HY-152733 Search Results


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Euchromatic histone-lysine methyltransferase (EHMT) inhibitors enhance IFN-γ-induced CXCL10 production in human and mouse neuroblastoma cell lines. (A) Western blots for H3K9me2 and β-actin of human and mouse NB cell lines treated with EHMT inhibitors <t>UNC-0638</t> and BIX-01294 for 96 hours (2 µM). Representative blots of n=3. (B) qRT-PCR analysis CXCL10 mRNA expression (normalized to UBC ) in SK-N-BE cells treated with UNC-0638 or BIX-01294 for 96 hours (2 µM) and stimulated with increasing concentrations of IFN-γ for the last 24 hours. Results from biological replicates (n=3). (C) Experimental setup as described in (B) but ELISA for CXCL10 protein level (pg mL −1 ) in culture supernatant. Results from biological replicates (n=3). (D) qRT-PCR analysis of CXCL10 mRNA expression (normalized to UBC ) and (E) ELISA for CXCL10 protein level (pg mL −1 ) in supernatant. IMR-32 cells treated with BIX-01294 for 96 hours and stimulated with IFN-γ (250 U mL −1 ) for the last 24 hours. Results from biological replicates (n=3). (F, G) as described in (D, E), but mNB-A1 cells. (H, I) and (J, K) as described in (D, E), but SH-SY5Y and SK-N-AS cells. Results from biological replicates (n=3). Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test. Error bars: mean±SD. IFN, interferon; NB, neuroblastoma.
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Euchromatic histone-lysine methyltransferase (EHMT) inhibitors enhance IFN-γ-induced CXCL10 production in human and mouse neuroblastoma cell lines. (A) Western blots for H3K9me2 and β-actin of human and mouse NB cell lines treated with EHMT inhibitors UNC-0638 and BIX-01294 for 96 hours (2 µM). Representative blots of n=3. (B) qRT-PCR analysis CXCL10 mRNA expression (normalized to UBC ) in SK-N-BE cells treated with UNC-0638 or BIX-01294 for 96 hours (2 µM) and stimulated with increasing concentrations of IFN-γ for the last 24 hours. Results from biological replicates (n=3). (C) Experimental setup as described in (B) but ELISA for CXCL10 protein level (pg mL −1 ) in culture supernatant. Results from biological replicates (n=3). (D) qRT-PCR analysis of CXCL10 mRNA expression (normalized to UBC ) and (E) ELISA for CXCL10 protein level (pg mL −1 ) in supernatant. IMR-32 cells treated with BIX-01294 for 96 hours and stimulated with IFN-γ (250 U mL −1 ) for the last 24 hours. Results from biological replicates (n=3). (F, G) as described in (D, E), but mNB-A1 cells. (H, I) and (J, K) as described in (D, E), but SH-SY5Y and SK-N-AS cells. Results from biological replicates (n=3). Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test. Error bars: mean±SD. IFN, interferon; NB, neuroblastoma.

Journal: Journal for Immunotherapy of Cancer

Article Title: Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma

doi: 10.1136/jitc-2020-001335

Figure Lengend Snippet: Euchromatic histone-lysine methyltransferase (EHMT) inhibitors enhance IFN-γ-induced CXCL10 production in human and mouse neuroblastoma cell lines. (A) Western blots for H3K9me2 and β-actin of human and mouse NB cell lines treated with EHMT inhibitors UNC-0638 and BIX-01294 for 96 hours (2 µM). Representative blots of n=3. (B) qRT-PCR analysis CXCL10 mRNA expression (normalized to UBC ) in SK-N-BE cells treated with UNC-0638 or BIX-01294 for 96 hours (2 µM) and stimulated with increasing concentrations of IFN-γ for the last 24 hours. Results from biological replicates (n=3). (C) Experimental setup as described in (B) but ELISA for CXCL10 protein level (pg mL −1 ) in culture supernatant. Results from biological replicates (n=3). (D) qRT-PCR analysis of CXCL10 mRNA expression (normalized to UBC ) and (E) ELISA for CXCL10 protein level (pg mL −1 ) in supernatant. IMR-32 cells treated with BIX-01294 for 96 hours and stimulated with IFN-γ (250 U mL −1 ) for the last 24 hours. Results from biological replicates (n=3). (F, G) as described in (D, E), but mNB-A1 cells. (H, I) and (J, K) as described in (D, E), but SH-SY5Y and SK-N-AS cells. Results from biological replicates (n=3). Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test. Error bars: mean±SD. IFN, interferon; NB, neuroblastoma.

Article Snippet: Either 2 µM of EHMT inhibitor UNC-0638 (Cayman Chemical, #10734), 3 µM of EZH2 inhibitor EPZ011989 (MedChemExpress, #HY-16986) or the combination of both (UNC-0638 2 µM plus EPZ011989 3 µM) were added to culture media for 7 days with media changes every 3 days.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Discordant effects of euchromatic histone-lysine methyltransferase (EHMT) inhibitors on growth inhibition and IFN-γ transcriptional responses dependent on MYCN status in human neuroblastoma cells (A) and (B) representative images from biological replicates (n=3) of stained culture dishes of MYCN -amplified and MYCN -non-amplified human NB cell lines treated with UNC-0638 and BIX-01249 at indicated concentrations for 96 hours. (C) and (D) Quantifications of the experiments described in (A) and (B). (E) CXCL10 and (F) hallmark interferon gamma response signature expression (log2) in human NB cells based on 3′mRNA-seq data and averaged values from biological duplicates. Left panels: Scatter plots comparing expressing expression in the presence of IFN-γ versus IFN-γ and UNC-0638. Vertical bars indicate log2 fold changes (Log2FCs). Right panels: Log2FCs replotted from scatter plots for statistical comparison. (G) Left panel: GSEA results from group comparison as indicated based on 3′mRNA-seq data described in (E) and (F). Right panel: GSEA plot for indicated gene set. (H) Log2 averaged expression gene set from (G) in NB cell lines stratified by MYCN status. Cell lines as indicated. Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test with logarithmic relative growth (%) values comparing groups MYCN -amplified versus MYCN -non-amplified at each concentration in (C) and (D). Two-sided unpaired ratio t-test in (E, F, H). Error bars: mean±SD. Boxplots: Boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median. ES, enrichments score; FDR, false discovery rate; FWER, family wise error rate; GSEA, gene set enrichment analysis; IFN, interferon; NB, neuroblastoma; NES, normalized ES.

Journal: Journal for Immunotherapy of Cancer

Article Title: Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma

doi: 10.1136/jitc-2020-001335

Figure Lengend Snippet: Discordant effects of euchromatic histone-lysine methyltransferase (EHMT) inhibitors on growth inhibition and IFN-γ transcriptional responses dependent on MYCN status in human neuroblastoma cells (A) and (B) representative images from biological replicates (n=3) of stained culture dishes of MYCN -amplified and MYCN -non-amplified human NB cell lines treated with UNC-0638 and BIX-01249 at indicated concentrations for 96 hours. (C) and (D) Quantifications of the experiments described in (A) and (B). (E) CXCL10 and (F) hallmark interferon gamma response signature expression (log2) in human NB cells based on 3′mRNA-seq data and averaged values from biological duplicates. Left panels: Scatter plots comparing expressing expression in the presence of IFN-γ versus IFN-γ and UNC-0638. Vertical bars indicate log2 fold changes (Log2FCs). Right panels: Log2FCs replotted from scatter plots for statistical comparison. (G) Left panel: GSEA results from group comparison as indicated based on 3′mRNA-seq data described in (E) and (F). Right panel: GSEA plot for indicated gene set. (H) Log2 averaged expression gene set from (G) in NB cell lines stratified by MYCN status. Cell lines as indicated. Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test with logarithmic relative growth (%) values comparing groups MYCN -amplified versus MYCN -non-amplified at each concentration in (C) and (D). Two-sided unpaired ratio t-test in (E, F, H). Error bars: mean±SD. Boxplots: Boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median. ES, enrichments score; FDR, false discovery rate; FWER, family wise error rate; GSEA, gene set enrichment analysis; IFN, interferon; NB, neuroblastoma; NES, normalized ES.

Article Snippet: Either 2 µM of EHMT inhibitor UNC-0638 (Cayman Chemical, #10734), 3 µM of EZH2 inhibitor EPZ011989 (MedChemExpress, #HY-16986) or the combination of both (UNC-0638 2 µM plus EPZ011989 3 µM) were added to culture media for 7 days with media changes every 3 days.

Techniques: Inhibition, Staining, Amplification, Expressing, Concentration Assay

Combined euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibition restores robust transcriptional responses to IFN-γ and CXCL10 chemokine production in MYCN -amplified human neuroblastoma cells. (A) Western blots H3K27me3 and β-actin in SK-N-BE and IMR-32 cells treated with different PRC2 and EZH2 inhibitors (all 3 µM) for 96 hours. Representative blots of n=3. (B) Western blots for H3K27me3, H3K9me2 and β-actin in SK-N-BE and IMR-32 cells treated with EHMT inhibitor UNC-0638 (2 µM), EZH2 inhibitor EPZ011989 (3 µM) or the combination of both drugs for 96 hours. Representative blots of n=3. (C) CXCL10 mRNA expression assessed by qRT-PCR in SK-N-BE NB cells treated as indicated. Results from biological replicates (n=3). (D) Heatmap visualizing the transcriptional response to IFN-γ in SK-N-BE and (E) IMR-32 cells treated with vehicle or UNC-0638, EPZ011989 or both for 7 days. IFN-γ (250 U/mL) was added for the last 24 hours. Experiments performed in biological replicates (n=3). (F, G) ELISA for CXCL10 protein level (pg/mL) in supernatants from SK-N-BE (F) and IMR-32 NB (G) cells treated as described in (C) and (D). Results from biological replicates (n=3). Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test. Error bars: mean±SD. IFN, interferon; NB, neuroblastoma.

Journal: Journal for Immunotherapy of Cancer

Article Title: Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma

doi: 10.1136/jitc-2020-001335

Figure Lengend Snippet: Combined euchromatic histone-lysine methyltransferase (EHMT) and EZH2 inhibition restores robust transcriptional responses to IFN-γ and CXCL10 chemokine production in MYCN -amplified human neuroblastoma cells. (A) Western blots H3K27me3 and β-actin in SK-N-BE and IMR-32 cells treated with different PRC2 and EZH2 inhibitors (all 3 µM) for 96 hours. Representative blots of n=3. (B) Western blots for H3K27me3, H3K9me2 and β-actin in SK-N-BE and IMR-32 cells treated with EHMT inhibitor UNC-0638 (2 µM), EZH2 inhibitor EPZ011989 (3 µM) or the combination of both drugs for 96 hours. Representative blots of n=3. (C) CXCL10 mRNA expression assessed by qRT-PCR in SK-N-BE NB cells treated as indicated. Results from biological replicates (n=3). (D) Heatmap visualizing the transcriptional response to IFN-γ in SK-N-BE and (E) IMR-32 cells treated with vehicle or UNC-0638, EPZ011989 or both for 7 days. IFN-γ (250 U/mL) was added for the last 24 hours. Experiments performed in biological replicates (n=3). (F, G) ELISA for CXCL10 protein level (pg/mL) in supernatants from SK-N-BE (F) and IMR-32 NB (G) cells treated as described in (C) and (D). Results from biological replicates (n=3). Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided unpaired t-test. Error bars: mean±SD. IFN, interferon; NB, neuroblastoma.

Article Snippet: Either 2 µM of EHMT inhibitor UNC-0638 (Cayman Chemical, #10734), 3 µM of EZH2 inhibitor EPZ011989 (MedChemExpress, #HY-16986) or the combination of both (UNC-0638 2 µM plus EPZ011989 3 µM) were added to culture media for 7 days with media changes every 3 days.

Techniques: Inhibition, Amplification, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

High-risk neuroblastomas with high euchromatic histone-lysine methyltransferase (EHMT) and EZH2 activity are characterized by MYCN amplification and a T-cell infiltration-poor tumor microenvironment. (A) Outline of bioinformatic strategy. (B, C, D) Generation of drug response signatures from overlap of differentially expressed genes in SK-N-BE and IMR-32 cells treated with UNC-0638 (B), EPZ011989 (C) or both drugs (D). (E) Exemplary visualization of calculation of EHMT activity scores for high-risk NB samples. (F, G) Exemplary heatmap visualization of expression of UNC-0638 drug response genes in high-risk NB samples ranked by increasing EHMT activity score. (H–J) Heatmaps visualizing immune contexture marker genes (eg, CD8A , CXCL10 ), EHMT2/1 , EZH2/1 and MYCN in high-risk NB samples ranked by increasing activity scores of EHMT (H), EZH2 (I) and EHMT+EZH2 (J). Pearson correlation coefficients are indicated besides the names of the transcripts. Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided t-test for Pearson product moment correlation coefficient. NB, neuroblastoma.

Journal: Journal for Immunotherapy of Cancer

Article Title: Druggable epigenetic suppression of interferon-induced chemokine expression linked to MYCN amplification in neuroblastoma

doi: 10.1136/jitc-2020-001335

Figure Lengend Snippet: High-risk neuroblastomas with high euchromatic histone-lysine methyltransferase (EHMT) and EZH2 activity are characterized by MYCN amplification and a T-cell infiltration-poor tumor microenvironment. (A) Outline of bioinformatic strategy. (B, C, D) Generation of drug response signatures from overlap of differentially expressed genes in SK-N-BE and IMR-32 cells treated with UNC-0638 (B), EPZ011989 (C) or both drugs (D). (E) Exemplary visualization of calculation of EHMT activity scores for high-risk NB samples. (F, G) Exemplary heatmap visualization of expression of UNC-0638 drug response genes in high-risk NB samples ranked by increasing EHMT activity score. (H–J) Heatmaps visualizing immune contexture marker genes (eg, CD8A , CXCL10 ), EHMT2/1 , EZH2/1 and MYCN in high-risk NB samples ranked by increasing activity scores of EHMT (H), EZH2 (I) and EHMT+EZH2 (J). Pearson correlation coefficients are indicated besides the names of the transcripts. Statistics: *p<0.05; **p<0.01; ***p<0.001; two-sided t-test for Pearson product moment correlation coefficient. NB, neuroblastoma.

Article Snippet: Either 2 µM of EHMT inhibitor UNC-0638 (Cayman Chemical, #10734), 3 µM of EZH2 inhibitor EPZ011989 (MedChemExpress, #HY-16986) or the combination of both (UNC-0638 2 µM plus EPZ011989 3 µM) were added to culture media for 7 days with media changes every 3 days.

Techniques: Activity Assay, Amplification, Expressing, Marker