RESUMO
The Hippo signaling pathway is commonly dysregulated in human cancer, which leads to a powerful tumor dependency on the YAP/TAZ transcriptional coactivators. Here, we used paralog co-targeting CRISPR screens to identify the kinases MARK2/3 as absolute catalytic requirements for YAP/TAZ function in diverse carcinoma and sarcoma contexts. Underlying this observation is direct MARK2/3-dependent phosphorylation of NF2 and YAP/TAZ, which effectively reverses the tumor suppressive activity of the Hippo module kinases LATS1/2. To simulate targeting of MARK2/3, we adapted the CagA protein from H. pylori as a catalytic inhibitor of MARK2/3, which we show can regress established tumors in vivo. Together, these findings reveal MARK2/3 as powerful co-dependencies of YAP/TAZ in human cancer; targets that may allow for pharmacology that restores Hippo pathway-mediated tumor suppression.
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The presence of basal lineage characteristics signifies hyperaggressive human adenocarcinomas of the breast, bladder and pancreas. However, the biochemical mechanisms that maintain this aberrant cell state are poorly understood. Here we performed marker-based genetic screens in search of factors needed to maintain basal identity in pancreatic ductal adenocarcinoma (PDAC). This approach revealed MED12 as a powerful regulator of the basal cell state in this disease. Using biochemical reconstitution and epigenomics, we show that MED12 carries out this function by bridging the transcription factor ΔNp63, a known master regulator of the basal lineage, with the Mediator complex to activate lineage-specific enhancer elements. Consistent with this finding, the growth of basal-like PDAC is hypersensitive to MED12 loss when compared to PDAC cells lacking basal characteristics. Taken together, our genetic screens have revealed a biochemical interaction that sustains basal identity in human cancer, which could serve as a target for tumor lineage-directed therapeutics.
Assuntos
Carcinoma Ductal Pancreático , Complexo Mediador , Neoplasias Pancreáticas , Fatores de Transcrição , Proteínas Supressoras de Tumor , Humanos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Linhagem da Célula/genética , Elementos Facilitadores GenéticosRESUMO
The presence of basal lineage characteristics signifies hyper-aggressive human adenocarcinomas of the breast, bladder, and pancreas. However, the biochemical mechanisms that maintain this aberrant cell state are poorly understood. Here we performed marker-based genetic screens in search of factors needed to maintain basal identity in pancreatic ductal adenocarcinoma (PDAC). This approach revealed MED12 as a powerful regulator of the basal cell state in this disease. Using biochemical reconstitution and epigenomics, we show that MED12 carries out this function by bridging the transcription factor p63, a known master regulator of the basal lineage, with the Mediator complex to activate lineage-specific enhancer elements. Consistent with this finding, the growth of basal-like PDAC is hypersensitive to MED12 loss when compared to classical PDAC. Taken together, our comprehensive genetic screens have revealed a biochemical interaction that sustains basal identity in human cancer, which could serve as a target for tumor lineage-directed therapeutics.
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Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.
Assuntos
Rabdomiossarcoma , Fator de Ligação a CCAAT/genética , Diferenciação Celular/genética , Aberrações Cromossômicas , Rabdomiossarcoma/genética , Fatores de TranscriçãoRESUMO
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.
Assuntos
Neoplasias , Rabdomiossarcoma Alveolar , Rabdomiossarcoma Embrionário , Biologia , Criança , DNA Helicases/genética , Humanos , Proteínas Nucleares/genética , Rabdomiossarcoma Alveolar/genética , Fatores de Transcrição/genéticaRESUMO
An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate-limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hypermethylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme. SIGNIFICANCE: We show how epigenetic silencing can provoke a nutrient dependency in AML by exploiting a synthetic lethality relationship between biosynthesis and transport of myo-inositol. Blocking the function of this solute carrier may have therapeutic potential in an epigenetically defined subset of AML.This article is highlighted in the In This Issue feature, p. 275.
Assuntos
Proteínas de Choque Térmico/genética , Inositol/biossíntese , Leucemia Mieloide Aguda/tratamento farmacológico , Simportadores/genética , Animais , Biologia do Desenvolvimento , Humanos , CamundongosRESUMO
In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time (15)N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [(15)N]Glu or (15)NH(4)(+) respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.
Assuntos
Glutamato Desidrogenase/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Glutamato Desidrogenase/genética , Ácido Glutâmico/biossíntese , Espectroscopia de Ressonância Magnética , Nitrogênio/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Compostos de Amônio Quaternário/metabolismo , Nicotiana/genéticaRESUMO
Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the beta-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the alpha-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the alpha-subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of (15)NH(4) or [(15)N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities.
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Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Nicotiana/enzimologia , Vitis/genética , Ânions/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glutamato Desidrogenase/genética , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/metabolismo , Subunidades Proteicas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Transcrição GênicaRESUMO
Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the alpha-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the alpha-subunit of GDH, increase in immunoreactive alpha-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy-mass spectrometry monitoring of (15)N-Glu, (15)N-Gln, and (15)N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of alpha-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive alpha-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces alpha-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/biossíntese , Prolina/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Aminação , Ânions , Fragmentação do DNA , Cromatografia Gasosa-Espectrometria de Massas , Glutamato Desidrogenase/genética , Dados de Sequência Molecular , Cloreto de Sódio/metabolismoRESUMO
Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (~51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.