Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 downregulates de novo DNA methyltransferase DNMT3A. A RNA-sequencing-based transcript expression of DNA methylating and demethylating enzymes in U87-EGFRvIII cells with siRNA against Scramble sequence or Rictor. Bar graph showed the expression level of de novo DNA methyltransferases including DNMT3A and DNMT3B in U87-EGFRvIII cells with siScramble or siRictor. KD, knockdown; ND, not detected; NS, not significant; RPKM, reads per kilobase per million mapped reads. B Transcript expression of DNMT3A gene in various types of cancers, based on TCGA datasets. GBM is highlighted in a red box. C Relative protein expression of DNMT3A in U87-EGFRvIII cells treated with drugs targeting mTORC2 substrates including Akt (Akti-1/2: 2.5 µM), SGK1 (GSK650394: 2.0 µM) and PKC-α (Bis-I: 10 µM) for 48 h

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: RNA Sequencing, Expressing, Sequencing, Knockdown

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 redistributes EZH2 in the DNMT3A promoter to suppress its expression. A Immunoblot detection of DNMT3A and H3 p.K27me3 in U87-EGFRvIII cells treated with GSK126 (EZH2 inhibitor: 2.5 µM) and GSKJ4 (JmjC inhibitor: 10 µM) for 48 h. B , C ChIP-qPCR analysis on H3 p.K27me3 ( B ) and EZH2 ( C ) enrichment in DNMT3A promoter regions of U87-EGFRvIII cells transfected with siRNAs against Scramble sequence or Rictor. D Immunoblot analyses of acetylated EZH2 (Ac-EZH2) in U87-EGFRvIII cells with shScramble or shRictor. Ac-K, acetylated-lysine; IB, immunoblotting; IP, immunoprecipitation. E Immunoblot analyses of acetylated EZH2 (Ac-EZH2) in U87 cells with overexpression of GFP or Rictor. Ac-K, acetylated-lysine; IB, immunoblotting; IP, immunoprecipitation. F Analyses on acetylation and redistribution of EZH2 on the DNMT3A promoter in U87-EGFRvIII cells with addition of TSA (1.0 µM) and acetate (10 mM) for 48 h. Ac, acetate. G mRNA expression of DNMT3A in U87-EGFRvIII cells treated by PP242 (mTORC1/C2 inhibitor: 5 uM) along with supplementation of TSA (1.0 µM) and acetate (10 mM) for 48 h. H mTORC2 drives protein acetylation to redistribute EZH2 into the DNMT3A promoter region, and increases H3 p.K27me3 to suppress the expression of DNMT3A in GBM. Ac, acetyl-group

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Expressing, Western Blot, ChIP-qPCR, Transfection, Sequencing, Immunoprecipitation, Over Expression

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2-induced DNA hypomethylation is dependent on the downregulation of DNMT3A. A Enzymatic activities of fractionated nuclear DNMT in U87-EGFRvIII cells with shScramble or shRictor. OD, optical density; U, unit of DNMT enzyme control. B RNA-sequencing analysis of potential DNMT3A target genes regarding cell proliferation and differentiation in U87-EGFRvIII cells with siScramble or siRictor. Note that mTORC2 activation (Scramble) upregulates proliferation-related genes, but downregulates differentiation-related genes. KD, knockdown. C A shift in global DNA methylation level, represented by LINE-1 methylation in Scramble- or Rictor-depleted U87-EGFRvIII cells, with concurrent knockdown of DNMT3A. D Immunohistochemistry for mTORC2 activation marker (p-Akt S473), DNMT3A and 5-mC in human GBM tissue (n = 21). The scatter plots showed the negative or positive correlation between DNMT3A and mTORC2 marker (p-Akt S473: upper panel) or 5-mC (lower panel) respectively, based on quantitative immunohistochemistry. Scale bars, 40 µm (for pAKT and DNMT3A) and 80 µm (for 5-mC)

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Control, RNA Sequencing, Activation Assay, Knockdown, DNA Methylation Assay, Methylation, Immunohistochemistry, Marker

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2-driven global DNA hypomethylation reprograms glutamatergic network in GBM. A Heatmap of the DNA methylation profile (Infinium HumanMethylation 850 K BeadChip) in U87-EGFRvIII cells with shScramble or shRictor. DMP, differential methylation probes; KD, knockdown; SD, standard deviation. B Differential DNA-methylated regions (DMRs) in U87-EGFRvIII cells with shScramble or shRictor, including CpG-islands. ExonBnd, exon boundaries; IGR, intergenic region; TSS, transcription start sites; UTR, untranslated region. C GO term analyses on David_RHyper10perGenes on mTORC2 inhibition.”Chemical synaptic transmission (GO:0007268)” suggest that mTORC2-dependent hypomethylator could regulate the expression of genes related to EAA metabolism. D mRNA expression of glutamate transporters (SLC1A1, SLC1A3, SLC1A6) in Rictor knockdown U87-EGFRvIII GBM cells. E Measurement of EAA (glutamate and aspartate) indicated that Rictor knockdown reduced intracellular glutamate (Glu) and aspartate (Asp) in U87-EGFRvIII GBM cells. Conc, concentration

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: DNA Methylation Assay, Methylation, Knockdown, Standard Deviation, Inhibition, Transmission Assay, Expressing, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: Reprogramming of glutamate metabolism drives invasive phenotypes in GBM. A Lower methylation signals by MeDIP-qPCR on the GRIA1 promoter and higher expression of GRIA1 transcripts were observed in U87 cells with lentivirus-mediated overexpression of human Rictor (Rictor OE) in comparison with control (GFP). Met, methylation (5-mC). B GBM tumors from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4) were probed by immunohistochemistry against GRIA1. Note intratumoral heterogeneity of GRIA1 immunoreactivity in accordance with the status of mTORC2 activation (pAkt) and 5-mC expression. Peri-necrotic area indicates pAkt_low/5-mC_high region, and non-necrotic to pAkt_high/5-mC_low region. Refer to Fig. B. Nec, necrosis. Scale bar, 20 µm. C Scratch assays using the co-culture of U87-EGFRvIII (GBM) cells with siRNA-mediated knockdown of GRIA1 and SH-SY5Y (neuroblastoma) cells. The area of gap was calculated 24 h after scratch. Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. D Knockdown of GRIA1 significantly ( p < 0.01) affected GBM cell migration in the co-culture of U87-EGFRvIII and SH-SY5Y cells. E Measurement of glutamate (Glu) indicated that GRIA1 knockdown increased extracellular Glu (reduced intracellular Glu) in U87-EGFRvIII GBM cells. Conc, concentration. F Wound healing/migration assay on the co-culture of SH-SY5Y neuroblastoma cells with U87-EGFRvIII GBM cells treated by PhTx-74 (GRIA1/GRIA2 inhibitor: 20 μM) or a combination of PhTx-74 (20 μM) with GSK2256098 (FAK inhibitor: 100 nM). Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. G PhTx-74 treatment increased extracellular Glu (reduced intracellular Glu), and FAK inhibitor decreased phosphorylation of FAK (Tyr397) in U87-EGFRvIII GBM cells. Conc, concentration. H TCGA datasets on overall survival and progression free survival of GBM cases stratified by the expression level of GRIA1 transcripts

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Methylation, Methylated DNA Immunoprecipitation, Expressing, Over Expression, Comparison, Control, Infection, Retroviral, Immunohistochemistry, Activation Assay, Co-Culture Assay, Knockdown, Software, Migration, Concentration Assay, Phospho-proteomics

Journal: Acta Neuropathologica Communications

Article Title: DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

doi: 10.1186/s40478-024-01750-x

Figure Lengend Snippet: mTORC2 activation correlates with global DNA hypomethylation phenotypes in RTK-mutated GBM. A Immunohistochemical (IHC) staining of human GBM tissue (n = 20) with antibodies against mutant EGFR (EGFRvIII) and a DNA methylation marker (5-mC). EGFR amplification was assessed by FISH with probes for EGFR (7p11.2, Red) and CEP7 (7p11.1-q11.1, Green). Scale bar, 40 µm. B Cerebral and brainstem tissue with GBM tumors was harvested from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4). Immunohistochemistry was performed on paraffin-embedded tissue sections against a DNA methylation marker (5-mC) and an mTORC2 marker (p-Akt S473). Nec, necrosis. Scale bars, 50 µm (upper panels) and 20 µm (lower panels). C Immunofluorescent staining of 5-mC in U87-EGFRvIII cells transfected with shRNAs against control sequence (scramble) or Rictor (shRictor#1). Scale bar, 10 µm. D Dot blot analysis of 5-mC in U87-EGFRvIII cells transfected with shScramble versus shRictor#1 (upper panel), or overexpressed (OE) with GFP versus Rictor (lower panel). Total DNA for each sample was determined by methylene blue staining. E Detection of global DNA methylation (ELISA-based assay), represented by methylation of LINE-1 retrotransposable elements in U87-EGFRvIII cells transfected with shScramble or shRictor. OD, optical density; STD, standard

Article Snippet: Lentiviral shRNA vectors targeting human Rictor and scramble sequences were obtained from Addgene (shRictor#1) and Santa Cruz (shRictor#2).

Techniques: Activation Assay, Immunohistochemical staining, Immunohistochemistry, Mutagenesis, DNA Methylation Assay, Marker, Amplification, Infection, Retroviral, Staining, Transfection, Control, Sequencing, Dot Blot, Enzyme-linked Immunosorbent Assay, Methylation