Fig 2: EZH2 deficiency decreases MYCN and MYC expression and transcriptional activity in neuroblastoma cells.a Immunoblot to detect MYCN or MYC levels upon EZH2 depletion with specific sgRNAs in MYCN-amplified (Kelly, SK-N-BE2 and SMS-KAN) and MYCN-nonamplified (SK-N-AS, SH-SY5Y and NBLS) neuroblastoma cells. Actin was used as a loading control. The experiments were independently repeated three times with similar results. b Heatmap presentation of genome-wide MYCN and pol II occupancies in MYCN-amplifed SK-N-BE2 cells analyzed by ChIP-Seq. c Genome browser views of MYCN occupancy in the NCL and PRMT5 loci in SK-N-BE2 cells with or without EZH2 depletion. d Binding of MYCN to the NCL or PRMT5 promoter was analyzed by ChIP-qPCR in SK-N-BE2 cells with or without EZH2 depletion. Averages of fold enrichment between anti-MYCN and the isotype IgG control are shown. Graph shows mean ± SD from triplicates; significance was determined by two-way ANOVA test followed by Tukey’s correction. e Real-time qPCR analysis of representative MYCN target genes in SK-N-BE2 cells upon EZH2 depletion. Graph shows mean ± SD from triplicates; significance was determined by unpaired two-tailed Student’s t-test. Source data are provided as a Source data file.

Fig 3: EZH2 regulates MYCN stabilization independent of its methyltransferase activity.a Analysis of MYCN polyubiquitination in the presence of WT EZH2 or the methyltransferase-inactive mutant (H689A). 293T cells were transfected with plasmids expressing epitope-tagged MYCN, ubiquitin, FBW7α and WT or H689A EZH2 for 48 h as shown. Ubiquitin-conjugated MYCN proteins were pulled down with SBP beads and subjected to immunoblot with ubiquitin antibody. b Comparison of MYCN protein levels in 293T cells expressing EZH2 WT or H689A. 293T cells were transfected with plasmids expressing epitope-tagged MYCN, FBW7α, and EZH2 (WT or H689A) for 48 h as shown. MYCN and other indicated proteins were analyzed by immunoblots. c Kelly cells upon shRNA depletion of endogenous EZH2 (targeting EZH2 3′UTR) were transduced with EZH2 WT or H689A. MYCN and other indicated proteins were analyzed by immunoblots. Histone H3 was used as a loading control. d Immunoblot of MYCN in the presence of various EZH2 inhibitors. Kelly cells were respectively treated with 5 μM of DZNep, GSK126, or EPZ6438 for 48 h before subjected to immunoblotting. e Co-immunoprecipitation (co-IP) to detect the protein-protein between MYCN and FBW7αΔF in the presence of EZH2 WT and H689A. 293T cells were transfected with plasmids expressing epitope-tagged MYCN, FBW7αΔF (F-box deletion mutant) and increasing amounts of EZH2 WT or H689A mutant for 48 h as shown. HA-MYCN-associated proteins were analyzed by immunoblot. f In vitro pull-down experiments to detect the protein–protein between MYCN and EZH2 (or FBW7α). Recombinant His-tagged MYCN was incubated with GST-tagged FBW7α and/or increasing amounts of EZH2. MYCN-associated proteins were precipitated by nickel beads and analyzed by immunoblotting. The experiments were independently repeated three times with similar results (a–f). Source data are provided as a Source data file.

Fig 4: Ezh2 conditional knockout effectively depletes MYCN and impedes tumor formation of MYCN-driven neuroblastoma.a, b Analysis of MYCN expression and cell proliferation upon shRNA depletion of murine Ezh2 (a) or administration of 5 μM of DZNep or GSK126 (b) in primary neuroblastoma cells from TH-MYCN+/+ mice. Data shown were obtained from averages (±SD) of technical triplicates; significance was determined by two-way ANOVA test followed by Tukey’s correction (a, b). c Kaplan–Meier survival plot of tumor-bearing TH-MYCN+/+ mice treated with DZNep (3 mg/kg) or GSK126 (50 mg/kg). Significant difference between the DZNep-treated group versus control was determined by log-rank test. d Immunohistochemistry detection of Ezh2, MYCN, and H3K27me3 upon DZNep or GSK126 treatment. Representative stains of indicated proteins are shown using paraffin-embedded tumor sections derived from (c). The scale bar represents 50 μm. e Immunoblots of the indicated proteins in primary sympathetic ganglia lysates from Ezh2-deleted TH-MYCN+/+ mice and age-matched controls. Histone H3 was used as a loading control. f Representative FDG-PET images of murine neuroblastoma (left) and quantification of the standard glucose uptake in Ezh2-deleted TH-MYCN+/+ mice and age-matched controls (n = 8 per group). Graph shows mean ± SD; significance was determined by unpaired two-tailed Student’s t-test. g Immunoblots of Ezh2 and MYCN in TH-MYCN+/+ primary neuroblastoma tumor lysates. h Correlation between Ezh2 and MYCN protein levels in mouse neuroblastoma samples as shown in (g). The experiments were independently repeated three times with similar results (d–e, g). Source data are provided as a Source data file.

Fig 5: Depletion of EZH2 reduces MYC(N) expression and suppresses growth of MYC(N)-driven small cell carcinoma cells and MYC-driven Burkitt’s lymphoma cells.a–c Analysis of MYCN or MYC expression and cell proliferation upon EZH2 depletion by specific sgRNAs or administration of 5 μM of DZNep or GSK126 in NCI-H526 (a), NCI-H82 (b), or Daudi (c) cells. Actin or H3 was used as a loading control. Data shown were obtained from averages (± SD) of technical triplicates; significance was determined by two-way ANOVA test followed by Tukey’s correction. The experiments were independently repeated three times with similar results (a–c). d, e Xenograft tumor growth assays were performed using NCI-H526 (d) and NCI-H82 (e) cells upon DZNep or GSK126 treatment. Images of subcutaneous tumors (n = 5 per group) under different treatments are shown. Data shown were obtained from averages (±SD) of technical triplicates; significance was determined by one-way ANOVA test followed by Tukey’s correction (d, e). Source data are provided as a Source data file.