RESUMO
BACKGROUND: Renal medullary carcinoma (RMC) is a highly aggressive cancer in need of new therapeutic strategies. The neddylation pathway can protect cells from DNA damage induced by the platinum-based chemotherapy used in RMC. We investigated if neddylation inhibition with pevonedistat will synergistically enhance antitumour effects of platinum-based chemotherapy in RMC. METHODS: We evaluated the IC50 concentrations of the neddylation-activating enzyme inhibitor pevonedistat in vitro in RMC cell lines. Bliss synergy scores were calculated using growth inhibition assays following treatment with varying concentrations of pevonedistat and carboplatin. Protein expression was assessed by western blot and immunofluorescence assays. The efficacy of pevonedistat alone or in combination with platinum-based chemotherapy was evaluated in vivo in platinum-naïve and platinum-experienced patient-derived xenograft (PDX) models of RMC. RESULTS: The RMC cell lines demonstrated IC50 concentrations of pevonedistat below the maximum tolerated dose in humans. When combined with carboplatin, pevonedistat demonstrated a significant in vitro synergistic effect. Treatment with carboplatin alone increased nuclear ERCC1 levels used to repair the interstrand crosslinks induced by platinum salts. Conversely, the addition of pevonedistat to carboplatin led to p53 upregulation resulting in FANCD2 suppression and reduced nuclear ERCC1 levels. The addition of pevonedistat to platinum-based chemotherapy significantly inhibited tumour growth in both platinum-naïve and platinum-experienced PDX models of RMC (p < .01). CONCLUSIONS: Our results suggest that pevonedistat synergises with carboplatin to inhibit RMC cell and tumour growth through inhibition of DNA damage repair. These findings support the development of a clinical trial combining pevonedistat with platinum-based chemotherapy for RMC.
Assuntos
Carcinoma Medular , Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológicoRESUMO
Neural tube defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to the cerebral folate deficiency syndrome by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole-genome sequencing (WGS) data of 140 isolated spina bifida cases and identified eight missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased the FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants downregulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD.
Assuntos
Defeitos do Tubo Neural , Proteínas Repressoras , Disrafismo Espinal , Animais , Feminino , Humanos , Camundongos , Gravidez , Receptor 1 de Folato/genética , Ácido Fólico , Mutação de Sentido Incorreto , Defeitos do Tubo Neural/genética , Células NIH 3T3 , Disrafismo Espinal/genética , Células HeLa , Proteínas Repressoras/genéticaRESUMO
Gene discovery efforts in autism spectrum disorder have identified heterozygous defects in chromatin remodeller genes, the 'readers, writers and erasers' of methyl marks on chromatin, as major contributors to this disease. Despite this advance, a convergent aetiology between these defects and aberrant chromatin architecture or gene expression has remained elusive. Recently, data have begun to emerge that chromatin remodellers also function directly on the cytoskeleton. Strongly associated with autism spectrum disorder, the SETD2 histone methyltransferase for example, has now been shown to directly methylate microtubules of the mitotic spindle. However, whether microtubule methylation occurs in post-mitotic cells, for example on the neuronal cytoskeleton, is not known. We found the SETD2 α-tubulin lysine 40 trimethyl mark occurs on microtubules in the brain and in primary neurons in culture, and that the SETD2 C-terminal SRI domain is required for binding and methylation of α-tubulin. A CRISPR knock-in of a pathogenic SRI domain mutation (Setd2SRI) that disables microtubule methylation revealed at least one wild-type allele was required in mice for survival, and while viable, heterozygous Setd2SRI/wtmice exhibited an anxiety-like phenotype. Finally, whereas RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) showed no concomitant changes in chromatin methylation or gene expression in Setd2SRI/wtmice, primary neurons exhibited structural deficits in axon length and dendritic arborization. These data provide the first demonstration that microtubules of neurons are methylated, and reveals a heterozygous chromatin remodeller defect that specifically disables microtubule methylation is sufficient to drive an autism-associated phenotype.
Assuntos
Ansiedade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Histonas/metabolismo , Metilação , Camundongos , FenótipoRESUMO
Epigenetic effectors "read" marks "written" on chromatin to regulate function and fidelity of the genome. Here, we show that this coordinated read-write activity of the epigenetic machinery extends to the cytoskeleton, with PBRM1 in the PBAF chromatin remodeling complex reading microtubule methyl marks written by the SETD2 histone methyltransferase. PBRM1 binds SETD2 methyl marks via BAH domains, recruiting PBAF components to the mitotic spindle. This read-write activity was required for normal mitosis: Loss of SETD2 methylation or pathogenic BAH domain mutations disrupt PBRM1 microtubule binding and PBAF recruitment and cause genomic instability. These data reveal PBRM1 functions beyond chromatin remodeling with domains that allow it to integrate chromatin and cytoskeletal activity via its acetyl-binding BD and methyl-binding BAH domains, respectively. Conserved coordinated activity of the epigenetic machinery on the cytoskeleton opens a previously unknown window into how chromatin remodeler defects can drive disease via both epigenetic and cytoskeletal dysfunction.
Assuntos
Microtúbulos , Leitura , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Citoesqueleto/metabolismo , Microtúbulos/metabolismoRESUMO
The methyltransferase SET domain-containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.
Assuntos
Actinas , Proteínas de Ligação ao GTP/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Lisina , Actinas/metabolismo , Citoesqueleto/metabolismo , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-TraducionalRESUMO
Neural tube defects (NTDs) are a group of severe congenital malformations caused by a failure of neural tube closure during early embryonic development. Although extensively investigated, the genetic etiology of NTDs remains poorly understood. FKBP8 is critical for proper mammalian neural tube closure. Fkbp8-/- mouse embryos showed posterior NTDs consistent with a diagnosis of spina bifida (SB). To date, no publication has reported any association between FKBP8 and human NTDs. Using Sanger sequencing on genomic DNA samples from 472 SB and 565 control samples, we identified five rare (MAF ≤ 0.001) deleterious variants in SB patients, while no rare deleterious variant was identified in the controls (P = 0.0191). p.Glu140* affected FKBP8 localization to the mitochondria and created a truncated form of the FKBP8 protein, thus impairing its interaction with BCL2 and ultimately leading to an increase in cellular apoptosis. p.Ser3Leu, p.Lys315Asn and p.Ala292Ser variants decreased FKBP8 protein level. p.Lys315Asn further increased the cellular apoptosis. RNA sequencing on anterior and posterior tissues isolated from Fkbp8-/- and wildtype mice at E9.5 and E10.5 showed that Fkbp8-/- embryos have an abnormal expression profile within tissues harvested at posterior sites, thus leading to a posterior NTD. Moreover, we found that Fkbp8 knockout mouse embryos have abnormal expression of Wnt3a and Nkx2.9 during the early stage of neural tube development, perhaps also contributing to caudal specific NTDs. These findings provide evidence that functional variants of FKBP8 are risk factors for SB, which may involve a novel mechanism by which Fkbp8 mutations specifically cause SB in mice.
Assuntos
Proteínas de Homeodomínio/genética , Disrafismo Espinal/genética , Proteínas de Ligação a Tacrolimo/genética , Fatores de Transcrição/genética , Proteína Wnt3A/genética , Animais , Apoptose/genética , Feminino , Predisposição Genética para Doença , Humanos , Recém-Nascido , Masculino , Camundongos , Camundongos Knockout , Malformações do Sistema Nervoso , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/patologia , Fatores de Risco , Disrafismo Espinal/patologiaRESUMO
Extensive studies that have sought causative mutation(s) for neural tube defects (NTDs) have yielded limited positive findings to date. One possible reason for this is that many studies have been confined to analyses of germline mutations and so may have missed other, non-germline mutations in NTD cases. We hypothesize that somatic mutations of planar polarity pathway (PCP) genes may play a role in the development of NTDs. Torrent™ Personal Genome Machine™ (PGM) sequencing was designed for selected PCP genes in paired DNA samples extracted from the tissues of lesion sites and umbilical cord from 48 cases. Sanger sequencing was used to validate the detected mutations. The source and distribution of the validated mutations in tissues from different germ layers were investigated. Subcellular location, western blotting, and luciferase assays were performed to better understand the effects of the mutations on protein localization, protein level, and pathway signaling. ix somatic mutations were identified and validated, which showed diverse distributions in different tissues. Three somatic mutations were novel/rare: CELSR1 p.Gln2125His, FZD6 p.Gln88Glu, and VANGL1 p.Arg374His. FZD6 p.Gln88Glu caused mislocalization of its protein from the cytoplasm to the nucleus, and disrupted the colocalization of CELSR1 and FZD6. This mutation affected non-canonical WNT signaling in luciferase assays. VANGL1 p.Arg374His impaired the co-localization of CELSR1 and VANGL1, increased the protein levels of VANGL1, and influenced cell migration. In all, 7/48 (14.5%) of the studied NTD cases contained somatic PCP mutations. Somatic mutations in PCP genes (e.g., FZD6 and VANGL1) are associated with human NTDs, and they may occur in different stages and regions during embryonic development, resulting in a varied distribution in fetal tissues/organs.
Assuntos
Caderinas/genética , Proteínas de Transporte/genética , Receptores Frizzled/genética , Proteínas de Membrana/genética , Mutação , Defeitos do Tubo Neural/genética , Tubo Neural/metabolismo , Transporte Ativo do Núcleo Celular/genética , Sequência de Aminoácidos , Animais , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Feto , Receptores Frizzled/metabolismo , Expressão Gênica , Genoma Humano , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/metabolismo , Tubo Neural/anormalidades , Defeitos do Tubo Neural/diagnóstico , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Gravidez , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sequenciamento Completo do GenomaRESUMO
Renal medullary carcinoma (RMC) is a highly lethal malignancy that mainly afflicts young individuals of African descent and is resistant to all targeted agents used to treat other renal cell carcinomas. Comprehensive genomic and transcriptomic profiling of untreated primary RMC tissues was performed to elucidate the molecular landscape of these tumors. We found that RMC was characterized by high replication stress and an abundance of focal copy-number alterations associated with activation of the stimulator of the cyclic GMP-AMP synthase interferon genes (cGAS-STING) innate immune pathway. Replication stress conferred a therapeutic vulnerability to drugs targeting DNA-damage repair pathways. Elucidation of these previously unknown RMC hallmarks paves the way to new clinical trials for this rare but highly lethal malignancy.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Medular/patologia , Carcinoma de Células Renais/patologia , Aberrações Cromossômicas , Replicação do DNA , Neoplasias Renais/patologia , Proteína SMARCB1/metabolismo , Adulto , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Medular/genética , Carcinoma Medular/imunologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Proliferação de Células , Estudos de Coortes , Variações do Número de Cópias de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína SMARCB1/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Loss of the gene von Hippel-Lindau (VHL) is associated with loss of primary cilia and is causally linked to elevated levels of Aurora kinase A (AURKA). We developed an image-based high-throughput screening (HTS) assay using a dual-labeling image analysis strategy that identifies both the cilium and the basal body. By using this strategy, we screened small-molecule compounds for the targeted rescue of cilia defects associated with VHL deficiency with high accuracy and reproducibility. Bexarotene was identified and validated as a positive regulator of the primary cilium. Importantly, the inability of an alternative retinoid X receptor (RXR) agonist to rescue ciliogenesis, in contrast to bexarotene, suggested that multiple bexarotene-driven mechanisms were responsible for the rescue. We found that bexarotene decreased AURKA expression in VHL-deficient cells, thereby restoring the ability of these cells to ciliate in the absence of VHL Finally, bexarotene treatment reduced the propensity of subcutaneous lesions to develop into tumors in a mouse xenograft model of renal cell carcinoma (RCC), with a concomitant decrease in activated AURKA, highlighting the potential of bexarotene treatment as an intervention strategy in the clinic to manage renal cystogenesis associated with VHL deficiency and elevated AURKA expression.
Assuntos
Aurora Quinase A/metabolismo , Bexaroteno/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Aurora Quinase A/genética , Linhagem Celular Tumoral , Cílios/efeitos dos fármacos , Cílios/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação/efeitos dos fármacos , Mutação/genética , Proteína Supressora de Tumor Von Hippel-Lindau/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase SETD2, which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic, Setd2-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In SETD2-inactivated human kidney cells, rescue with a pathogenic SETD2 mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the SETD2 tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability.Significance: Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. Cancer Res; 78(12); 3135-46. ©2018 AACR.
Assuntos
Carcinoma de Células Renais/genética , Cromossomos Humanos Par 3/genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias Renais/genética , Microtúbulos/metabolismo , Animais , Carcinogênese/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Fibroblastos , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Haploinsuficiência , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Neoplasias Renais/patologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/patologia , Lisina/metabolismo , Metilação , Camundongos , Micronúcleos com Defeito CromossômicoRESUMO
OBJECTIVE: This study analyzes the social determinants associated with the overweight or obesity prevalence of 85 elementary schools during the 2010-11 academic year in a predominantly Hispanic school district. METHODS: A binomial logistic regression is used to analyze the aggregate overweight or obesity rate of a school by the percent of Hispanic students in each school, selected school and neighborhood characteristics, and its geographical location. RESULTS: The proportion of Hispanic enrollment more readily explains a school's aggregate overweight or obesity rate than social determinants or spatial location. Number of fast food establishments and the academic ranking of a school appear to slightly impact the aggregate prevalence rate. Spatial location of school is not a significant factor, controlling for other determinants. CONCLUSIONS: An elementary school's overall overweight or obesity rate provides a valuable health indicator to study the social determinants of obesity among Hispanics and other students within a local neighborhood.
Assuntos
Índice de Massa Corporal , Sobrepeso/epidemiologia , Obesidade Infantil/epidemiologia , Distância Psicológica , Instituições Acadêmicas , Determinantes Sociais da Saúde , Criança , Estudos Transversais , Feminino , Hispânico ou Latino/estatística & dados numéricos , Humanos , Masculino , New Mexico/epidemiologia , Sobrepeso/diagnóstico , Sobrepeso/psicologia , Obesidade Infantil/diagnóstico , Obesidade Infantil/etnologia , Obesidade Infantil/psicologia , Prevalência , Serviços de Saúde EscolarRESUMO
The spindle assembly checkpoint (SAC) delays mitotic progression until all sister chromatid pairs achieve bi-orientation, and while the SAC can maintain mitotic arrest for extended periods, moderate delays in mitotic progression have significant effects on the resulting daughter cells. Here we show that when retinal-pigmented epithelial (RPE1) cells experience mitotic delay, there is a time-dependent increase in centrosome fragmentation and centriole disengagement. While most cells with disengaged centrioles maintain spindle bipolarity, clustering of disengaged centrioles requires the kinesin-14, HSET. Centrosome fragmentation and precocious centriole disengagement depend on separase and anaphase-promoting complex/cyclosome (APC/C) activity, which also triggers the acquisition of distal appendage markers on daughter centrioles and the loss of procentriolar markers. Together, these results suggest that moderate delays in mitotic progression trigger the initiation of centriole licensing through centriole disengagement, at which point the ability to maintain spindle bipolarity becomes a function of HSET-mediated spindle pole clustering.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Mitose , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Linhagem Celular , Centríolos/genética , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
C2-aryl- and C2-alkyl-7-deazahypoxanthines as analogues of marine alkaloid rigidins were prepared utilizing novel synthetic methods developed for the construction of the pyrrolo[2,3-d]pyrimidine ring system. The new compounds exhibited sub-micromolar to nanomolar antiproliferative potencies against a panel of cell lines including in vitro models for drug-resistant tumors, such as glioblastoma, melanoma and non-small-cell lung cancer. A selected representative C2-methyl-7-deazahypoxanthine was found to inhibit microtubule dynamics in cancer cells, lending evidence for tubulin targeting as a mode of action for these compounds in cancer cells. The results of the docking studies utilizing the colchicine site on ß-tubulin were consistent with the observed structure-activity relationship data, including an important finding that derivatization at C2 with linear alkyl groups leads to the retention of activity, thus permitting the attachment of a biotin-containing linker for the subsequent proteomics assays. Because many microtubule-targeting compounds are successfully used to fight cancer in the clinic, the reported antitubulin rigidin analogues have significant potential as new anticancer agents.
Assuntos
Alcaloides/química , Hipoxantinas/química , Moduladores de Tubulina/química , Moduladores de Tubulina/síntese química , Tubulina (Proteína)/química , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Hipoxantinas/síntese química , Hipoxantinas/toxicidade , Células MCF-7 , Microscopia de Vídeo , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Pirimidinas/química , Pirróis/química , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/toxicidadeRESUMO
We developed synthetic chemistry to access the marine alkaloid rigidins and over 40 synthetic analogues based on the 7-deazaxanthine, 7-deazaadenine, 7-deazapurine, and 7-deazahypoxanthine skeletons. Analogues based on the 7-deazahypoxanthine skeleton exhibited nanomolar potencies against cell lines representing cancers with dismal prognoses, tumor metastases, and multidrug resistant cells. Studies aimed at elucidating the mode(s) of action of the 7-deazahypoxanthines in cancer cells revealed that they inhibited in vitro tubulin polymerization and disorganized microtubules in live HeLa cells. Experiments evaluating the effects of the 7-deazahypoxanthines on the binding of [(3)H]colchicine to tubulin identified the colchicine site on tubulin as the most likely target for these compounds in cancer cells. Because many microtubule-targeting compounds are successfully used to fight cancer in the clinic, we believe the new chemical class of antitubulin agents represented by the 7-deazahypoxanthine rigidin analogues have significant potential as new anticancer agents.
Assuntos
Alcaloides/química , Produtos Biológicos/química , Tubulina (Proteína)/efeitos dos fármacos , Alcaloides/farmacologia , Produtos Biológicos/farmacologia , Colchicina/química , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Tubulina (Proteína)/químicaRESUMO
A variant structural skeleton of epipodophyllotoxin was synthesized and found to rival the natural cyclolignan in antiproliferative and microtubule destabilizing properties. This discovery leads to a new structural class of tubulin targeting agents.