Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene–amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc–mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522–33. ©2017 AACR.

Neuroblastoma is the most commonly diagnosed pediatric solid tumor in early childhood (1). Neuroblastoma arises from neural crest cells and is characterized by variable clinical behavior, from spontaneous regression to inexorable progression (2). Adverse clinical prognostic factors include age > 18 months at diagnosis, advanced disease stage, and amplification of the MYCN oncogene, which encodes the N-Myc oncoprotein (3–5).

N-Myc is expressed during embryogenesis in the nervous system (6, 7). N-Myc induces neuroblastoma by regulating the expression of genes important for cell differentiation, malignant transformation, and cell proliferation (8). Like its analogue c-Myc oncoprotein, N-Myc activates target gene transcription by forming a heterodimer with MAX, and this heterodimer in turn recruits a range of cofactors that alter chromatin structure, such as histone H3 lysine 79 (H3K79) dimethylation (9–11).

DOT1L is a unique histone methyltransferase as it is the only known histone methyltransferase that catalyzes monomethylation (me), dimethylation (me2), and trimethylation (me3) at the H3K79 position (12, 13). Quantitative chromatin immunoprecipitation studies of H3K79 methylation across the human genome reveal that H3K79me and H3K79me2 are linked to active gene transcription (14–16). DOT1L is involved in the oncogenesis of several leukemia subtypes characterized by chromosomal translocations involving the mixed lineage leukemia (MLL) oncogene. DOT1L forms a protein complex with the MLL fusion proteins, and DOT1L-mediated H3K79 methylation is responsible for maintaining an open chromatin state around MLL fusion protein target oncogenes (17, 18).

Crucially, H3K79me2 is essential for c-Myc binding to its target gene promoters (10, 11). Here, we investigated the role of DOT1L-mediated H3K79 methylation in N-Myc–induced target gene transcription, neuroblastoma cell proliferation in vitro and tumor progression in vivo, and examined whether DOT1L expression in human neuroblastoma tissues independently predicted patient prognosis.

Cell culture

Neuroblastoma BE(2)-C, NBL-S, SK-N-FI, and HEK-293 cells were cultured in DMEM supplemented with 10% FCS. Kelly cells were grown in RPMI1640 supplemented with 10% FCS (19, 20). BE(2)-C and NBL-S cells were provided by Barbara Spengler (Fordham University, New York, NY) and Dr. Susan Cohn (Northwestern University, Chicago, IL), respectively 20 years ago. HEK-293 cells were obtained from the ATCC 20 years ago. Kelly and SK-N-FI cells were obtained from the European Collection of Cell Cultures and Sigma Aldrich in 2010. The identity of cell lines was verified in 2010, 2014, and 2015 by small tandem repeat profiling conducted at the Garvan Institute of Medical Research (Darlinghurst, New South Wales, Australia) or Cellbank Australia.

Protein coimmunoprecipitation assays

HEK293 human embryonic cells were transiently cotransfected with an N-Myc-expression pcDNA3.1 construct, together with an empty vector or Flag-DOT1L-expression pcDNA3.1 construct for 48 hours. Protein was extracted from the cells and coimmunoprecipitation was carried out using an anti-FLAG antibody (Abcam) or a mouse IgG as a negative control, followed by immunoblot analysis.

GST pull-down assays

Different N-Myc protein fragments were cloned into the pGEX-2T construct, in frame with N-terminal GST. The constructs were transformed into BL-21 E.Coli and IPTG was used for induction of T7-driven transcription. After purification, equal amount of GST-N-Myc protein fragments were immobilized onto glutathione-agarose beads (Sigma). HEK-293T cells were transfected with Flag-DOT1L expression construct, and nuclear protein from the cells was incubated with equal amount of different GST-N-Myc protein fragments immobilized onto glutathione agarose beads. Pulled-down complexes were analyzed by immunoblot with a monoclonal anti-Flag antibody (Santa Cruz Biotechnology), and Ponceau staining detected by ChemiDoc MP (Bio-Rad) was used as loading controls.

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation (ChIP) assays were performed with mouse anti-N-Myc antibody (Merck Millipore), rabbit anti-H3K79me2 antibody (Abcam), rabbit and mouse control IgG (Santa Cruz Biotechnology), followed by PCR with primers designed to cover the core promoter regions of the DOT1L, ODC1, and E2F2 genes containing Myc-responsive element E-Boxes or remote negative control regions. Fold enrichment of the DOT1L, E2F2, and ODC1 gene core promoter regions by the anti-N-Myc or anti-H3K79me2 antibody was calculated by dividing cycle threshold of PCR products from the DOT1L, E2F2, and ODC1 gene core promoter regions by cycle threshold of PCR products from the negative control region, relative to input.

Luciferase reporter assays

Modulation of DOT1L promoter activity by N-Myc was analyzed by luciferase assays. The DOT1L gene core promoter containing the E-Box (−389 bp to +50 bp relative to transcription start site) was cloned into the pLightSwitch_Prom construct (SwitchGear Genomics). BE(2)-C cells were cotransfected with control siRNA or N-Myc siRNA-1, together with Go Clone Promoter Control construct plus the pLightSwitch_Prom construct expressing empty vector or the DOT1L core promoter. Luciferase activities were measured with a LightSwitch Dual Assay System Kit (SwitchGear Genomics), and normalized according to Go Clone Promoter Control construct according to the manufacturer's instructions.

Establishment of doxycycline-inducible control shRNA and DOT1L shRNA expression constructs and neuroblastoma cell lines stably expressing the constructs

The lentiviral doxycycline-inducible GFP-IRES-shRNA FH1tUTG construct from Dr. Marco Herold (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; ref. 21) was used to generate control shRNA- or DOT1L shRNA–expressing construct and neuroblastoma cell lines. DOT1L shRNA and scrambled control shRNA sequences were selected from previously published work (22) and cloned into the FH1tUTG construct. The doxycycline -inducible GFP-IRES-control shRNA or DOT1L shRNA FH1tUTG construct was transfected into 293T cells. The viral media were collected and used to infect BE(2)-C and Kelly neuroblastoma cells over 72 hours with polybrene (Santa Cruz Biotechnology). GFP-based cell sorting was performed for selecting cells with high GFP protein expression.

In vivo mouse experiments

Animal experiments were approved by the Animal Care and Ethics Committee of UNSW Australia, and the animals' care was in accord with institutional guidelines. Female Balb/c nude mice aged 5 to 6 weeks were injected subcutaneously while under anesthesia with either 2 × 106 doxycycline -inducible DOT1L shRNA BE(2)-C cells or 8 × 106 doxycycline -inducible DOT1L shRNA Kelly cells into the right flank. Mice were fed with 10% sucrose water with or with doxycycline at 2 mg/mL when tumors reached 0.005 cm3 in volume. Tumor development was monitored and tumor volume calculated using (length × width × height)/2. Mice were culled when tumor volume reached 1 cm3, and tumor tissues were snap-frozen and analyzed by immunoblotting for DOT1L, ODC1, and E2F2 protein expression.

Patient tumor sample analysis

DOT1L, N-Myc, ODC1, and E2F2 mRNA expression was examined in 88 (Versteeg dataset; ref. 23) and 476 (Kocak dataset; refs.24, 25) human neuroblastoma samples in the publicly available gene expression datasets (http://r2.amc.nl). Correlation between DOT1L and N-Myc, ODC1 as well as E2F2 expression in human neuroblastoma tissues was analyzed with Pearson correlation. Probability of survival was investigated according to the method of Kaplan and Meier and two-sided log-rank tests (26). Multivariable Cox regression analyses were performed after inclusion of disease stage, age at diagnosis, MYCN amplification status, N-Myc and DOT1L expression levels. Probabilities of survival and HR were provided with 95% confidence intervals. Proportionality was confirmed by visual inspection of the plots of log[2log(S(time))] versus log(time), which were observed to remain parallel (27).

Statistical analysis

For statistical analysis, experiments were conducted three times. Data were analyzed with Prism 6 software (GraphPad) and presented as mean ± SE. Differences were analyzed for significance using ANOVA among groups or two-sided t test for two groups. All statistical tests were two-sided. A P value of less than 0.05 was considered statistically significant.

N-Myc upregulates DOT1L expression by binding to the DOT1L gene promoter

Myc proteins induce gene transcription by binding to canonical and noncanonical E-boxes at target gene promoters (9). Our bioinformatics analysis identified a noncanonical E-box (CACGCG) located −288 bp upstream of the DOT1L gene transcription start site. We therefore examined whether N-Myc modulated DOT1L gene expression in neuroblastoma cells. As shown in Fig. 1A and B, transfection of N-Myc–overexpressing BE(2)-C and Kelly neuroblastoma cells with N-Myc siRNA-1 or N-Myc siRNA-2 efficiently knocked down N-Myc mRNA and protein expression, and reduced DOT1L mRNA and protein expression. Consistently, withdrawal of tetracycline from cell culture media of SHEP-Tet/21N cells, which are stably transfected with a tetracycline withdrawal–inducible N-Myc expression construct, increased N-Myc and DOT1L mRNA and protein expression (Fig. 1C).

Figure 1.

N-Myc upregulates DOT1L expression by binding to the DOT1L gene promoter. A and B, BE(2)-C (A) and Kelly (B) neuroblastoma cells were transfected with control siRNA, N-Myc siRNA-1, or N-Myc siRNA-2 for 48 hours, followed by RT-PCR and immunoblot analyses of N-Myc and DOT1L mRNA and protein expression. C, SHEP-Tet/21N cells were treated with tetracycline (2 μg/mL) or vehicle control for 72 hours. RT-PCR and immunoblot analyses were conducted on N-Myc and DOT1L expression. D, Schematic representation of the DOT1L gene promoter. TSS, transcription start site. E, ChIP assays were performed with a control IgG or N-Myc antibody (Ab), followed by PCR with primers targeting a remote negative control region, the 5′ untranscribed region (Amplicon A), the E-Box region (Amplicon B), or the intron 1 region (Amplicons C and D) of the DOT1L gene in BE(2)-C cells. Fold enrichment of the DOT1L gene promoter regions was calculated as the difference in cycle thresholds obtained with the anti-N-Myc Ab compared with the control IgG, relative to input. F, Luciferase assays were performed in BE(2)-C cells after cotransfection with control siRNA or N-Myc siRNA-1, together with Go Clone Promoter Control construct plus pLightSwitch_Prom construct expressing empty vector or DOT1L gene core promoter. Error bars, SEs. *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively.

Figure 1.

N-Myc upregulates DOT1L expression by binding to the DOT1L gene promoter. A and B, BE(2)-C (A) and Kelly (B) neuroblastoma cells were transfected with control siRNA, N-Myc siRNA-1, or N-Myc siRNA-2 for 48 hours, followed by RT-PCR and immunoblot analyses of N-Myc and DOT1L mRNA and protein expression. C, SHEP-Tet/21N cells were treated with tetracycline (2 μg/mL) or vehicle control for 72 hours. RT-PCR and immunoblot analyses were conducted on N-Myc and DOT1L expression. D, Schematic representation of the DOT1L gene promoter. TSS, transcription start site. E, ChIP assays were performed with a control IgG or N-Myc antibody (Ab), followed by PCR with primers targeting a remote negative control region, the 5′ untranscribed region (Amplicon A), the E-Box region (Amplicon B), or the intron 1 region (Amplicons C and D) of the DOT1L gene in BE(2)-C cells. Fold enrichment of the DOT1L gene promoter regions was calculated as the difference in cycle thresholds obtained with the anti-N-Myc Ab compared with the control IgG, relative to input. F, Luciferase assays were performed in BE(2)-C cells after cotransfection with control siRNA or N-Myc siRNA-1, together with Go Clone Promoter Control construct plus pLightSwitch_Prom construct expressing empty vector or DOT1L gene core promoter. Error bars, SEs. *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively.

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We next performed ChIP assays with an anti-N-Myc antibody or control IgG and real-time PCR with primers targeting a negative control region, the E-box region located −288 bp upstream of the DOT1L transcription start site (Amplicon B), 5′ untranscribed region located approximately −600 to −800 bp upstream (Amplicon A), or intron 1 regions located approximately 100 to 180 bp (Amplicon C) or 350 to 500 bp (Amplicon D) downstream (Fig. 1D). The ChIP assays showed that the N-Myc antibody immunoprecipitated the E-box region (Amplicon B) 7-fold higher than the negative control region (Fig. 1E). Amplicons A, C, and D located to either side of amplicon B displayed much lower enrichment by the N-Myc antibody (Fig. 1E). Importantly, luciferase assays showed that transfection with the pLightSwitch_Prom construct encoding the DOT1L gene core promoter containing the E-Box led to high luciferase activity, compared with the empty vector pLightSwitch_Prom construct, and that knocking down N-Myc largely blocked the luciferase activity (Fig. 1F). Taken together, the data suggest that N-Myc upregulates DOT1L gene expression via binding to the DOT1L gene promoter.

DOT1L-mediated H3K79 methylation is required for N-Myc protein binding to target gene promoters

DOT1L forms protein complexes with MLL fusion proteins, and DOT1L-mediated histone H3K79 methylation is essential for MLL fusion protein–mediated leukemogenic gene transcription and leukaemogenesis (28, 29). H3K79me2 has been shown to be essential for c-Myc protein binding at c-Myc target gene promoters (10, 11). We therefore examined whether DOT1L formed a protein complex with N-Myc, and whether DOT1L is required for histone H3K79me2 and N-Myc protein binding at N-Myc target gene promoters.

HEK293 cells were cotransfected with an N-Myc expression construct, together with a construct encoding empty vector or Flag-tagged DOT1L. Immunoprecipitation with an anti-Flag antibody pulled-down N-Myc protein in cells cotransfected with the DOT1L and the N-Myc expression constructs, but did not pull-down N-Myc protein in cells transfected with the N-Myc expression construct alone (Fig. 2A). Furthermore, different N-Myc protein fragments (1-86, 82-254, 249-358, 336-400 and 400-464 amino acids) were cloned into the pGEX-2T construct, in frame with N-terminal GST. GST pull-down assays showed that DOT1L protein bound to the N-Myc 82-254 amino acid fragment (Fig. 2B). The data demonstrate that DOT1L protein directly binds to the Myc Box II region of N-Myc protein.

Figure 2.

DOT1L-mediated H3K79 methylation is required for N-Myc protein binding to target gene promoters. A, HEK293 cells were cotransfected with N-Myc-expression pcDNA3.1 construct, together with empty vector or Flag-DOT1L-expression pcDNA3.1 construct for 48 hours. Protein was extracted from the cells and coimmunoprecipitation (IP) was carried out using an anti-FLAG antibody (Ab) or a mouse IgG as a negative control. Immunoblot analysis was carried out using an anti-N-Myc Ab and anti-DOT1L Ab. B, HEK-293T cells were transfected with Flag-DOT1L expression construct. Nuclear protein extract from the cells was incubated with equal amount of different GST-N-Myc protein fragments immobilized onto glutathione agarose beads, followed by immunoblot with an anti-Flag antibody. As loading controls, Ponceau stained images were detected by ChemiDoc MP. Numbers on the left refer to molecular weights. C and D, BE(2)-C cells were transfected with control siRNA, DOT1L siRNA-1, or DOT1L siRNA-2 for 72 hours. ChIP assays were performed with a control IgG, anti-histone H3K79me2 (C), or anti-N-Myc Ab (D), followed by PCR with primers targeting the E-box regions of the ODC1 and E2F2 gene promoters or the DNA region −4,000 bp upstream of the E2F2 gene transcription start site as the negative control. Error bars, SE. * and ** indicate P < 0.05 and 0.01, respectively.

Figure 2.

DOT1L-mediated H3K79 methylation is required for N-Myc protein binding to target gene promoters. A, HEK293 cells were cotransfected with N-Myc-expression pcDNA3.1 construct, together with empty vector or Flag-DOT1L-expression pcDNA3.1 construct for 48 hours. Protein was extracted from the cells and coimmunoprecipitation (IP) was carried out using an anti-FLAG antibody (Ab) or a mouse IgG as a negative control. Immunoblot analysis was carried out using an anti-N-Myc Ab and anti-DOT1L Ab. B, HEK-293T cells were transfected with Flag-DOT1L expression construct. Nuclear protein extract from the cells was incubated with equal amount of different GST-N-Myc protein fragments immobilized onto glutathione agarose beads, followed by immunoblot with an anti-Flag antibody. As loading controls, Ponceau stained images were detected by ChemiDoc MP. Numbers on the left refer to molecular weights. C and D, BE(2)-C cells were transfected with control siRNA, DOT1L siRNA-1, or DOT1L siRNA-2 for 72 hours. ChIP assays were performed with a control IgG, anti-histone H3K79me2 (C), or anti-N-Myc Ab (D), followed by PCR with primers targeting the E-box regions of the ODC1 and E2F2 gene promoters or the DNA region −4,000 bp upstream of the E2F2 gene transcription start site as the negative control. Error bars, SE. * and ** indicate P < 0.05 and 0.01, respectively.

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We next examined whether knocking down DOT1L expression reduced H3K79me2 and N-Myc protein binding at the gene promoters of ODC1 and E2F2, two known Myc target genes (30–32). BE(2)-C cells were transfected with control siRNA, DOT1L siRNA-1, or DOT1L siRNA-2 for 72 hours. ChIP assays were performed with an anti-H3K79me2 antibody, anti-N-Myc antibody or control IgG, followed by PCR with primers targeting a negative control region or E-Box regions of the ODC1 or E2F2 gene promoters. The assays showed that the H3K79me2 antibody and the anti-N-Myc antibody immunoprecipitated the E-Box regions of both the ODC1 and E2F2 gene promoters in cells transfected with scrambled control siRNA. Compared to the scrambled control siRNA, both DOT1L siRNAs reduced the presence of H3K79me2 and N-Myc protein binding at the E-Box regions (Fig. 2C and D). These findings together suggest that DOT1L and N-Myc form a protein complex at the N-Myc target gene promoters, and that DOT1L-mediated H3K79 methylation is required for N-Myc binding to Myc-binding motifs present at N-Myc target gene promoters.

DOT1L activates the expression of the N-Myc target genes ODC1 and E2F2

We next examined whether DOT1L regulated the expression of the Myc target genes ODC1 and E2F2 in human neuroblastoma cells. BE(2)-C and Kelly cells were transfected with control siRNA, N-Myc siRNA-1, N-Myc siRNA-2, DOT1L siRNA-1, or DOT1L siRNA-2. Consistent with literature (30–32), RT-PCR and immunoblot analyses showed that transfection with N-Myc siRNAs reduced ODC1 and E2F2 mRNA and protein expression (Supplementary Fig. S1). RT-PCR and immunoblot analyses showed that transfection with DOT1L siRNAs efficiently knocked down DOT1L mRNA and protein expression, and significantly reduced ODC1 and E2F2 mRNA and protein expression (Fig. 3A and B).

Figure 3.

DOT1L activates the expression of the N-Myc target genes ODC1 and E2F2. A and B, BE(2)-C (A) and Kelly (B) neuroblastoma cells were transfected with control siRNA, DOT1L siRNA-1, or DOT1L siRNA-2 for 96 hours, followed by RT-PCR and immunoblot analyses of DOT1L, ODC1 and E2F2 mRNA, and protein expression. C and D, Doxycycline-inducible control (Cont) shRNA or DOT1L shRNA BE(2)-C (C) and Kelly (D) cells were treated with vehicle control or 2 μg/mL doxycycline for 72 hours, followed by acid extraction of histone proteins and immunoblot analyses with anti-histone H3 and anti-H3K79me2 antibodies. E and F, Doxycycline-inducible control shRNA or DOT1L shRNA BE(2)-C (E) and Kelly (F) cells were treated with vehicle control or 2 μg/mL doxycycline for 96 hours, followed by RT-PCR and immunoblot analyses of DOT1L, ODC1, and E2F2 expression. Error bars, SEs. **, ***, and **** indicate P < 0.01, 0.001, and 0.0001, respectively.

Figure 3.

DOT1L activates the expression of the N-Myc target genes ODC1 and E2F2. A and B, BE(2)-C (A) and Kelly (B) neuroblastoma cells were transfected with control siRNA, DOT1L siRNA-1, or DOT1L siRNA-2 for 96 hours, followed by RT-PCR and immunoblot analyses of DOT1L, ODC1 and E2F2 mRNA, and protein expression. C and D, Doxycycline-inducible control (Cont) shRNA or DOT1L shRNA BE(2)-C (C) and Kelly (D) cells were treated with vehicle control or 2 μg/mL doxycycline for 72 hours, followed by acid extraction of histone proteins and immunoblot analyses with anti-histone H3 and anti-H3K79me2 antibodies. E and F, Doxycycline-inducible control shRNA or DOT1L shRNA BE(2)-C (E) and Kelly (F) cells were treated with vehicle control or 2 μg/mL doxycycline for 96 hours, followed by RT-PCR and immunoblot analyses of DOT1L, ODC1, and E2F2 expression. Error bars, SEs. **, ***, and **** indicate P < 0.01, 0.001, and 0.0001, respectively.

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To further demonstrate that DOT1L regulates Myc target gene expression, we established BE(2)-C and Kelly cells stably expressing doxycycline -inducible control shRNA or DOT1L shRNA using the GFP-IRES-shRNA expression FH1tUTG construct (21). The cells were then treated with vehicle control or doxycycline or 72 hours, followed by histone protein extraction. Immunoblot analysis confirmed that doxycycline treatment considerably reduced histone H3K79 dimethylation in doxycycline -inducible DOT1L shRNA, but not control shRNA, BE(2)-C and Kelly cells (Fig. 3C and D). We then treated the doxycycline-inducible control shRNA or DOT1L shRNA BE(2)-C and Kelly cells with vehicle control or doxycycline for 96 hours, followed by RNA and protein extraction. RT-PCR and immunoblot analyses showed that treatment with doxycycline efficiently knocked down DOT1L mRNA and protein expression, and also effectively reduced ODC1 and E2F2 mRNA and protein expression in both BE(2)-C and Kelly cells (Fig. 3E and F). In comparison, knocking down N-Myc or DOT1L did not have an effect on the expression of the non-Myc target genes HIF1α and VEGF (Supplementary Fig. S2A and S2B). We next performed differential gene expression studies with Affymetrix microarray in doxycycline-inducible control shRNA and DOT1L shRNA BE(2)-C cells after treatment with vehicle control or doxycycline. Gene-set enrichment analysis showed that genes with E-Boxes at promoters were possibly enriched among those downregulated by DOT1L shRNA (Supplementary Table S1).

Taken together, the data suggest that DOT1L induces histone H3K79 dimethylation and activates the transcription of the N-Myc target genes ODC1 and E2F2.

DOT1L is required for MYCN-amplified neuroblastoma cell proliferation

Upregulation of ODC1 has been well-documented to contribute to N-Myc–mediated neuroblastoma cell proliferation (31). Next, we examined the effect of DOT1L and its target E2F2 upon neuroblastoma cell proliferation. BE(2)-C and Kelly neuroblastoma cells were transfected with control siRNA, DOT1L siRNA-1, DOT1L siRNA-2, E2F2 siRNA-1, or E2F2 siRNA-2 for 96 hours. Alamar blue assays showed that transfection with DOT1L siRNAs or E2F2 siRNAs reduced the numbers of BE(2)-C and Kelly cells (Fig. 4A and B).

Figure 4.

DOT1L is required for MYCN-amplified neuroblastoma cell proliferation. A and B, BE(2)-C and Kelly cells were transfected with control siRNA, DOT1L siRNA-1, DOT1L siRNA-2 (A), E2F2 siRNA-1, or E2F2 siRNA-2 (B) for 96 hours, followed by Alamar blue assays. C, Doxycycline-inducible control shRNA or DOT1L shRNA BE(2)-C and Kelly cells were treated with vehicle control or 2 μg/mL doxycycline for 96 hours, followed by Alamar blue assays. D, BE(2)-C and Kelly cells were treated with control or 1.25 μmol/L SGC0946 for 72 hours, 96 hours, or 7 days, followed by immunoblot analysis of H3K79me2 and total H3 proteins. E–H, BE(2)-C and Kelly (E and F) and MYCN-nonamplified NBL-S and SK-N-FI (G and H) cells were treated with control or SGC0946 for 7 days, followed by RT-PCR analysis of E2F2 gene expression (E and G) or Alamar blue assays (F and H). Error bars, SE. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively.

Figure 4.

DOT1L is required for MYCN-amplified neuroblastoma cell proliferation. A and B, BE(2)-C and Kelly cells were transfected with control siRNA, DOT1L siRNA-1, DOT1L siRNA-2 (A), E2F2 siRNA-1, or E2F2 siRNA-2 (B) for 96 hours, followed by Alamar blue assays. C, Doxycycline-inducible control shRNA or DOT1L shRNA BE(2)-C and Kelly cells were treated with vehicle control or 2 μg/mL doxycycline for 96 hours, followed by Alamar blue assays. D, BE(2)-C and Kelly cells were treated with control or 1.25 μmol/L SGC0946 for 72 hours, 96 hours, or 7 days, followed by immunoblot analysis of H3K79me2 and total H3 proteins. E–H, BE(2)-C and Kelly (E and F) and MYCN-nonamplified NBL-S and SK-N-FI (G and H) cells were treated with control or SGC0946 for 7 days, followed by RT-PCR analysis of E2F2 gene expression (E and G) or Alamar blue assays (F and H). Error bars, SE. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively.

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To further demonstrate that DOT1L is required for neuroblastoma cell proliferation, we treated doxycycline -inducible control shRNA or DOT1L shRNA BE(2)-C and Kelly cells with vehicle control or doxycycline. Alamar blue assays showed that doxycycline treatment had minimal effect on cell proliferation in doxycycline -inducible control shRNA BE(2)-C and Kelly cells, but significantly reduced cell proliferation in doxycycline -inducible DOT1L shRNA BE(2)-C and Kelly cells (Fig. 4C). In addition, cell-cycle analysis showed that knocking down DOT1L expression with doxycycline did not increase the percentage of cells at the sub-G1 phase in doxycycline -inducible DOT1L shRNA BE(2)-C and Kelly cells (Supplementary Fig. S3A and S3B).

Small-molecule DOT1L inhibitors, such as SGC0946, are promising novel anticancer agents (33–35). We next treated MYCN gene–amplified BE(2)-C and Kelly cells with vehicle control or 1.25 μmol/L SGC0946 for 72 hours, 96 hours, or 7 days. Immunoblot analyses confirmed that treatment with SGC0946 led to reduction in H3K79me2 72 hours posttreatment and more significantly 7 days posttreatment (Fig. 4D). RT-PCR analysis showed that SGC0946 reduced the expression of the DOT1L and N-Myc target gene E2F2 (Fig. 4E), and Alamar blue assays confirmed that SGC0946 dose-dependently reduced BE(2)-C and Kelly cells' proliferation (Fig. 4F). In contrast, treatment with SGC0946 did not reduce E2F2 gene expression and did not have an effect on cell proliferation in MYCN gene nonamplified NBL-S and SK-N-FI neuroblastoma cells (Fig. 4G and H). Taken together, the data suggest that DOT1L is required for MYCN-amplified neuroblastoma cell proliferation, and that DOT1L inhibitors are effective anticancer agents against MYCN-amplified neuroblastoma.

DOT1L is required for neuroblastoma tumor growth in vivo

We next examined whether DOT1L is required for neuroblastoma tumor growth in vivo. Doxycycline -inducible DOT1L shRNA BE(2)-C and Kelly cells were injected into the flanks of Balb/c nude mice. Once tumors were palpable, the mice were divided into doxycycline or vehicle control treatment subgroups, and fed with water with or without doxycycline. Tumor growth was monitored and mice culled once tumor volume reached 1 cm3.

The doxycycline treatment subgroup displayed slower tumor growth in comparison with the control treatment subgroup for both doxycycline -inducible DOT1L shRNA BE(2)-C and Kelly cell xenografts (Fig. 5A). Kaplan–Meier survival analysis showed that doxycycline treatment, compared with control treatment, resulted in a longer median survival in mice xenografted with doxycycline-inducible DOT1L shRNA BE(2)-C or Kelly cells by 2-fold (Fig. 5B).

Figure 5.

DOT1L is required for neuroblastoma tumor growth in vivo. A and B, Doxycycline-inducible DOT1L shRNA BE(2)-C and Kelly cells were xenografted into nude mice. Once tumors reached 0.005 cm3 in volume, the mice were divided into doxycycline and vehicle control subgroups, and fed with 10% sucrose water with or without 2 mg/mL doxycycline (DOX). A, Tumor growth was measured every two days using calipers, and mice culled when tumor volume reached 1 cm3. B, Kaplan–Meier survival curves show the probability of overall survival of the mice. P value was obtained from two-sided log-rank test. C and D, Protein was extracted from the tumors from the mice and subjected to immunoblot analysis of DOT1L, ODC1, and E2F2 protein expression.

Figure 5.

DOT1L is required for neuroblastoma tumor growth in vivo. A and B, Doxycycline-inducible DOT1L shRNA BE(2)-C and Kelly cells were xenografted into nude mice. Once tumors reached 0.005 cm3 in volume, the mice were divided into doxycycline and vehicle control subgroups, and fed with 10% sucrose water with or without 2 mg/mL doxycycline (DOX). A, Tumor growth was measured every two days using calipers, and mice culled when tumor volume reached 1 cm3. B, Kaplan–Meier survival curves show the probability of overall survival of the mice. P value was obtained from two-sided log-rank test. C and D, Protein was extracted from the tumors from the mice and subjected to immunoblot analysis of DOT1L, ODC1, and E2F2 protein expression.

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At the conclusion of the in vivo experiment, mice were culled, tumor tissues snap-frozen, and protein extracted. Immunoblot analysis showed that both the doxycycline-inducible DOT1L shRNA BE(2)-C and Kelly tumors displayed a decrease in DOT1L protein expression as a result of doxycycline treatment, compared with the control treatment (Fig. 5C and D for immunoblot gels; Supplementary Fig. S4A and S4B for protein quantification). Protein expression of the N-Myc target genes, ODC1 and E2F2, in both doxycycline-inducible DOT1L shRNA BE(2)-C and Kelly xenograft tumors, was also reduced in the doxycycline treatment group, compared with the control treatment group (Fig. 5C and D for immunoblot gels; Supplementary Fig. S4A and S4B for protein quantification). Taken together, knocking down DOT1L gene expression with doxycycline reduces ODC1 and E2F2 protein expression, reduces neuroblastoma tumor progression, and improves mouse survival in vivo.

High levels of DOT1L gene expression in human neuroblastoma tissues independently predict poor patient prognosis

To assess clinical relevance of DOT1L expression in human neuroblastoma tissues, we examined DOT1L gene expression in human neuroblastoma tissues in the publicly available Versteeg (23) and Kocak (24, 25) microarray gene expression–patient prognosis datasets, downloaded from the R2 platform (http://r2.amc.nl; last accessed on August 26, 2014). Two-sided Pearson correlation study showed that DOT1L mRNA expression positively correlated to N-Myc mRNA expression in the 88 human neuroblastoma tissues of the Versteeg dataset and in the 476 human neuroblastoma tissues of the Kocak dataset (Fig. 6A). In addition, DOT1L mRNA expression positively correlated to ODC1 and E2F2 mRNA expression in human neuroblastoma tissues (Supplementary Fig. S5).

Figure 6.

High levels of DOT1L gene expression in human neuroblastoma tissues independently predict poor patient prognosis. A, Two-sided Pearson correlation was employed to analyze correlation between DOT1L and N-Myc mRNA expression in 88 and 476 human neuroblastoma samples in the publicly available microarray gene expression Versteeg and Kocak datasets downloaded from the R2 platform (http://r2.amc.nl). B and C, Kaplan–Meier curves showed the probability of overall survival of patients according to DOT1L mRNA expression levels in the 88 and 476 neuroblastoma patients in the Versteeg and Kocak datasets, using the median (B) or upper quartile (C) of DOT1L expression level as the cut-off point, respectively. D, Kaplan–Meier curves show the probability of patient overall survival according to the DOT1L mRNA expression level in the 72 MYCN-amplified neuroblastoma samples in the large Kocak dataset, using the upper quartile of DOT1L expression level as the cut-off point.

Figure 6.

High levels of DOT1L gene expression in human neuroblastoma tissues independently predict poor patient prognosis. A, Two-sided Pearson correlation was employed to analyze correlation between DOT1L and N-Myc mRNA expression in 88 and 476 human neuroblastoma samples in the publicly available microarray gene expression Versteeg and Kocak datasets downloaded from the R2 platform (http://r2.amc.nl). B and C, Kaplan–Meier curves showed the probability of overall survival of patients according to DOT1L mRNA expression levels in the 88 and 476 neuroblastoma patients in the Versteeg and Kocak datasets, using the median (B) or upper quartile (C) of DOT1L expression level as the cut-off point, respectively. D, Kaplan–Meier curves show the probability of patient overall survival according to the DOT1L mRNA expression level in the 72 MYCN-amplified neuroblastoma samples in the large Kocak dataset, using the upper quartile of DOT1L expression level as the cut-off point.

Close modal

Using the median and the upper quartile DOT1L mRNA expression as the cut-off points, Kaplan–Meier survival analysis showed that high DOT1L mRNA expression levels in neuroblastoma tissues were associated with poor patient prognosis in both the Versteeg and the Kocak datasets (Fig. 6B and C). In addition, high levels of DOT1L mRNA expression in the 72 MYCN-amplified neuroblastoma tissues were positively associated with poor patient overall survival in the large Kocak dataset (Fig. 6D). Importantly, multivariable Cox regression analysis showed that high levels of DOT1L expression in neuroblastoma tissues strongly associated with reduced patient overall survival and event-free survival, independent of disease stage, age at diagnosis, N-Myc mRNA expression level, and MYCN amplification status, the current most important prognostic markers for neuroblastoma patients (1), using the median or upper quartile of DOT1L mRNA expression as the cut-off points (Table 1), or using the exact value of N-Myc and DOT1L mRNA expression levels (Supplementary Table S2). Taken together, the data suggest that high levels of DOT1L expression in human neuroblastoma tissues independently predict poor patient prognosis.

Table 1.

Multivariable Cox regression analysis of DOT1L expression in tumor tissues as a factor prognostic for outcome in 476 neuroblastoma patientsa

Event-free survivalOverall survival
FactorsHR (95% CI)PHR (95% CI)P
High DOT1L expression (median level as cutoff) 1.90 (1.315–2.753) 0.0001 1.81 (1.076–3.045) 0.025 
MYCN amplification 2.03 (1.397–2.949) 0.0002 4.52 (2.885–7.080) 4.5E−11 
Age > 18 months 1.07 (0.749–1.529) 0.710 1.60 (1.000–2.563) 0.050 
Stages III & IVb 1.05 (1.032–1.072) 2.1E−7 1.07 (1.041–1.108) 8.0E−6 
High DOT1L expression (upper quartile as cutoff) 1.96 (1.329–2.903) 0.001 1.97 (1.180–3.281) 0.010 
MYCN amplification 1.69 (1.110–2.575) 0.015 3.70 (2.230–6.139) 4.1E−7 
Age > 18 months 1.02 (0.710–1.473) 0.904 1.54 (0.953–2.475) 0.078 
Stages III & IVb 1.05 (1.034–1.074) 5.5E−8 1.08 (1.043–1.111) 4.0E−6 
Event-free survivalOverall survival
FactorsHR (95% CI)PHR (95% CI)P
High DOT1L expression (median level as cutoff) 1.90 (1.315–2.753) 0.0001 1.81 (1.076–3.045) 0.025 
MYCN amplification 2.03 (1.397–2.949) 0.0002 4.52 (2.885–7.080) 4.5E−11 
Age > 18 months 1.07 (0.749–1.529) 0.710 1.60 (1.000–2.563) 0.050 
Stages III & IVb 1.05 (1.032–1.072) 2.1E−7 1.07 (1.041–1.108) 8.0E−6 
High DOT1L expression (upper quartile as cutoff) 1.96 (1.329–2.903) 0.001 1.97 (1.180–3.281) 0.010 
MYCN amplification 1.69 (1.110–2.575) 0.015 3.70 (2.230–6.139) 4.1E−7 
Age > 18 months 1.02 (0.710–1.473) 0.904 1.54 (0.953–2.475) 0.078 
Stages III & IVb 1.05 (1.034–1.074) 5.5E−8 1.08 (1.043–1.111) 4.0E−6 

aDOT1L expression was considered high or low in relation to the median or upper quartile DOT1L expression in all 476 tumors analyzed. HRs were calculated as the antilogs of the regression coefficients in the proportional hazards regression. Multivariable Cox regression analysis was carried out by including the above listed four factors into the Cox regression model. P value was obtained using two-sided log-rank test.

bTumor stage was classified as favorable (International Neuroblastoma Staging System stages I, II, and IVS) or unfavorable (International Neuroblastoma Staging System stages III and IV).

Like its analogue c-Myc, N-Myc exerts tumorigenic effects in part by binding to Myc-responsive element E-boxes at target gene promoters and consequently activating oncogenic gene expression (9). In this study, we have identified a noncanonical E-Box upstream of the DOT1L gene transcription start site, and demonstrated that N-Myc directly binds to the DOT1L gene core promoter region containing the E-Box and upregulates DOT1L promoter activity, DOT1L mRNA and protein expression in neuroblastoma cells. The data suggest that N-Myc upregulates DOT1L gene expression by binding to its gene promoter.

By analyzing 35 histone marks, Guccione and colleagues have shown that histone H3K4 trimethylation and H3K79 dimethylation at Myc-responsive elements of target gene promoters are strict prerequisites for Myc-induced transcriptional activation (10). Thomas and colleagues and we have recently shown that the histone H3K4 trimethylation presenter WDR5 binds to N-Myc and c-Myc proteins, and that WDR5-mediated histone H3K4 trimethylation plays an essential role in Myc-mediated target gene transcription (36, 37). However, the mechanism through which histone H3K79 is dimethylated during Myc-induced transcriptional activation, is unknown.

DOT1L is the only histone methyltransferase that catalyzes methylation at the H3K79 position (12, 13). DOT1L is well-known to form protein complexes with MLL fusion proteins and plays a critical role in MLL-mediated transcriptional activation and leukemogenesis (28, 29). Most recently, DOT1L has been found to complex with c-Myc, leading to transcriptional activation of the epithelial–mesenchymal transition regulator genes (38). In this study, we have found that knocking down DOT1L gene expression with siRNAs or doxycycline -inducible shRNA reduces the expression of well-known Myc target genes, including ODC1 and E2F2 (30–32). Knocking down DOT1L gene expression reduces H3K79 dimethylation at ODC1 and E2F2 gene promoter regions, and reduces N-Myc protein binding at the ODC1 and E2F2 gene promoter regions containing Myc-responsive element E-Boxes. Protein coimmunoprecipitation and GST pull-known assays demonstrate that DOT1L protein directly binds to N-Myc protein. Taken together, our data indicate that DOT1L and N-Myc form a protein complex at N-Myc target gene promoters, resulting in H3K79 dimethylation and transcriptional activation of N-Myc target genes including ODC1 and E2F2.

Upregulation of ODC1 contributes to N-Myc–mediated neuroblastoma cell proliferation and tumorigenesis (31). E2F2 is involved in DNA synthesis (39), and loss of E2F2 results in reduced tumor incidence in a mouse model of c-Myc–induced breast cancer (40). In this study, we have demonstrated that suppression of E2F2 or DOT1L gene expression reduces neuroblastoma cell proliferation. The data suggest that DOT1L is required for neuroblastoma cell proliferation through activating the transcription of N-Myc target genes, such as ODC1 and E2F2.

In this study, we have found that DOT1L expression correlates with N-Myc, ODC1, and E2F2 expression in primary human neuroblastoma tissues, and that a high level of DOT1L expression in tumor tissues correlates with poor neuroblastoma patient survival, independent of disease stage, age at the time of diagnosis, and MYCN amplification status, the current most important prognostic markers for neuroblastoma patients (1). Importantly, knocking down DOT1L gene expression in mice xenografted with neuroblastoma cells reduces ODC1 and E2F2 expression in tumor tissues and reduces tumor growth. The data suggest that DOT1L plays a critical role in N-Myc–mediated neuroblastoma by increasing the expression of oncogenic genes, such as ODC1 and E2F2, and that DOT1L is a therapeutic target for neuroblastoma.

Several small-molecule DOT1L inhibitors, including EPZ004777, EPZ5676, and SGC0946 have recently been reported (33–35). EPZ004777 displays specificity against DOT1L compared with a panel of eight other histone methyltransferases, and reduces MLL-rearranged leukaemogenesis (28, 34). Modifications to EPZ00477 lead to the synthesis of EPZ5676 (33) and SGC0946 (35) with improved efficacy and in vivo availability. In this study, we have found that treatment with SGC0946 reduces H3K79 methylation and DOT1L target gene expression, and results in growth inhibition in MYCN-amplified, but not MYCN nonamplified neuroblastoma cells. The data suggest that DOT1L inhibitors are potential anticancer agents against MYCN-amplified neuroblastoma.

No potential conflicts of interest were disclosed.

Conception and design: M. Wong, A.E.L.Tee, G. Milazzo, G. Perini, C.J. Scarlett, T. Liu

Development of methodology: M. Wong, A.E.L.Tee, G. Milazzo, Y. Sun, D. Jing, C.J. Scarlett

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Wong, J.L. Bell, B. Atmadibrata, P.Y. Liu, S. Hüttelmaier, J. Wang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Wong, A.E.L. Tee, J.L. Bell, R.C. Poulos, B. Atmadibrata, Y. Sun, N. Ho, D. Ling, P.Y. Liu, X.D. Zhang, J.W.H. Wong, G. Perini

Writing, review, and/or revision of the manuscript: M. Wong, A.E.L. Tee, Y. Sun, N. Ho, P. Polly, G. Perini, C.J. Scarlett, T. Liu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.E.L.Tee, B. Atmadibrata, Y. Sun, D. Ling

Study supervision: P. Polly, T. Liu

We thank Dr. Marco Herold at Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, for providing the FH1tUTG construct. Children's Cancer Institute Australia is affiliated with University of New South Wales, Australia and Sydney Children's Hospitals Network.

This work was supported by National Health & Medical Research Council Australia. G. Perini is supported by a grant of the Italian Association for Cancer Research (AIRC-IG15182) and T. Liu is the recipient of an Australian Research Council Future Fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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