression. Using the CRISPR/Cas9-editing system, we generated a novel cell line in which expression of eIF4AII was eliminated. The absence of eIF4AII does not affect cell viability, proliferation, or global mRNA translation. Importantly, we show that eIF4AII is dispensable for miRNA-mediated gene silencing.


Assuntos
Fator de Iniciação 4A em Eucariotos/fisiologia , Inativação Gênica/fisiologia , MicroRNAs/fisiologia , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Camundongos , Células NIH 3T3
8.
Immunity ; 42(1): 41-54, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25607458

RESUMO

Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Adaptação Fisiológica/imunologia , Animais , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Metabolismo Energético , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Imunomodulação , Ativação Linfocitária/genética , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Biossíntese de Proteínas/genética
9.
PLoS One ; 9(1): e87237, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24466343

RESUMO

Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.


Assuntos
Diferenciação Celular , Fator de Iniciação 4A em Eucariotos/metabolismo , Regulação da Expressão Gênica , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Animais , Células Cultivadas , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/genética , Imunofluorescência , Immunoblotting , Imunoprecipitação , Camundongos , Proteína MyoD/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
ACS Chem Biol ; 8(7): 1519-27, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23614532

RESUMO

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.


Assuntos
Benzofuranos/metabolismo , Fator de Iniciação 4F em Eucariotos/química , Fator de Iniciação 4F em Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Benzofuranos/química , Sítios de Ligação , Modelos Biológicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Triterpenos/química , Triterpenos/metabolismo
11.
Methods Enzymol ; 511: 437-61, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22713332

RESUMO

The RNA helicases eIF4AI and eIF4AII play key roles in recruiting ribosomes to mRNA templates during eukaryotic translation initiation. Small molecule inhibitors of eIF4AI and eIF4AII have been useful for chemically dissecting their role in translation in vitro and in vivo. Here, we describe a screen performed on a small focused library of kinase inhibitors to identify a novel helicase inhibitor. We describe assays that have been critical for characterizing novel RNA helicase inhibitors.


Assuntos
Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Antracenos , Benzofuranos/farmacologia , Compostos de Epóxi/farmacologia , Humanos , Macrolídeos/farmacologia , Perileno/análogos & derivados , Perileno/farmacologia , Esteróis/farmacologia , Tiazóis/farmacologia
12.
RNA ; 18(7): 1373-84, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22589333

RESUMO

The recruitment of ribosomes to eukaryotic cellular mRNAs requires the activity of two prototypic RNA helicases, eukaryotic initiation factor (eIF) 4AI and eIF4AII. The eIF4A isoforms are highly conserved, are thought to be functionally interchangeable, and are directed to the 5' m(7)GpppN cap structure of mRNAs during translation initiation by virtue of their assembly into eIF4F, a heterotrimeric complex that also harbors the eIF4E cap binding protein and eIF4G scaffolding unit. During the course of RNA interference experiments aimed at investigating the respective roles of eIF4AI and eIF4AII in translation, we uncovered a cellular response pathway whereby suppression of eIF4AI increases transcription of the eIF4AII gene, leading to elevated eIF4AII mRNA and protein levels. Inhibition of eIF4AI suppresses protein synthesis, and although eIF4AII protein levels increase above and beyond what should be sufficient to compensate for the decrease in eIF4AI levels, there is no corresponding rescue of translation or of the block on cellular proliferation that occurs upon eIF4AI suppression. These results were phenocopied using the small molecule eIF4A inhibitor hippuristanol. Taken together, our results indicate that eIF4AI and eIF4AII expression appear linked and that the two protein isoforms exhibit functional differences.


Assuntos
Fator de Iniciação 4A em Eucariotos/metabolismo , Transcrição Gênica/fisiologia , Adulto , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Próstata/efeitos dos fármacos , Próstata/metabolismo , Isoformas de Proteínas/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Esteróis/farmacologia , Testículo/efeitos dos fármacos , Testículo/metabolismo , Timo/efeitos dos fármacos , Timo/metabolismo , Transcrição Gênica/efeitos dos fármacos
13.
PLoS One ; 4(4): e5223, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19401772

RESUMO

BACKGROUND: Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. METHODOLOGY/PRINCIPAL FINDINGS: Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. CONCLUSIONS/SIGNIFICANCE: Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Triterpenos/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/prevenção & controle , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transplante Heterólogo , Triterpenos/química
14.
Anal Biochem ; 384(1): 180-8, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18926791

RESUMO

Protein-RNA interactions are involved in all facets of RNA biology. The identification of small molecules that selectively block such bimolecular interactions could provide insight into previously unexplored steps of gene regulation. Such is the case for regulation of eukaryotic protein synthesis where interactions between messenger RNA (mRNA) and several eukaryotic initiation factors govern the recruitment of 40S ribosomes (and associated factors) to mRNA templates during the initiation phase. We have designed simple fluorescence polarization-based high-throughput screening assays that query the binding of several translation factors to RNA and found that the mixed inhibitor p-chloromercuribenzoate interferes with poly(A) binding protein-RNA interaction.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Polarização de Fluorescência/métodos , RNA/metabolismo , Fatores de Iniciação em Eucariotos/análise , Humanos , Proteínas de Ligação a Poli(A)/metabolismo , RNA/análise , Ácido p-Cloromercurobenzoico/química
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