RESUMO
Angiogenesis and MYC expression associate with poor outcome in diffuse large B-cell lymphoma (DLBCL). MYC promotes neo-vasculature development but whether its deregulation in DLBCL contributes to angiogenesis is unclear. Examination of this relationship may uncover novel pathogenic regulatory circuitry as well as anti-angiogenic strategies in DLBCL. Here, we show that MYC expression positively correlates with vascular endothelial growth factor (VEGF) expression and angiogenesis in primary DLBCL biopsies, independently of dual expressor status or cell-of-origin classification. We found that MYC promotes VEGFA expression, a correlation that was validated in large datasets of mature B-cell tumours. Using DLBCL cell lines and patient-derived xenograft models, we identified the second messenger cyclic-AMP (cAMP) as a potent suppressor of MYC expression, VEGFA secretion and angiogenesis in DLBCL in normoxia. In hypoxia, cAMP switched targets and suppressed hypoxia-inducible factor 1α, a master regulator of VEGFA/angiogenesis in low oxygen environments. Lastly, we used the phosphodiesterase 4b (Pde4b) knockout mouse to demonstrate that the cAMP/PDE4 axis exercises additional anti-angiogenesis by directly targeting the lymphoma microenvironment. In conclusion, MYC could play a direct role in DLBCL angiogenesis, and modulation of cAMP levels, which can be achieved with clinical grade PDE4 inhibitors, has cell and non-cell autonomous anti-angiogenic activity in DLBCL.
Assuntos
AMP Cíclico , Subunidade alfa do Fator 1 Induzível por Hipóxia , Linfoma Difuso de Grandes Células B , Proteínas Proto-Oncogênicas c-myc , Monofosfato de Adenosina , Animais , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The characterization of immunoglobulin heavy chain (IGH) translocations provides information on the diagnosis and guides therapeutic decisions in mature B-cell malignancies while enhancing our understanding of normal and malignant B-cell biology. However, existing methodologies for the detection of IGH translocations are labor intensive, often require viable cells, and are biased toward known IGH fusions. To overcome these limitations, we developed a capture sequencing strategy for the identification of IGH rearrangements at nucleotide level resolution and tested its capabilities as a diagnostic and discovery tool in 78 primary diffuse large B-cell lymphomas (DLBCLs). We readily identified IGH-BCL2, IGH-BCL6, IGH-MYC, and IGH-CCND1 fusions and discovered IRF8, EBF1, and TNFSF13 (APRIL) as novel IGH partners in these tumors. IRF8 and TNFSF13 expression was significantly higher in lymphomas with IGH rearrangements targeting these loci. Modeling the deregulation of IRF8 and EBF1 in vitro defined a lymphomagenic profile characterized by up-regulation of AID and/or BCL6, down-regulation of PRMD1, and resistance to apoptosis. Using a capture sequencing strategy, we discovered the B-cell relevant genes IRF8, EBF1, and TNFSF13 as novel targets for IGH deregulation. This methodology is poised to change how IGH translocations are identified in clinical settings while remaining a powerful tool to uncover the pathogenesis of B-cell malignancies.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Fatores Reguladores de Interferon/genética , Linfoma de Células B/genética , Proteínas de Fusão Oncogênica/genética , Transativadores/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Sequência de Bases , Linhagem Celular Tumoral , Biblioteca Gênica , Rearranjo Gênico do Linfócito B/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Fatores Reguladores de Interferon/fisiologia , Dados de Sequência Molecular , Transativadores/fisiologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/fisiologia , Estudos de Validação como AssuntoRESUMO
Constitutive activation of the NF-κB pathway is associated with diffuse large B-cell lymphoma (DLBCL) pathogenesis, but whether microRNA dysfunction can contribute to these events remains unclear. Starting from an integrative screening strategy, we uncovered that the negative NF-κB regulator TNFAIP3 is a direct target of miR-125a and miR-125b, which are commonly gained and/or overexpressed in DLBCL. Ectopic expression of these microRNAs in multiple cell models enhanced K63-linked ubiquitination of proximal signaling complexes and elevated NF-κB activity, leading to aberrant expression of its transcriptional targets and the development of a proproliferative and antiapoptotic phenotype in malignant B cells. Concordantly, genetic inhibition of miR-125a/miR-125b blunted NF-κB signals, whereas rescue assays and genetic modulation of a TNFAIP3-null model defined the essential role of the TNFAIP3 targeting on miR-125a/miR-125b-mediated lymphomagenesis. Importantly, miR-125a/mir-125b effects on TNFAIP3 expression and NF-κB activity were confirmed in a well-characterized cohort of primary DLBCLs. Our data delineate a unique epigenetic model for aberrant activation of the NF-κB pathway in cancer and provide a coherent mechanism for the role of these miRNAs in immune cell activation and hematopoiesis. Further, as miR-125b is a direct NF-κB transcriptional target, our results suggest the presence of a positive self-regulatory loop whereby termination of TNFAIP3 function by miR-125 could strengthen and prolong NF-κB activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Imunoprecipitação , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não Paramétricas , Frações Subcelulares , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
The mechanism by which interferon regulatory factor 8 (IRF8) mutation contributes to lymphomagenesis is unknown. We modeled IRF8 variants in B cell lymphomas and found that they affected the expression of regulators of antigen presentation. Expression of IRF8 mutants in murine B cell lymphomas suppressed CD4, but not CD8, activation elicited by antigen presentation and downmodulated CD74 and human leukocyte antigen (HLA) DM, intracellular regulators of antigen peptide processing/loading in the major histocompatibility complex (MHC) II. Concordantly, mutant IRF8 bound less efficiently to the promoters of these genes. Mice harboring IRF8 mutant lymphomas displayed higher tumor burden and remodeling of the tumor microenvironment, typified by depletion of CD4, CD8, and natural killer cells, increase in regulatory T cells and T follicular helper cells. Deconvolution of bulk RNA sequencing data from IRF8-mutant human diffuse large B cell lymphoma (DLBCL) recapitulated part of the immune remodeling detected in mice. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.
Assuntos
Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B , Antígenos de Histocompatibilidade Classe II , Fatores Reguladores de Interferon , Mutação , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Animais , Apresentação de Antígeno/imunologia , Apresentação de Antígeno/genética , Humanos , Camundongos , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Microambiente Tumoral/imunologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Linhagem Celular Tumoral , Evasão Tumoral/genética , Regulação Neoplásica da Expressão GênicaRESUMO
In diffuse large B-cell lymphoma (DLBCL), the transcription factor IRF8 is the target of a series of potentially oncogenic events, including, chromosomal translocation, focal amplification, and super-enhancer perturbations. IRF8 is also frequently mutant in DLBCL, but how these variants contribute to lymphomagenesis is unknown. We modeled IRF8 mutations in DLBCL and found that they did not meaningfully impact cell fitness. Instead, IRF8 mutants, mapping either to the DNA-binding domain (DBD) or c-terminal tail, displayed diminished transcription activity towards CIITA, a direct IRF8 target. In primary DLBCL, IRF8 mutations were mutually exclusive with mutations in genes involved in antigen presentation. Concordantly, expression of IRF8 mutants in murine B cell lymphomas uniformly suppressed CD4, but not CD8, activation elicited by antigen presentation. Unexpectedly, IRF8 mutation did not modify MHC CII expression on the cell surface, rather it downmodulated CD74 and HLA- DM, intracellular regulators of antigen peptide processing/loading in the MHC CII complex. These changes were functionally relevant as, in comparison to IRF8 WT, mice harboring IRF8 mutant lymphomas displayed a significantly higher tumor burden, in association with a substantial remodeling of the tumor microenvironment (TME), typified by depletion of CD4, CD8, Th1 and NK cells, and increase in T-regs and Tfh cells. Importantly, the clinical and immune phenotypes of IRF8-mutant lymphomas were rescued in vivo by ectopic expression of CD74. Deconvolution of bulk RNAseq data from primary human DLBCL recapitulated part of the immune remodeling detected in mice and pointed to depletion of dendritic cells as another feature of IRF8 mutant TME. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.
RESUMO
Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2Δ or fum1Δ) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ~8-fold in the sdh2Δ mutant and ~4-fold in the fum1Δ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2Δ and fum1Δ strains also exhibited 3-4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway. Co-disruption of the SFC1 gene encoding the mitochondrial succinate/fumarate transporter resulted in higher relative mitochondrial levels of succinate and in substantial reductions of Cit2 expression in sdh2Δsfc1Δ and fum1Δsfc1Δ strains as compared with sdh2Δ and fum1Δ strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2Δ and fum1Δ strains. A defect (rtg1Δ) in the yeast retrograde response pathway, which controls expression of several mitochondrial proteins and Cit2, eliminated expression of Cit2 and reduced expression of NAD-specific isocitrate dehydrogenase (Idh) and aconitase (Aco1) in parental, sdh2Δ, and fum1Δ strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2Δ and fum1Δ strains, of fumarate in the fum1Δ strain, and citrate in an idhΔ strain. Thus, the retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in the parental strain and for metabolite accumulation in TCA cycle mutants.
Assuntos
Ciclo do Ácido Cítrico , Mitocôndrias/metabolismo , Mutação , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Transporte Biológico Ativo/fisiologia , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Técnicas de Silenciamento de Genes , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismoRESUMO
Mitochondria can function as signaling organelles, and part of this output leads to epigenetic remodeling. The full extent of this far-reaching interplay remains undefined. Here, we show that MYC transcriptionally activates IDH2 and increases alpha-ketoglutarate (αKG) levels. This regulatory step induces the activity of αKG-dependent DNA hydroxylases and RNA demethylases, thus reducing global DNA and RNA methylation. MYC, in a IDH2-dependent manner, also promotes the nuclear accumulation of TET1-TET2-TET3, FTO and ALKBH5. Notably, this subcellular movement correlated with the ability of MYC, in an IDH2-dependent manner, and, unexpectedly, of αKG to directly induce O-GlcNAcylation. Concordantly, modulation of the activity of OGT and OGA, enzymes that control the cycling of this non-canonical mono-glycosylation, largely recapitulated the effects of the MYC-IDH2-αKG axis on the subcellular movement of DNA and RNA demethylases. Together, we uncovered a hitherto unsuspected crosstalk between MYC, αKG and O-GlcNAcylation which could influence the epigenome and epitranscriptome homeostasis.
Assuntos
Metilação de DNA , RNA , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Mitocôndrias/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD(+) binding sites, and four AMP binding sites, only half of the sites for each ligand can be measured in binding assays. On the basis of a potential interaction between side chains of Cys-150 residues in IDH2 subunits in each tetramer of the enzyme, ligand binding assays of wild-type (IDH1/IDH2) and IDH1/IDH2(C150S) octameric enzymes were conducted in the presence of dithiothreitol. These assays demonstrated the presence of eight isocitrate and four AMP binding sites for the wild-type enzyme in the presence of dithiothreitol and for the IDH1/IDH2(C150S) enzyme in the absence or presence of this reagent, suggesting that interactions between sulfhydryl side chains of IDH2 Cys-150 residues limit access to these sites. However, only two NAD(+) sites could be measured for either enzyme. A tetrameric form of IDH (an IDH1(G15D)/IDH2 mutant enzyme) demonstrated half-site binding for isocitrate (two sites) in the absence of dithiothreitol and full-site binding (four sites) in the presence of dithiothreitol. Only one NAD(+) site could be measured for the tetramer under both conditions. In the context of the structure of the enzyme, these results suggest that an observed asymmetry between heterotetramers in the holoenzyme contributes to interactions between IDH2 Cys-150 residues and to half-site binding of isocitrate, but that a form of negative cooperativity may limit access to apparently equivalent NAD(+) binding sites.
Assuntos
Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Monofosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Diamida/farmacologia , Dissulfetos/química , Ditiotreitol/farmacologia , Isocitrato Desidrogenase/genética , Isocitratos/metabolismo , Cinética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , NAD/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation with an IDH1 G15D or IDH1 D168K residue substitution produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited â¼50% reductions in V(max) and in cooperativity with respect to isocitrate relative to those of the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated (-5)IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfide bond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit of one tetramer to the IDH2 Cys-150 residues in the other tetramer.
Assuntos
Isocitrato Desidrogenase/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Diamida/química , Dissulfetos/química , Isocitrato Desidrogenase/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Subunidades Proteicas/química , Saccharomyces cerevisiae/genéticaRESUMO
Cyclic-AMP (cAMP) exerts suppressive effects in the innate and adaptive immune system. The PD-1/PD-L1 immune checkpoint downregulates T-cell activity. Here, we examined if these two immunosuppressive nodes intersect. Using normal and malignant lymphocytes from humans, and the phosphodiesterase 4b (Pde4b) knockout mouse, we found that cAMP induces PD-L1 transcription and protein expression. Mechanistically, we discovered that the cAMP effectors PKA and CREB induce the transcription/secretion of IL-10, IL-8, and IL-6, which initiate an autocrine loop that activates the JAK/STAT pathway and ultimately increase PD-L1 expression in the cell surface. This signaling axis is disarmed at two specific nodes in subsets of diffuse large B-cell lymphoma, which may help explain the variable PD-L1 expression in these tumors. In vivo, we found that despite its immunosuppressive attributes, the PDE4 inhibitor roflumilast did not decrease the clinical activity of checkpoint inhibitors, an important clinical observation given the approved use of these agents in multiple diseases. In summary, we discovered that PD-L1 induction is a part of the repertoire of immunosuppressive actions mediated by cAMP, defined a cytokine-mediated autocrine loop that executes this action and, reassuringly, showed that PDE4 inhibition does not antagonize immune checkpoint blockade in an in vivo syngeneic lymphoma model.
Assuntos
Antígeno B7-H1/genética , AMP Cíclico/genética , Tolerância Imunológica/genética , Aminopiridinas/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Ciclopropanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Tolerância Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Camundongos , Camundongos Knockout , Inibidores da Fosfodiesterase 4/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
In mature B-cell malignancies, chromosomal translocations often juxtapose an oncogenic locus to the regulatory regions of the immunoglobulin genes. These genomic rearrangements can associate with specific clinical/pathological sub-entities and inform diagnosis and treatment decisions. Recently, we characterized the t(14;16)(q32;q24) in diffuse large B-cell lymphoma (DLBCL), and showed that it targets the transcription factor IRF8, which is also somatically mutated in ~10% of DLBCLs. IRF8 regulates innate and adaptive immune responses mediated by myeloid/monocytic and lymphoid cells. While the role of IRF8 in human myeloid/dendritic-cell disorders is well established, less is known of its contribution to the pathogenesis of mature B-cell malignancies. To address this knowledge gap, we generated the Eµ-Irf8 mouse model, which mimics the IRF8 deregulation associated with t(14;16) of DLBCL. Eµ-Irf8 mice develop normally and display peripheral blood cell parameters within normal range. However, Eµ-Irf8 mice accumulate pre-pro-B-cells and transitional B-cells in the bone marrow and spleen, respectively, suggesting that the physiological role of Irf8 in B-cell development is amplified. Notably, in Eµ-Irf8 mice, the lymphomagenic Irf8 targets Aicda and Bcl6 are overexpressed in mature B-cells. Yet, the incidence of B-cell lymphomas is not increased in the Eµ-Irf8 model, even though their estimated survival probability is significantly lower than that of WT controls. Together, these observations suggest that the penetrance on the Irf8-driven phenotype may be incomplete and that introduction of second genetic hit, a common strategy in mouse models of lymphoma, may be necessary to uncover the pro-lymphoma phenotype of the Eµ-Irf8 mice.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Fatores Reguladores de Interferon/genética , Linfoma de Células B/mortalidade , Proteínas de Fusão Oncogênica/genética , Animais , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Feminino , Humanos , Linfoma de Células B/genética , Masculino , Camundongos , Análise de SobrevidaRESUMO
Mitochondrial D2HGDH and L2HGDH catalyze the oxidation of D-2-HG and L-2-HG, respectively, into αKG. This contributes to cellular homeostasis in part by modulating the activity of αKG-dependent dioxygenases. Signals that control the expression/activity of D2HGDH/L2HGDH are presumed to broadly influence physiology and pathology. Using cell and mouse models, we discovered that MYC directly induces D2HGDH and L2HGDH transcription. Furthermore, in a manner suggestive of D2HGDH, L2HGDH, and αKG dependency, MYC activates TET enzymes and RNA demethylases, and promotes their nuclear localization. Consistent with these observations, in primary B cell lymphomas MYC expression positively correlated with enhancer hypomethylation and overexpression of lymphomagenic genes. Together, these data provide additional evidence for the role of mitochondria metabolism in influencing the epigenome and epitranscriptome, and imply that in specific contexts wild-type TET enzymes could demethylate and activate oncogenic enhancers.
Assuntos
Oxirredutases do Álcool/genética , Epigenoma , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-myc/genética , Ativação Transcricional , Animais , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Transcriptoma , Células Tumorais CultivadasRESUMO
The tricarboxylic acid cycle NAD+-specific isocitrate dehydrogenase (IDH) of Saccharomyces cerevisiae is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. Recent structural analyses revealed the close proximity of Cys-150 residues from IDH2 in adjacent heterodimers, and features of the structure for the ligand-free enzyme suggested that formation of a disulfide bond between these residues might stabilize an inactive form of the enzyme. We constructed two mutant forms of IDH, one containing a C150S substitution in IDH2 and the other containing C56S/C242S substitutions in IDH2 leaving Cys-150 as the sole cysteine residue. Treatment of the affinity-purified enzymes with diamide resulted in the formation of disulfide bonds and in decreased activities for the wild-type and C56S/C242S enzymes. Both effects were reversible by the addition of dithiothreitol. Diamide had no effect on the C150S mutant enzyme, suggesting that Cys-150 is essential for the formation of a disulfide bond that inhibits IDH activity. Diamide-induced formation of the Cys-150 disulfide bond was also observed in vivo for yeast transformants expressing the wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. Finally, natural formation of the Cys-150 disulfide bond with a concomitant decrease in cellular IDH activity was observed during the stationary phase for the parental strain and for transformants expressing wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. A reduction in viability for the latter strain suggests that a decrease in IDH activity is important for metabolic changes in stationary phase cells.
Assuntos
Dissulfetos/metabolismo , Isocitrato Desidrogenase/química , Saccharomyces cerevisiae/enzimologia , Catálise , Cisteína/química , Dissulfetos/química , Regulação para Baixo/genética , Estabilidade Enzimática/genética , Ligação de Hidrogênio , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , NAD/biossíntese , NAD/química , Saccharomyces cerevisiae/genéticaRESUMO
Yeast mutants lacking mitochondrial NAD(+)-specific isocitrate dehydrogenase (idhDelta) or aconitase (aco1Delta) were found to share several growth phenotypes as well as patterns of specific protein expression that differed from the parental strain. These shared properties of idhDelta and aco1Delta strains were eliminated or moderated by co-disruption of the CIT1 gene encoding mitochondrial citrate synthase. Gas chromatography/mass spectrometry analyses indicated a particularly dramatic increase in cellular citrate levels in idhDelta and aco1Delta strains, whereas citrate levels were substantially lower in idhDeltacit1Delta and aco1Deltacit1Delta strains. Exogenous addition of citrate to parental strain cultures partially recapitulated effects of high endogenous levels of citrate in idhDelta and aco1Delta strains. Finally, effects of elevated cellular citrate in idhDelta and aco1Delta mutant strains were partially alleviated by addition of iron or by an increase in pH of the growth medium, suggesting that detrimental effects of citrate are due to elevated levels of the ionized form of this metabolite.
Assuntos
Aconitato Hidratase/metabolismo , Citrato (si)-Sintase/metabolismo , Isocitrato Desidrogenase/metabolismo , Saccharomyces cerevisiae/enzimologia , Aconitato Hidratase/genética , Western Blotting , Cromatografia Gasosa-Espectrometria de Massas , Isocitrato Desidrogenase/genética , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Purpose: Aberrant activation of the B-cell receptor (BCR) is implicated in the pathogenesis of mature B-cell tumors, a concept validated in part by the clinical success of inhibitors of the BCR-related kinases BTK (Bruton's tyrosine kinase) and PI3Kδ. These inhibitors have limitations, including the paucity of complete responses, acquired resistance, and toxicity. Here, we examined the mechanism by which the cyclic-AMP/PDE4 signaling axis suppresses PI3K, toward identifying a novel mechanism-based combinatorial strategy to attack BCR-dependency in mature B-cell malignancies.Experimental Design: We used in vitro and in vivo diffuse large B-cell lymphoma (DLBCL) cell lines and primary chronic lymphocytic leukemia (CLL) samples to preclinically evaluate the effects of the combination of the FDA-approved phosphodiesterase 4 (PDE4) inhibitor roflumilast and idelalisib on cell survival and tumor growth. Genetic models of gain- and loss-of-function were used to map multiple signaling intermediaries downstream of the BCR.Results: Roflumilast elevates the intracellular levels of cyclic-AMP and synergizes with idelalisib in suppressing tumor growth and PI3K activity. Mechanistically, we show that roflumilast suppresses PI3K by inhibiting BCR-mediated activation of the P85 regulatory subunit, distinguishing itself from idelalisib, an ATP-competitive inhibitor of the catalytic P110 subunit. Using genetic models, we linked the PDE4-regulated modulation of P85 activation to the oncogenic kinase SYK.Conclusions: These data demonstrate that roflumilast and idelalisib suppress PI3K by distinct mechanisms, explaining the basis for their synergism, and suggest that the repurposing of PDE4 inhibitors to treat BCR-dependent malignancies is warranted. Clin Cancer Res; 24(5); 1103-13. ©2017 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Inibidores da Fosfodiesterase 4/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/metabolismo , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Ciclopropanos/farmacologia , Ciclopropanos/uso terapêutico , Reposicionamento de Medicamentos , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Nus , Inibidores da Fosfodiesterase 4/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Quinazolinonas/farmacologia , Quinazolinonas/uso terapêutico , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: In this study, we aimed to validate our extensive preclinical data on phosphodiesterase 4 (PDE4) as actionable target in B-cell malignancies. Our specific objectives were to determine the safety, pharmacokinetics, and pharmacodynamics (PI3K/AKT activity), as well as to capture any potential antitumor activity of the PDE4 inhibitor roflumilast in combination with prednisone in patients with advanced B-cell malignancies.Experimental Design: Single-center, exploratory phase Ib open-label, nonrandomized study. Roflumilast (500 mcg PO) was given daily for 21 days with prednisone on days 8 to 14. Additional 21-day cycles were started if patients tolerated cycle 1 and had at least stable disease.Results: Ten patients, median age 65 years with an average of three prior therapies, were enrolled. The median number of cycles administered was 4 (range, 1-13). Treatment was well tolerated; the most common ≥grade 2 treatment-related adverse events were fatigue, anorexia (≥25%), and transient ≥ grade 2 neutropenia (30%). Treatment with roflumilast as a single agent significantly suppressed PI3K activity in the 77% of patients evaluated; on average, patients with PI3K/AKT suppression stayed in trial for 156 days (49-315) versus 91 days (28-139 days) for those without this biomarker response. Six of the nine evaluable patients (66%) had partial response or stable disease. The median number of days in trial was 105 days (range, 28-315).Conclusions: Repurposing the PDE4 inhibitor roflumilast for treatment of B-cell malignancies is safe, suppresses the oncogenic PI3K/AKT kinases, and may be clinically active. Clin Cancer Res; 23(5); 1186-92. ©2016 AACR.
Assuntos
Aminopiridinas/administração & dosagem , Benzamidas/administração & dosagem , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Neoplasias/tratamento farmacológico , Inibidores da Fosfodiesterase 4/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminopiridinas/efeitos adversos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Benzamidas/efeitos adversos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/efeitos dos fármacos , Ciclopropanos/administração & dosagem , Ciclopropanos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/genética , Inibidores da Fosfodiesterase 4/efeitos adversos , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
Angiogenesis associates with poor outcome in diffuse large B-cell lymphoma (DLBCL), but the contribution of the lymphoma cells to this process remains unclear. Addressing this knowledge gap may uncover unsuspecting proangiogenic signaling nodes and highlight alternative antiangiogenic therapies. Here, we identify the second messenger cyclic-AMP (cAMP) and the enzyme that terminates its activity, phosphodiesterase 4B (PDE4B), as regulators of B-cell lymphoma angiogenesis. We first show that cAMP, in a PDE4B-dependent manner, suppresses PI3K/AKT signals to downmodulate vascular endothelial growth factor (VEGF) secretion and vessel formation in vitro. Next, we create a novel mouse model that combines the lymphomagenic Myc transgene with germline deletion of Pde4b. We show that lymphomas developing in a Pde4b-null background display significantly lower microvessel density (MVD) in association with lower VEGF levels and PI3K/AKT activity. We recapitulate these observations by treating lymphoma-bearing mice with the FDA-approved PDE4 inhibitor, Roflumilast. Lastly, we show that primary human DLBCLs with high PDE4B expression display significantly higher MVD. Here, we defined an unsuspected signaling circuitry in which the cAMP generated in lymphoma cells downmodulates PI3K/AKT and VEGF secretion to negatively influence vessel development in the microenvironment. These data identify PDE4 as an actionable antiangiogenic target in DLBCL.
Assuntos
Linfócitos B/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/enzimologia , Neovascularização Patológica/enzimologia , Aminopiridinas/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Benzamidas/farmacologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Ciclopropanos/farmacologia , Modelos Animais de Doenças , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Microambiente Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In biological processes, the balance between positive and negative inputs is critical for an effective physiological response and to prevent disease. A case in point is the germinal center (GC) reaction, wherein high mutational and proliferation rates are accompanied by an obligatory suppression of the DNA repair machinery. Understandably, when the GC reaction goes awry, loss of immune cells or lymphoid cancer ensues. Here, we detail the functional interactions that make microRNA 155 (miR-155) a key part of this process. Upon antigen exposure, miR-155(-/-) mature B cells displayed significantly higher double-strand DNA break (DSB) accumulation and p53 activation than their miR-155(+/+) counterparts. Using B cell-specific knockdown strategies, we confirmed the role of the miR-155 target Aicda (activation-induced cytidine deaminase) in this process and, in combination with a gain-of-function model, unveiled a previously unappreciated role for Socs1 in directly modulating p53 activity and the DNA damage response in B lymphocytes. Thus, miR-155 controls the outcome of the GC reaction by modulating its initiation (Aicda) and termination (Socs1/p53 response), suggesting a mechanism to explain the quantitative defect in germinal center B cells found in mice lacking or overexpressing this miRNA.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Citidina Desaminase/imunologia , MicroRNAs/imunologia , Proteínas Supressoras da Sinalização de Citocina/imunologia , Proteína Supressora de Tumor p53/imunologia , Animais , Apoptose , Linfócitos B/metabolismo , Pontos de Checagem do Ciclo Celular , Células Cultivadas , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , Feminino , Deleção de Genes , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Histonas/imunologia , Masculino , Camundongos , MicroRNAs/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Regulação para CimaRESUMO
Isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate (α-KG). In cancer, mutant IDH1/2 reduces α-KG to D2-hydroxyglutarate (D2-HG) disrupting α-KG-dependent dioxygenases. However, the physiological relevance of controlling the interconversion of D2-HG into α-KG, mediated by D2-hydroxyglutarate dehydrogenase (D2HGDH), remains obscure. Here we show that wild-type D2HGDH elevates α-KG levels, influencing histone and DNA methylation, and HIF1α hydroxylation. Conversely, the D2HGDH mutants that we find in diffuse large B-cell lymphoma are enzymatically inert. D2-HG is a low-abundance metabolite, but we show that it can meaningfully elevate α-KG levels by positively modulating mitochondrial IDH activity and inducing IDH2 expression. Accordingly, genetic depletion of IDH2 abrogates D2HGDH effects, whereas ectopic IDH2 rescues D2HGDH-deficient cells. Our data link D2HGDH to cancer and describe an additional role for the enzyme: the regulation of IDH2 activity and α-KG-mediated epigenetic remodelling. These data further expose the intricacies of mitochondrial metabolism and inform on the pathogenesis of D2HGDH-deficient diseases.