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1.
Sci Adv ; 7(29)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34261657

RESUMO

Histone H3K27M is a driving mutation in diffuse intrinsic pontine glioma (DIPG), a deadly pediatric brain tumor. H3K27M reshapes the epigenome through a global inhibition of PRC2 catalytic activity and displacement of H3K27me2/3, promoting oncogenesis of DIPG. As a consequence, a histone modification H3K36me2, antagonistic to H3K27me2/3, is aberrantly elevated. Here, we investigate the role of H3K36me2 in H3K27M-DIPG by tackling its upstream catalyzing enzymes (writers) and downstream binding factors (readers). We determine that NSD1 and NSD2 are the key writers for H3K36me2. Loss of NSD1/2 in H3K27M-DIPG impedes cellular proliferation and tumorigenesis by disrupting tumor-promoting transcriptional programs. Further, we demonstrate that LEDGF and HDGF2 are the main readers mediating the protumorigenic effects downstream of NSD1/2-H3K36me2. Treatment with a chemically modified peptide mimicking endogenous H3K36me2 dislodges LEDGF/HDGF2 from chromatin and specifically inhibits the proliferation of H3K27M-DIPG. Our results indicate a functional pathway of NSD1/2-H3K36me2-LEDGF/HDGF2 as an acquired dependency in H3K27M-DIPG.

2.
J Biol Chem ; 296: 100798, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34022221

RESUMO

GPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF-CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM.


Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , AMP Cíclico/metabolismo , Glioblastoma/metabolismo , Humanos , Proteólise , Receptores Acoplados a Proteínas G/química , Células Tumorais Cultivadas
3.
Neurooncol Adv ; 2(1): vdaa053, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32642706

RESUMO

Background: Glioma is a family of primary brain malignancies with limited treatment options and in need of novel therapies. We previously demonstrated that the adhesion G protein-coupled receptor GPR133 (ADGRD1) is necessary for tumor growth in adult glioblastoma, the most advanced malignancy within the glioma family. However, the expression pattern of GPR133 in other types of adult glioma is unknown. Methods: We used immunohistochemistry in tumor specimens and non-neoplastic cadaveric brain tissue to profile GPR133 expression in adult gliomas. Results: We show that GPR133 expression increases as a function of WHO grade and peaks in glioblastoma, where all tumors ubiquitously express it. Importantly, GPR133 is expressed within the tumor bulk, as well as in the brain-infiltrating tumor margin. Furthermore, GPR133 is expressed in both isocitrate dehydrogenase (IDH) wild-type and mutant gliomas, albeit at higher levels in IDH wild-type tumors. Conclusion: The fact that GPR133 is absent from non-neoplastic brain tissue but de novo expressed in glioma suggests that it may be exploited therapeutically.

4.
Front Oncol ; 9: 417, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31165048

RESUMO

The identification of heterozygous neomorphic isocitrate dehydrogenase (IDH) mutations across multiple cancer types including both solid and hematologic malignancies has revolutionized our understanding of oncogenesis in these malignancies and the potential for targeted therapeutics using small molecule inhibitors. The neomorphic mutation in IDH generates an oncometabolite product, 2-hydroxyglutarate (2HG), which has been linked to the disruption of metabolic and epigenetic mechanisms responsible for cellular differentiation and is likely an early and critical contributor to oncogenesis. In the past 2 years, two mutant IDH (mutIDH) inhibitors, Enasidenib (AG-221), and Ivosidenib (AG-120), have been FDA-approved for IDH-mutant relapsed or refractory acute myeloid leukemia (AML) based on phase 1 safety and efficacy data and continue to be studied in trials in hematologic malignancies, as well as in glioma, cholangiocarcinoma, and chondrosarcoma. In this review, we will summarize the molecular pathways and oncogenic consequences associated with mutIDH with a particular emphasis on glioma and AML, and systematically review the development and preclinical testing of mutIDH inhibitors. Existing clinical data in both hematologic and solid tumors will likewise be reviewed followed by a discussion on the potential limitations of mutIDH inhibitor monotherapy and potential routes for treatment optimization using combination therapy.

5.
Nat Commun ; 10(1): 754, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765700

RESUMO

Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.


Assuntos
Genes Virais/genética , Herpesvirus Humano 1/genética , Nanoporos , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Linhagem Celular , Células Cultivadas , Células Epiteliais/virologia , Fibroblastos/virologia , Genoma Viral/genética , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Neurônios/citologia , Neurônios/virologia , RNA Viral/genética , Proteínas Virais/genética
6.
Neurosurg Clin N Am ; 30(1): 17-25, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30470401

RESUMO

Advances in genome sequencing have elucidated the genetics of low-grade glioma. Available evidence indicates a neomorphic mutation in isocitrate dehydrogenase (IDH) initiates gliomagenesis. Mutant IDH produces the oncometabolite 2-hydroxyglutarate, which inhibits enzymes that demethylate genomic DNA and histones. Recent findings by the authors and others suggest the ensuing hypermethylation alters chromatin conformation and the transcription factor landscape in brain progenitor cells, leading to a block in differentiation and tumor initiation. Work in preclinical models has identified selective metabolic and molecular vulnerabilities of low-grade glioma. These new concepts will trigger a wave of innovative clinical trials in the near future.


Assuntos
Neoplasias Encefálicas/genética , Diferenciação Celular/genética , Glioma/genética , Isocitrato Desidrogenase/genética , Mutação/genética , Neoplasias Encefálicas/patologia , Metilação de DNA/genética , Glioma/patologia , Humanos
7.
Methods Mol Biol ; 1741: 227-237, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29392705

RESUMO

Gliomas are malignant primary tumors of the central nervous system. Their cell-of-origin is thought to be a neural progenitor or stem cell that acquires mutations leading to oncogenic transformation. Thanks to advances in human stem cell biology, it has become possible to derive human cell types that represent putative cells-of-origin in vitro and model the gliomagenesis process by systematically introducing genetic alterations in these human cells. Here, we present methods to derive human neural stem cells (NSCs) from human embryonic stem cells (hESCs). Because these NSCs are genetically unmodified at baseline, they can be used as a cellular platform to study the effects of individual oncogenic hits in a well-controlled manner in the backdrop of a human genetic background. We also present some key applications of these NSCs, which include their transduction with lentiviral vectors, their neuroglial differentiation and xenografting methods into immunocompromised mice to assess in vivo behavior.


Assuntos
Desdiferenciação Celular , Glioma/patologia , Células-Tronco Embrionárias Humanas/citologia , Modelos Biológicos , Células-Tronco Neurais , Animais , Astrócitos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Xenoenxertos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lentivirus/genética , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia
8.
J Am Soc Nephrol ; 29(4): 1154-1164, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29371417

RESUMO

Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P<0.05) observed in controls and increased the mitochondria membrane potential (P<0.05), maximal respiratory capacity (P<0.05), and intracellular ATP levels (P<0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemia-reperfusion injury and mimics ischemic preconditioning.


Assuntos
Precondicionamento Isquêmico , Isocitrato Desidrogenase/genética , Rim/irrigação sanguínea , Trifosfato de Adenosina/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Creatinina/sangue , Vetores Genéticos/administração & dosagem , Injeções Intravenosas , Isocitrato Desidrogenase/fisiologia , Túbulos Renais Proximais/citologia , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Recidiva , Transfecção , Regulação para Cima
9.
Cell Rep ; 21(5): 1267-1280, 2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29091765

RESUMO

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.


Assuntos
Isocitrato Desidrogenase/genética , Fatores de Transcrição SOXB1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Nuclear Ligada ao X/genética , Animais , Apoptose , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Fator de Ligação a CCCTC/metabolismo , Diferenciação Celular , Células Cultivadas , Metilação de DNA , Epigênese Genética , Humanos , Isocitrato Desidrogenase/metabolismo , Camundongos , Camundongos SCID , Gradação de Tumores , Invasividade Neoplásica , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Interferência de RNA , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Proteína Nuclear Ligada ao X/antagonistas & inibidores , Proteína Nuclear Ligada ao X/metabolismo
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