RESUMO
Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.
Assuntos
Endorribonucleases , MicroRNAs , RNA Mensageiro , Humanos , Genes jun/genética , Genes myc/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Endorribonucleases/química , Endorribonucleases/metabolismo , TranscriptomaRESUMO
Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.
Assuntos
Genes Supressores de Tumor , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tristetraprolina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estabilidade de RNA , RNA Mensageiro/químicaRESUMO
Group 3 innate lymphoid cells (ILC3s) are RORγT+ lymphocytes that are predominately enriched in mucosal tissues and produce IL-22 and IL-17A. They are the innate counterparts of Th17 cells. While Th17 lymphocytes utilize unique metabolic pathways in their differentiation program, it is unknown whether ILC3s make similar metabolic adaptations. We employed single-cell RNA sequencing and metabolomic profiling of intestinal ILC subsets to identify an enrichment of polyamine biosynthesis in ILC3s, converging on the rate-limiting enzyme ornithine decarboxylase (ODC1). In vitro and in vivo studies demonstrated that exogenous supplementation with the polyamine putrescine or its biosynthetic substrate, ornithine, enhanced ILC3 production of IL-22. Conditional deletion of ODC1 in ILC3s impaired mouse antibacterial defense against Citrobacter rodentium infection, which was associated with a decrease in anti-microbial peptide production by the intestinal epithelium. Furthermore, in a model of anti-CD40 colitis, deficiency of ODC1 in ILC3s markedly reduced the production of IL-22 and severity of inflammatory colitis. We conclude that ILC3-intrinsic polyamine biosynthesis facilitates efficient defense against enteric pathogens as well as exacerbates autoimmune colitis, thus representing an attractive target to modulate ILC3 function in intestinal disease.
Assuntos
Colite , Infecções por Enterobacteriaceae , Camundongos , Animais , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Interleucina-17 , Ornitina Descarboxilase/genética , Imunidade Inata , Putrescina , Colite/genética , Infecções por Enterobacteriaceae/genética , Células Th17/metabolismo , Ornitina , Antibacterianos , Interleucina 22RESUMO
The unique amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] is exclusively formed on the translational regulator eukaryotic initiation factor 5A (eIF5A) via a process coined hypusination. Hypusination is mediated by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH), and hypusinated eIF5A (eIF5AHyp) promotes translation elongation by alleviating ribosome pauses at amino acid motifs that cause structural constraints, and it also facilitates translation initiation and termination. Accordingly, eIF5AHyp has diverse biological functions that rely on translational control of its targets. Homozygous deletion of Eif5a, Dhps, or Dohh in mice leads to embryonic lethality, and heterozygous germline variants in EIF5A and biallelic variants in DHPS and DOHH are associated with rare inherited neurodevelopmental disorders, underscoring the importance of the hypusine circuit for embryonic and neuronal development. Given the pleiotropic effects of eIF5AHyp, a detailed understanding of the cell context-specific intrinsic roles of eIF5AHyp and of the chronic versus acute effects of eIF5AHyp inhibition is necessary to develop future strategies for eIF5AHyp-targeted therapy to treat various human health problems. Here, we review the most recent studies documenting the intrinsic roles of eIF5AHyp in different tissues/cell types under normal or pathophysiological conditions and discuss these unique aspects of eIF5AHyp-dependent translational control.
Assuntos
Fator de Iniciação de Tradução Eucariótico 5A , Lisina , Fatores de Iniciação de Peptídeos , Proteínas de Ligação a RNA , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Lisina/metabolismo , Lisina/análogos & derivados , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Biossíntese de Proteínas , CamundongosRESUMO
We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Melanoma , Síndromes Mielodisplásicas , Células Supressoras Mieloides , Animais , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Humanos , Melanoma/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos TRESUMO
Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53 mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. In this study, we characterized the immunological features of the malignant clone and alterations in the immune microenvironment in patients with TP53-mutant and wild-type MDS or sAML. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of patients with TP53 mutations, which is associated with MYC upregulation and marked downregulation of MYC's negative regulator miR-34a, a p53 transcription target. Notably, patients with TP53 mutations display significantly reduced numbers of bone marrow-infiltrating OX40+ cytotoxic T cells and helper T cells, as well as decreased ICOS+ and 4-1BB+ natural killer cells. Further, highly immunosuppressive regulatory T cells (Tregs) (ie, ICOShigh/PD-1-) and myeloid-derived suppressor cells (PD-1low) are expanded in cases with TP53 mutations. Finally, a higher proportion of bone marrow-infiltrating ICOShigh/PD-1- Treg cells is a highly significant independent predictor of overall survival. We conclude that the microenvironment of TP53 mutant MDS and sAML has an immune-privileged, evasive phenotype that may be a primary driver of poor outcomes and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly defined subpopulation.
Assuntos
Leucemia Mieloide Aguda , Mutação , Síndromes Mielodisplásicas , Células Supressoras Mieloides/imunologia , Linfócitos T Reguladores/imunologia , Proteína Supressora de Tumor p53 , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Terapia de Imunossupressão , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Masculino , MicroRNAs/genética , MicroRNAs/imunologia , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/patologia , Células Supressoras Mieloides/patologia , RNA Neoplásico/genética , RNA Neoplásico/imunologia , Linfócitos T Reguladores/patologia , Proteína Supressora de Tumor p53/imunologiaRESUMO
Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known "neosubstrates," such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Metabolismo Energético/genética , Ativação Linfocitária/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Imunomodulação/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The MDM2 homolog MDMX oncoprotein is indispensable for inhibition of p53 during normal embryonic development and malignant transformation, yet how MDMX harnesses p53 functions is unclear. In addition to a canonical N-terminal p53-binding domain, recent work suggests the central acidic domain of MDMX regulates p53 interaction through intramolecular mimicry and engages in second-site interaction with the p53 core domain in vitro. To test the physiological relevance of these interactions, we generated an MDMX knockin mouse having substitutions in a conserved WW motif necessary for these functions (W201S/W202G). Notably, MDMXSG cells have normal p53 level but increased p53 DNA binding and target gene expression, and rapidly senesce. In vivo, MDMXSG inhibits early-phase disease in Eµ-Myc transgenic mice but accelerates the onset of lethal lymphoma and shortens overall survival. Therefore, MDMX is an important regulator of p53 DNA binding, which complements the role of MDM2 in regulating p53 level. Furthermore, the results suggest that the WW motif has dual functions that regulate p53 and inhibit Myc-driven lymphomas independent of p53.
Assuntos
Carcinogênese/metabolismo , Linfoma/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinogênese/genética , Transformação Celular Neoplásica , Feminino , Genes myc , Humanos , Linfoma/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.
Assuntos
Acetiltransferases/deficiência , Ataxia/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/deficiência , Células de Purkinje/enzimologia , Acetiltransferases/genética , Animais , Apoptose/fisiologia , Ataxia/genética , Ataxia/patologia , Cerebelo/enzimologia , Cerebelo/patologia , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Células de Purkinje/patologia , Poliamina OxidaseRESUMO
Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms.
Assuntos
Infecções por Enterobacteriaceae/imunologia , Infecções por Helicobacter/imunologia , Histonas/metabolismo , Macrófagos/imunologia , Ornitina Descarboxilase/imunologia , Animais , Linhagem Celular , Citrobacter rodentium , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/patologia , Citocinas/imunologia , Infecções por Enterobacteriaceae/patologia , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Gastrite/imunologia , Gastrite/patologia , Infecções por Helicobacter/patologia , Helicobacter pylori , Humanos , Ativação de Macrófagos , Masculino , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Ornitina Descarboxilase/genética , Putrescina/metabolismoRESUMO
PURPOSE: Most prostate cancer in African American men lacks the ETS (E26 transforming specific) family fusion event (ETS-). We aimed to establish clinically relevant biomarkers in African American men by studying ETS dependent gene expression patterns to identified race specific genes predictive of outcomes. MATERIALS AND METHODS: Two multicenter cohorts of a total of 1,427 men were used for the discovery and validation (635 and 792 men, respectively) of race specific predictive biomarkers. We used false discovery rate adjusted q values to identify race and ETS dependent genes which were differentially expressed in African American men who experienced biochemical recurrence within 5 years. Principal component modeling along with survival analysis was done to assess the accuracy of the gene panel in predicting recurrence. RESULTS: We identified 3,047 genes which were differentially expressed based on ETS status. Of these genes 362 were differentially expressed in a race specific manner (false discovery rate 0.025 or less). A total of 81 genes were race specific and over expressed in African American men who experienced biochemical recurrence. The final gene panel included APOD, BCL6, EMP1, MYADM, SRGN and TIMP3. These genes were associated with 5-year biochemical recurrence (HR 1.97, 95% CI 1.27-3.06, p = 0.002) and they improved the predictive accuracy of clinicopathological variables only in African American men (60-month time dependent AUC 0.72). CONCLUSIONS: In an effort to elucidate biological features associated with prostate cancer aggressiveness in African American men we identified ETS dependent biomarkers predicting early onset biochemical recurrence only in African American men. Thus, these ETS dependent biomarkers representing ideal candidates for biomarkers of aggressive disease in this patient population.
Assuntos
Negro ou Afro-Americano/genética , Neoplasias da Próstata/genética , Idoso , Biomarcadores Tumorais/genética , Estudos de Coortes , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Prognóstico , Proteínas Proto-Oncogênicas c-ets/genéticaRESUMO
Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Autofagia/fisiologia , Proteínas de Ciclo Celular/fisiologia , Chaperoninas/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Chaperoninas/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4' positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4' hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure-activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.
Assuntos
Antibióticos Antineoplásicos/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Lactamas Macrocíclicas/metabolismo , Lactamas/metabolismo , Macrolídeos/metabolismo , Tiazóis/metabolismo , Tionas/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidade , Vias Biossintéticas , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Inativação Gênica , Humanos , Hidroxilação , Lactamas/química , Lactamas/toxicidade , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/toxicidade , Macrolídeos/química , Macrolídeos/toxicidade , Estrutura Molecular , Família Multigênica , Estereoisomerismo , Streptomyces/genética , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/toxicidade , Tionas/química , Tionas/toxicidadeRESUMO
Despite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1ß (IL-1ß) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and ß-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear ß-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention.
Assuntos
Inflamassomos/metabolismo , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Calgranulina B/metabolismo , Tamanho Celular , Ensaio de Unidades Formadoras de Colônias , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Camundongos Transgênicos , Mutação/genética , NADPH Oxidases/metabolismo , Fenótipo , Piroptose , Espécies Reativas de Oxigênio/metabolismo , beta Catenina/metabolismoRESUMO
Chimeric oncoproteins created by chromosomal translocations are among the most common genetic mutations associated with tumorigenesis. Malignant mucoepidermoid salivary gland tumors, as well as a growing number of solid epithelial-derived tumors, can arise from a recurrent t (11, 19)(q21;p13.1) translocation that generates an unusual chimeric cAMP response element binding protein (CREB)-regulated transcriptional coactivator 1 (CRTC1)/mastermind-like 2 (MAML2) (C1/M2) oncoprotein comprised of two transcriptional coactivators, the CRTC1 and the NOTCH/RBPJ coactivator MAML2. Accordingly, the C1/M2 oncoprotein induces aberrant expression of CREB and NOTCH target genes. Surprisingly, here we report a gain-of-function activity of the C1/M2 oncoprotein that directs its interactions with myelocytomatosis oncogene (MYC) proteins and the activation of MYC transcription targets, including those involved in cell growth and metabolism, survival, and tumorigenesis. These results were validated in human mucoepidermoid tumor cells that harbor the t (11, 19)(q21;p13.1) translocation and express the C1/M2 oncoprotein. Notably, the C1/M2-MYC interaction is necessary for C1/M2-driven cell transformation, and the C1/M2 transcriptional signature predicts other human malignancies having combined involvement of MYC and CREB. These findings suggest that such gain-of-function properties may also be manifest in other oncoprotein fusions found in human cancer and that agents targeting the C1/M2-MYC interface represent an attractive strategy for the development of effective and safe anticancer therapeutics in tumors harboring the t (11, 19) translocation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 19/genética , Proteínas de Ligação a DNA/química , Redes Reguladoras de Genes , Genes myc , Células HEK293 , Humanos , Camundongos , Tumor Mucoepidermoide/genética , Tumor Mucoepidermoide/metabolismo , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas de Fusão Oncogênica/química , Domínios e Motivos de Interação entre Proteínas , Ratos , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/metabolismo , Transativadores , Fatores de Transcrição/química , Translocação GenéticaRESUMO
Myc oncogenic transcription factors (c-Myc, N-Myc, and L-Myc) coordinate the control of cell growth, division, and metabolism. In cancer, Myc overexpression is often associated with aggressive disease, which is in part due to the destruction of select targets by the ubiquitin-proteasome system (eg, SCF(Skp2)-directed destruction of the Cdk inhibitor p27(Kip1)). We reasoned that Myc would also regulate SUMOylation, a related means of posttranslational modification of proteins, and that this circuit would play essential roles in Myc-dependent tumorigenesis. Here, we report marked increases in the expression of genes that encode regulators and components of the SUMOylation machinery in mouse and human Myc-driven lymphomas, resulting in hyper-SUMOylation in these tumors. Further, inhibition of SUMOylation by genetic means disables Myc-induced proliferation, triggering G2/M cell-cycle arrest, polyploidy, and apoptosis. Using genetically defined cell models and conditional expression systems, this response was shown to be Myc specific. Finally, in vivo loss-of-function and pharmacologic studies demonstrated that inhibition of SUMOylation provokes rapid regression of Myc-driven lymphoma. Thus, targeting SUMOylation represents an attractive therapeutic option for lymphomas with MYC involvement.
Assuntos
Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ácidos Anacárdicos/farmacologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma de Células B/genética , Camundongos , Camundongos Transgênicos , Poliploidia , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Sumoilação/efeitos dos fármacos , Transcrição Gênica , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
The unique amino acid hypusine is present in only two proteins in eukaryotic cells, eukaryotic translation initiation factor 5A-1 (eIF5A1), and eIF5A2, where it is covalently linked to the lysine-50 residue of these proteins via a post-translational modification coined hypusination. This unique modification is directed by two highly conserved and essential enzymes, deoxyhypusine synthase (DHPS), and deoxyhypusine hydroxylase (DOHH), which selectively use the polyamine spermidine as a substrate to generate hypusinated eIF5A. Notably, elevated levels of polyamines are a hallmark of most tumor types, and increased levels of polyamines can also be detected in the urine and blood of cancer patients. Further, in-clinic agents that block the function of key biosynthetic enzymes in the polyamine pathway markedly impair tumor progression and maintenance of the malignant state. Thus, the polyamine pathway is attractive as a prognostic, prevention and therapeutic target. As we review, recent advances in our understanding of the specific functions of hypusinated eIF5A and its role in tumorigenesis suggest that the polyamine-hypusine circuit is a high priority target for cancer therapeutics.
Assuntos
Poliaminas Biogênicas/biossíntese , Lisina/análogos & derivados , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Animais , Humanos , Lisina/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Sempervirine and analogues were synthesized using a route featuring Sonogashira and Larock Pd-catalyzed reactions. Structure-activity relationships were investigated using three human cancer cell lines. 10-Fluorosempervirine is the most potently cytotoxic member of the family yet described.
Assuntos
Antineoplásicos/síntese química , Paládio/química , Alcaloides de Triptamina e Secologanina/síntese química , Alcaloides de Triptamina e Secologanina/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Alcaloides de Triptamina e Secologanina/química , Relação Estrutura-AtividadeRESUMO
The death receptor ligand TRAIL has shown remarkable promise as an anticancer agent. However, TRAIL signaling also activates NF-kappaB, which induces the antiapoptotic regulators Mcl-1 and cIAP2, thus compromising its efficacy. In this issue of Cancer Cell, El-Deiry and colleagues explore pathways that disrupt TRAIL-induced survival signaling and show that the Myc oncoprotein and the Raf kinase inhibitor Sorafenib sensitize otherwise TRAIL-resistant colon cancer cells by effectively reducing NF-kappaB-mediated transcription of Mcl-1. These findings suggest that combining TRAIL with agents that disrupt NF-kappaB regulation or binding or those that directly destabilize or disable Mcl-1 will have therapeutic benefit.