The Histone Methyltransferase DOT1L Promotes Neuroblastoma ... · Tumor and Stem Cell Biology The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription

Tumor and Stem Cell Biology

The Histone Methyltransferase DOT1L PromotesNeuroblastoma by Regulating Gene TranscriptionMatthew Wong, Andrew E.L. Tee1, Giorgio Milazzo2, Jessica L. Bell3,Rebecca C. Poulos4, Bernard Atmadibrata1, Yuting Sun1, Duohui Jing1, Nicholas Ho1,Dora Ling1, Pei Yan Liu1, Xu Dong Zhang5, Stefan H€uttelmaier3, Jason W.H.Wong4,Jenny Wang1,6, Patsie Polly7, Giovanni Perini2, Christopher J. Scarlett8, and Tao Liu1,6

Abstract

Myc oncoproteins exert tumorigenic effects by regulatingexpression of target oncogenes. Histone H3 lysine 79 (H3K79)methylation atMyc-responsive elements of target gene promotersis a strict prerequisite for Myc-induced transcriptional activation,and DOT1L is the only known histone methyltransferase thatcatalyzes H3K79 methylation. Here, we show that N-Myc upre-gulates DOT1L mRNA and protein expression by binding to theDOT1L gene promoter. shRNA-mediated depletion of DOT1Lreduced mRNA and protein expression of N-Myc target genesODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79methylation and N-Myc protein binding at the ODC1 and E2F2gene 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 neuroblas-toma cells stably expressing doxycycline-inducible DOT1LshRNA, ablatingDOT1L expressionwith doxycycline significantlyreduced ODC1 and E2F2 expression, reduced tumor progression,and improved overall survival. In addition, high levels of DOT1Lgene expression in human neuroblastoma tissues correlated withhigh levels of MYCN, ODC1, and E2F2 gene expression andindependently correlated with poor patient survival. Takentogether, our results identify DOT1L as a novel cofactor in N-Myc–mediated transcriptional activation of target genes andneuroblastoma oncogenesis. Furthermore, they characterizeDOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522–33. �2017 AACR.

IntroductionNeuroblastoma is the most commonly diagnosed pediatric

solid tumor in early childhood (1). Neuroblastoma arises fromneural crest cells and is characterized by variable clinical behavior,from spontaneous regression to inexorable progression (2).Adverse clinical prognostic factors include age > 18 months atdiagnosis, advanced disease stage, and amplificationof theMYCNoncogene, which encodes the N-Myc oncoprotein (3–5).

N-Myc is expressed during embryogenesis in the nervoussystem (6, 7). N-Myc induces neuroblastoma by regulatingthe expression of genes important for cell differentiation,malignant transformation, and cell proliferation (8). Like itsanalogue c-Myc oncoprotein, N-Myc activates target gene tran-scription by forming a heterodimer with MAX, and this hetero-dimer in turn recruits a range of cofactors that alter chromatinstructure, such as histone H3 lysine 79 (H3K79) dimethylation(9–11).

DOT1L is a unique histone methyltransferase as it is the onlyknown histone methyltransferase that catalyzes monomethyla-tion (me), dimethylation (me2), and trimethylation (me3) at theH3K79 position (12, 13). Quantitative chromatin immunopre-cipitation studies of H3K79 methylation across the humangenome reveal thatH3K79me andH3K79me2 are linked to activegene transcription (14–16).DOT1L is involved in theoncogenesisof several leukemia subtypes characterized by chromosomaltranslocations involving themixed lineage leukemia (MLL) onco-gene. DOT1L forms a protein complex with the MLL fusionproteins, and DOT1L-mediated H3K79 methylation is responsi-ble for maintaining an open chromatin state around MLL fusionprotein target oncogenes (17, 18).

Crucially, H3K79me2 is essential for c-Myc binding to itstarget gene promoters (10, 11). Here, we investigated the role ofDOT1L-mediated H3K79 methylation in N-Myc–induced tar-get gene transcription, neuroblastoma cell proliferation in vitroand tumor progression in vivo, and examined whether DOT1Lexpression in human neuroblastoma tissues independentlypredicted patient prognosis.

1Children's Cancer Institute Australia for Medical Research, University of NewSouth Wales, Sydney, Australia. 2Department of Pharmacy and Biotechnology,University of Bologna, Bologna, Italy. 3Institute of Molecular Medicine, MartinLuther University, ZAMED, Halle, Germany. 4Prince of Wales Clinical School andLowy Cancer Research Centre, University of New South Wales, Sydney, NewSouth Wales, Australia. 5School of Biomedical Sciences and Pharmacy, Univer-sity of Newcastle, Newcastle, Australia. 6Centre for Childhood Cancer Research,UNSW Medicine, University of New South Wales, Sydney, New South Wales,Australia. 7Department of Pathology and Mechanisms of Disease and Transla-tional Research, University of New South Wales, Sydney, New South Wales,Australia. 8School of Environmental & Life Sciences, University of Newcastle,Ourimbah, New South Wales, Australia.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Tao Liu, Children's Cancer Institute Australia, LowyCancer Research Centre, University of New South Wales, Kensington, Sydney,New South Wales 2031, Australia. Phone: 612-9385-1935; Fax: 612-9662-6584;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1663

�2017 American Association for Cancer Research.

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Materials and MethodsCell culture

Neuroblastoma BE(2)-C, NBL-S, SK-N-FI, and HEK-293 cellswere cultured in DMEM supplemented with 10% FCS. Kelly cellswere 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. Kellyand SK-N-FI cells were obtained from the European Collection ofCell Cultures and Sigma Aldrich in 2010. The identity of cell lineswas verified in 2010, 2014, and 2015 by small tandem repeatprofiling conducted at the Garvan Institute of Medical Research(Darlinghurst, NewSouthWales, Australia) orCellbankAustralia.

Protein coimmunoprecipitation assaysHEK293 human embryonic cells were transiently cotransfected

with an N-Myc-expression pcDNA3.1 construct, together with anempty vector or Flag-DOT1L-expression pcDNA3.1 construct for48 hours. Protein was extracted from the cells and coimmuno-precipitation was carried out using an anti-FLAG antibody(Abcam) or a mouse IgG as a negative control, followed byimmunoblot analysis.

GST pull-down assaysDifferent N-Myc protein fragments were cloned into the pGEX-

2T construct, in frame with N-terminal GST. The constructs weretransformed into BL-21 E.Coli and IPTGwas used for induction ofT7-driven transcription. After purification, equal amount of GST-N-Myc protein fragments were immobilized onto glutathione-agarose beads (Sigma).HEK-293T cellswere transfectedwith Flag-DOT1L expression construct, and nuclear protein from the cellswas incubatedwith equal amount of differentGST-N-Myc proteinfragments immobilized onto glutathione agarose beads. Pulled-down complexes were analyzed by immunoblot with a mono-clonal anti-Flag antibody (Santa Cruz Biotechnology), and Pon-ceau staining detected by ChemiDoc MP (Bio-Rad) was used asloading controls.

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) assays were per-

formed with mouse anti-N-Myc antibody (Merck Millipore),rabbit anti-H3K79me2 antibody (Abcam), rabbit and mousecontrol IgG (Santa Cruz Biotechnology), followed by PCR withprimers designed to cover the core promoter regions of theDOT1L, ODC1, and E2F2 genes containing Myc-responsive ele-ment E-Boxes or remote negative control regions. Fold enrich-ment of theDOT1L, E2F2, andODC1 gene core promoter regionsby the anti-N-Myc or anti-H3K79me2 antibody was calculated bydividing cycle threshold of PCR products from the DOT1L, E2F2,andODC1 gene core promoter regions by cycle threshold of PCRproducts from the negative control region, relative to input.

Luciferase reporter assaysModulation of DOT1L promoter activity by N-Myc was ana-

lyzed by luciferase assays. The DOT1L gene core promoter con-taining the E-Box (�389 bp to þ50 bp relative to transcriptionstart site) was cloned into the pLightSwitch_Prom construct(SwitchGear Genomics). BE(2)-C cells were cotransfected withcontrol siRNA or N-Myc siRNA-1, together with Go Clone Pro-

moter Control construct plus the pLightSwitch_Prom constructexpressing empty vector or the DOT1L core promoter. Luciferaseactivities weremeasuredwith a LightSwitchDual Assay SystemKit(SwitchGear Genomics), and normalized according to Go ClonePromoter Control construct according to the manufacturer'sinstructions.

Establishment of doxycycline-inducible control shRNA andDOT1L shRNA expression constructs and neuroblastoma celllines stably expressing the constructs

The lentiviral doxycycline-inducible GFP-IRES-shRNAFH1tUTG construct from Dr. Marco Herold (Walter and ElizaHall Institute of Medical Research, Melbourne, Australia; ref. 21)was used to generate control shRNA- or DOT1L shRNA–expres-sing construct and neuroblastoma cell lines. DOT1L shRNA andscrambled control shRNA sequences were selected from previ-ously published work (22) and cloned into the FH1tUTG con-struct. The doxycycline -inducible GFP-IRES-control shRNA orDOT1L shRNA FH1tUTG construct was transfected into 293Tcells. The viral media were collected and used to infect BE(2)-Cand Kelly neuroblastoma cells over 72 hours with polybrene(Santa Cruz Biotechnology). GFP-based cell sorting was per-formed for selecting cells with high GFP protein expression.

In vivo mouse experimentsAnimal experiments were approved by the Animal Care and

Ethics Committee of UNSW Australia, and the animals' carewas in accord with institutional guidelines. Female Balb/c nudemice aged 5 to 6 weeks were injected subcutaneously whileunder anesthesia with either 2 � 106 doxycycline -inducibleDOT1L shRNA BE(2)-C cells or 8 � 106 doxycycline -inducibleDOT1L shRNA Kelly cells into the right flank. Mice were fedwith 10% sucrose water with or with doxycycline at 2 mg/mLwhen tumors reached 0.005 cm3 in volume. Tumor develop-ment was monitored and tumor volume calculated using(length � width � height)/2. Mice were culled when tumorvolume reached 1 cm3, and tumor tissues were snap-frozen andanalyzed by immunoblotting for DOT1L, ODC1, and E2F2protein expression.

Patient tumor sample analysisDOT1L, N-Myc, ODC1, and E2F2mRNA expressionwas exam-

ined in 88 (Versteeg dataset; ref. 23) and 476 (Kocak dataset;refs.24, 25) human neuroblastoma samples in the publicly avail-able gene expression datasets (http://r2.amc.nl). Correlationbetween DOT1L and N-Myc, ODC1 as well as E2F2 expressionin human neuroblastoma tissues was analyzed with Pearsoncorrelation. Probability of survival was investigated according tothe method of Kaplan and Meier and two-sided log-rank tests(26). Multivariable Cox regression analyses were performed afterinclusion of disease stage, age at diagnosis, MYCN amplificationstatus, N-Myc and DOT1L expression levels. Probabilities ofsurvival and HR were provided with 95% confidence intervals.Proportionality was confirmed by visual inspection of the plots oflog[2log(S(time))] versus log(time), which were observed toremain parallel (27).

Statistical analysisFor statistical analysis, experiments were conducted three

times. Data were analyzed with Prism 6 software (GraphPad)

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and presented as mean � SE. Differences were analyzed forsignificance using ANOVA among groups or two-sided t test fortwo groups. All statistical tests were two-sided. A P value of lessthan 0.05 was considered statistically significant.

ResultsN-Myc upregulates DOT1L expression by binding to theDOT1Lgene promoter

Myc proteins induce gene transcription by binding to canon-ical 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 genetranscription start site. We therefore examined whether N-Mycmodulated DOT1L gene expression in neuroblastoma cells. Asshown in Fig. 1A and B, transfection of N-Myc–overexpressingBE(2)-C and Kelly neuroblastoma cells with N-Myc siRNA-1 orN-Myc siRNA-2 efficiently knocked down N-Myc mRNA andprotein expression, and reduced DOT1L mRNA and proteinexpression. Consistently, withdrawal of tetracycline from cellculture media of SHEP-Tet/21N cells, which are stably trans-fected with a tetracycline withdrawal–inducible N-Myc expres-sion construct, increased N-Myc and DOT1L mRNA and proteinexpression (Fig. 1C).

We next performed ChIP assays with an anti-N-Myc antibodyor control IgG and real-time PCR with primers targeting anegative control region, the E-box region located �288 bpupstream of the DOT1L transcription start site (Amplicon B),50 untranscribed region located approximately �600 to �800bp upstream (Amplicon A), or intron 1 regions located approx-imately 100 to 180 bp (Amplicon C) or 350 to 500 bp(Amplicon D) downstream (Fig. 1D). The ChIP assays showedthat 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 ofamplicon B displayed much lower enrichment by the N-Mycantibody (Fig. 1E). Importantly, luciferase assays showed thattransfection with the pLightSwitch_Prom construct encodingthe DOT1L gene core promoter containing the E-Box led to highluciferase activity, compared with the empty vector pLightS-witch_Prom construct, and that knocking down N-Myc largelyblocked the luciferase activity (Fig. 1F). Taken together, the datasuggest that N-Myc upregulates DOT1L gene expression viabinding to the DOT1L gene promoter.

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

DOT1L forms protein complexes with MLL fusion proteins,andDOT1L-mediated histoneH3K79methylation is essential forMLL fusion protein–mediated leukemogenic gene transcriptionand leukaemogenesis (28, 29). H3K79me2 has been shown to beessential for c-Myc protein binding at c-Myc target gene promoters(10, 11). We therefore examined whether DOT1L formed aprotein complex with N-Myc, and whether DOT1L is requiredfor histone H3K79me2 and N-Myc protein binding at N-Myctarget gene promoters.

HEK293 cells were cotransfected with an N-Myc expressionconstruct, together with a construct encoding empty vector orFlag-tagged DOT1L. Immunoprecipitation with an anti-Flag anti-body pulled-down N-Myc protein in cells cotransfected with theDOT1L and the N-Myc expression constructs, but did not pull-down N-Myc protein in cells transfected with the N-Myc expres-

sion construct alone (Fig. 2A). Furthermore, different N-Mycprotein fragments (1-86, 82-254, 249-358, 336-400 and 400-464 amino acids) were cloned into the pGEX-2T construct, inframe with N-terminal GST. GST pull-down assays showed thatDOT1L protein bound to the N-Myc 82-254 amino acid fragment(Fig. 2B). The data demonstrate thatDOT1Lprotein directly bindsto the Myc Box II region of N-Myc protein.

We next examined whether knocking down DOT1L expressionreduced H3K79me2 and N-Myc protein binding at the genepromoters 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 assayswere performed with an anti-H3K79me2 antibody, anti-N-Mycantibody or control IgG, followed by PCRwith primers targeting anegative control regionor E-Box regions of theODC1orE2F2 genepromoters. The assays showed that the H3K79me2 antibody andthe anti-N-Myc antibody immunoprecipitated the E-Box regionsof both the ODC1 and E2F2 gene promoters in cells transfectedwith scrambled control siRNA. Compared to the scrambledcontrol siRNA, both DOT1L siRNAs reduced the presence ofH3K79me2 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 genepromoters.

DOT1L activates the expression of the N-Myc targetgenes ODC1 and E2F2

We next examined whether DOT1L regulated the expression ofthe Myc target genes ODC1 and E2F2 in human neuroblastomacells. BE(2)-C and Kelly cells were transfected with control siRNA,N-Myc siRNA-1, N-Myc siRNA-2, DOT1L siRNA-1, or DOT1LsiRNA-2. Consistent with literature (30–32), RT-PCR and immu-noblot analyses showed that transfection with N-Myc siRNAsreduced ODC1 and E2F2 mRNA and protein expression (Sup-plementary Fig. S1). RT-PCR and immunoblot analyses showedthat transfection with DOT1L siRNAs efficiently knocked downDOT1L mRNA and protein expression, and significantly reducedODC1 and E2F2 mRNA and protein expression (Fig. 3A and B).

To further demonstrate that DOT1L regulates Myc target geneexpression, we established BE(2)-C and Kelly cells stably expres-sing doxycycline -inducible control shRNA or DOT1L shRNAusing the GFP-IRES-shRNA expression FH1tUTG construct(21). The cells were then treated with vehicle control or doxycy-cline or 72 hours, followed by histone protein extraction. Immu-noblot analysis confirmed that doxycycline treatment consider-ably reduced histone H3K79 dimethylation in doxycycline-inducible DOT1L shRNA, but not control shRNA, BE(2)-C andKelly cells (Fig. 3C and D). We then treated the doxycycline-inducible control shRNA or DOT1L shRNA BE(2)-C and Kellycells with vehicle control or doxycycline for 96 hours, followed byRNA and protein extraction. RT-PCR and immunoblot analysesshowed that treatmentwith doxycycline efficiently knocked downDOT1L mRNA and protein expression, and also effectivelyreduced ODC1 and E2F2 mRNA and protein expression in bothBE(2)-C and Kelly cells (Fig. 3E and F). In comparison, knockingdownN-Myc orDOT1Ldid not have an effect on the expression ofthe non-Myc target genes HIF1a and VEGF (Supplementary Fig.S2A and S2B). We next performed differential gene expressionstudies with Affymetrix microarray in doxycycline-inducible

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Figure 1.

N-Myc upregulates DOT1L expression by binding to theDOT1L gene promoter.A and B, BE(2)-C (A) and Kelly (B) neuroblastoma cells were transfected with controlsiRNA, 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 mg/mL) or vehicle control for 72 hours. RT-PCR and immunoblot analyses were conducted on N-Myc andDOT1L expression.D, Schematic representation of theDOT1L gene promoter. TSS, transcription start site. E, ChIP assayswere performedwith a control IgG or N-Mycantibody (Ab), followed by PCR with primers targeting a remote negative control region, the 50 untranscribed region (Amplicon A), the E-Boxregion (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 wascalculated as the difference in cycle thresholds obtained with the anti-N-Myc Ab compared with the control IgG, relative to input. F, Luciferase assays wereperformed in BE(2)-C cells after cotransfection with control siRNA or N-Myc siRNA-1, together with Go Clone Promoter Control construct plus pLightSwitch_Promconstruct expressing empty vector or DOT1L gene core promoter. Error bars, SEs. � , �� , and �� indicate P < 0.05, 0.01, and 0.001, respectively.






control shRNA and DOT1L shRNA BE(2)-C cells after treatmentwith vehicle control or doxycycline. Gene-set enrichment analysisshowed that genes with E-Boxes at promoters were possiblyenriched among those downregulated by DOT1L shRNA (Sup-plementary Table S1).

Taken together, the data suggest that DOT1L induces histoneH3K79 dimethylation and activates the transcription of the N-Myc target genes ODC1 and E2F2.

DOT1L is required for MYCN-amplified neuroblastomacell proliferation

Upregulation of ODC1 has been well-documented to contrib-ute to N-Myc–mediated neuroblastoma cell proliferation (31).Next, we examined the effect of DOT1L and its target E2F2 uponneuroblastoma cell proliferation. BE(2)-C and Kelly neuroblas-toma 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 siRNAsor E2F2 siRNAs reduced the numbers of BE(2)-C and Kelly cells(Fig. 4A and B).

To further demonstrate that DOT1L is required for neuro-blastoma cell proliferation, we treated doxycycline -induciblecontrol shRNA or DOT1L shRNA BE(2)-C and Kelly cells withvehicle control or doxycycline. Alamar blue assays showedthat doxycycline treatment had minimal effect on cell prolif-eration in doxycycline -inducible control shRNA BE(2)-C andKelly cells, but significantly reduced cell proliferation in doxy-cycline -inducible DOT1L shRNA BE(2)-C and Kelly cells (Fig.4C). In addition, cell-cycle analysis showed that knockingdown DOT1L expression with doxycycline did not increase thepercentage of cells at the sub-G1 phase in doxycycline -induc-ible DOT1L shRNA BE(2)-C and Kelly cells (SupplementaryFig. S3A and S3B).

Small-molecule DOT1L inhibitors, such as SGC0946, arepromising novel anticancer agents (33–35). We next treatedMYCN gene–amplified BE(2)-C and Kelly cells with vehiclecontrol or 1.25 mmol/L SGC0946 for 72 hours, 96 hours, or 7days. Immunoblot analyses confirmed that treatment withSGC0946 led to reduction in H3K79me2 72 hours posttreatmentand more significantly 7 days posttreatment (Fig. 4D). RT-PCRanalysis showed that SGC0946 reduced the expression of theDOT1L and N-Myc target gene E2F2 (Fig. 4E), and Alamar blueassays confirmed that SGC0946 dose-dependently reduced BE(2)-C and Kelly cells' proliferation (Fig. 4F). In contrast, treatmentwith SGC0946 did not reduce E2F2 gene expression and did nothave an effect on cell proliferation in MYCN gene nonamplifiedNBL-S and SK-N-FI neuroblastoma cells (Fig. 4G and H). Takentogether, the data suggest that DOT1L is required for MYCN-amplified neuroblastoma cell proliferation, and that DOT1Linhibitors are effective anticancer agents againstMYCN-amplifiedneuroblastoma.

DOT1L is required for neuroblastoma tumor growth in vivoWe next examined whether DOT1L is required for neuro-

blastoma tumor growth in vivo. Doxycycline -inducible DOT1LshRNA BE(2)-C and Kelly cells were injected into the flanks ofBalb/c nude mice. Once tumors were palpable, the mice weredivided into doxycycline or vehicle control treatment sub-groups, and fed with water with or without doxycycline. Tumorgrowth was monitored and mice culled once tumor volumereached 1 cm3.

Figure 2.

DOT1L-mediated H3K79 methylation is required for N-Myc protein binding totarget gene promoters. A, HEK293 cells were cotransfected withN-Myc-expression pcDNA3.1 construct, together with empty vector or Flag-DOT1L-expression pcDNA3.1 construct for 48 hours. Protein was extractedfrom the cells and coimmunoprecipitation (IP) was carried out using ananti-FLAG antibody (Ab) or a mouse IgG as a negative control. Immunoblotanalysis 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 ofdifferent GST-N-Myc protein fragments immobilized onto glutathioneagarose beads, followed by immunoblot with an anti-Flag antibody. Asloading controls, Ponceau stained images were detected by ChemiDoc MP.Numbers on the left refer to molecular weights. C and D, BE(2)-C cells weretransfected with control siRNA, DOT1L siRNA-1, or DOT1L siRNA-2 for 72hours. ChIP assays were performed with a control IgG, anti-histoneH3K79me2 (C), or anti-N-Myc Ab (D), followed by PCR with primerstargeting the E-box regions of the ODC1 and E2F2 gene promoters or theDNA region �4,000 bp upstream of the E2F2 gene transcription start site asthe negative control. Error bars, SE. � and �� indicate P < 0.05 and 0.01,respectively.

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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 controlsiRNA, DOT1L siRNA-1, or DOT1L siRNA-2 for 96 hours, followed by RT-PCR and immunoblot analyses of DOT1L, ODC1 and E2F2 mRNA, and proteinexpression. 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 mg/mLdoxycycline 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 shRNABE(2)-C (E) and Kelly (F) cells were treated with vehicle control or 2 mg/mL doxycycline for 96 hours,followedbyRT-PCR and immunoblot analyses ofDOT1L,ODC1, and E2F2 expression. Error bars, SEs. �� , �� , and �� indicateP<0.01, 0.001, and0.0001, respectively.






Figure 4.

DOT1L is required forMYCN-amplified neuroblastoma cell proliferation. A and B, BE(2)-C and Kelly cells were transfected with control siRNA, DOT1L siRNA-1, DOT1LsiRNA-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 andKelly cellswere treatedwith vehicle control or 2mg/mL doxycycline for 96 hours, followed byAlamar blue assays.D,BE(2)-C andKelly cellswere treatedwith controlor 1.25 mmol/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) andMYCN-nonamplifiedNBL-S and SK-N-FI (G andH) cellswere treatedwith control or SGC0946 for 7 days, followedbyRT-PCR analysis of E2F2gene expression (E andG) 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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The doxycycline treatment subgroup displayed slowertumor growth in comparison with the control treatment sub-group for both doxycycline -inducible DOT1L shRNA BE(2)-C

and Kelly cell xenografts (Fig. 5A). Kaplan–Meier survivalanalysis showed that doxycycline treatment, compared withcontrol treatment, resulted in a longer median survival in mice

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, themice were divided into doxycycline and vehicle control subgroups, and fed with 10% sucrose water with or without 2mg/mL doxycycline (DOX). A, Tumor growth was measured every two days using calipers, and mice culled when tumor volume reached 1 cm3. B, Kaplan–Meiersurvival curves show theprobability of overall survival of themice.P valuewasobtained from two-sided log-rank test.C andD,Proteinwas extracted from the tumorsfrom the mice and subjected to immunoblot analysis of DOT1L, ODC1, and E2F2 protein expression.






xenografted with doxycycline-inducible DOT1L shRNA BE(2)-C or Kelly cells by 2-fold (Fig. 5B).

At the conclusion of the in vivo experiment, mice were culled,tumor tissues snap-frozen, and protein extracted. Immunoblotanalysis showed that both the doxycycline-inducible DOT1LshRNA BE(2)-C and Kelly tumors displayed a decrease inDOT1L protein expression as a result of doxycycline treatment,compared with the control treatment (Fig. 5C and D forimmunoblot gels; Supplementary Fig. S4A and S4B for proteinquantification). Protein expression of the N-Myc target genes,ODC1 and E2F2, in both doxycycline-inducible DOT1L shRNABE(2)-C and Kelly xenograft tumors, was also reduced in thedoxycycline treatment group, compared with the control treat-

ment group (Fig. 5C and D for immunoblot gels; Supplemen-tary Fig. S4A and S4B for protein quantification). Takentogether, knocking down DOT1L gene expression with doxy-cycline reduces ODC1 and E2F2 protein expression, reducesneuroblastoma tumor progression, and improves mouse sur-vival in vivo.

High levels of DOT1L gene expression in humanneuroblastoma tissues independently predict poorpatient prognosis

To assess clinical relevance of DOT1L expression in humanneuroblastoma tissues, we examined DOT1L gene expressioninhumanneuroblastoma tissues in thepublicly availableVersteeg

Figure 6.

High levels of DOT1L gene expression in humanneuroblastoma tissues independently predict poorpatient prognosis.A, Two-sided Pearson correlationwasemployed to analyze correlation between DOT1L and N-Myc mRNA expression in 88 and 476 humanneuroblastoma samples in the publicly availablemicroarray gene expression Versteeg and Kocakdatasets downloaded from the R2 platform (http://r2.amc.nl). B and C, Kaplan–Meier curves showed theprobability of overall survival of patients according toDOT1L mRNA expression levels in the 88 and 476neuroblastoma patients in the Versteeg and Kocakdatasets, using the median (B) or upper quartile (C) ofDOT1L expression level as the cut-off point, respectively.D, Kaplan–Meier curves show the probability of patientoverall survival according to the DOT1L mRNAexpression level in the 72 MYCN-amplifiedneuroblastoma samples in the large Kocak dataset, usingthe upper quartile of DOT1L expression level asthe cut-off point.

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(23) and Kocak (24, 25) microarray gene expression–patientprognosis datasets, downloaded from the R2 platform (http://r2.amc.nl; last accessed on August 26, 2014). Two-sided Pearsoncorrelation study showed that DOT1L mRNA expression posi-tively correlated to N-Myc mRNA expression in the 88 humanneuroblastoma tissues of the Versteeg dataset and in the 476human neuroblastoma tissues of the Kocak dataset (Fig. 6A). Inaddition, DOT1L mRNA expression positively correlated toODC1 and E2F2 mRNA expression in human neuroblastomatissues (Supplementary Fig. S5).

Using the median and the upper quartile DOT1L mRNAexpression as the cut-off points, Kaplan–Meier survival analysisshowed that high DOT1L mRNA expression levels in neuroblas-toma tissues were associated with poor patient prognosis in boththe Versteeg and the Kocak datasets (Fig. 6B and C). In addition,high levels of DOT1L mRNA expression in the 72 MYCN-ampli-fied neuroblastoma tissues were positively associated with poorpatient overall survival in the large Kocak dataset (Fig. 6D).Importantly, multivariable Cox regression analysis showed thathigh levels of DOT1L expression in neuroblastoma tissues strong-ly associated with reduced patient overall survival and event-freesurvival, independent of disease stage, age at diagnosis, N-MycmRNA expression level, and MYCN amplification status, thecurrent most important prognostic markers for neuroblastomapatients (1), using themedian or upper quartile of DOT1LmRNAexpression as the cut-off points (Table 1), or using the exact valueof N-Myc and DOT1L mRNA expression levels (SupplementaryTable S2). Taken together, the data suggest that high levels ofDOT1L expression in human neuroblastoma tissues indepen-dently predict poor patient prognosis.

DiscussionLike its analogue c-Myc, N-Myc exerts tumorigenic effects in

part by binding to Myc-responsive element E-boxes at target genepromoters and consequently activating oncogenic gene expres-sion (9). In this study, we have identified a noncanonical E-Boxupstream of the DOT1L gene transcription start site, and dem-onstrated that N-Myc directly binds to the DOT1L gene corepromoter region containing the E-Box and upregulates DOT1Lpromoter activity, DOT1L mRNA and protein expression inneuroblastoma cells. The data suggest that N-Myc upregulatesDOT1L gene expression by binding to its gene promoter.

By analyzing 35 histone marks, Guccione and colleagues haveshown that histone H3K4 trimethylation and H3K79 dimethyla-

tion atMyc-responsive elements of target gene promoters are strictprerequisites for Myc-induced transcriptional activation (10).Thomas and colleagues and we have recently shown that thehistone H3K4 trimethylation presenter WDR5 binds to N-Mycand c-Myc proteins, and that WDR5-mediated histone H3K4trimethylation plays an essential role inMyc-mediated target genetranscription (36, 37). However, the mechanism through whichhistone H3K79 is dimethylated during Myc-induced transcrip-tional activation, is unknown.

DOT1L is the only histone methyltransferase that catalyzesmethylation at the H3K79 position (12, 13). DOT1L is well-known to form protein complexes with MLL fusion proteins andplays a critical role in MLL-mediated transcriptional activationand leukemogenesis (28, 29). Most recently, DOT1L has beenfound to complex with c-Myc, leading to transcriptional activa-tion of the epithelial–mesenchymal transition regulator genes(38). In this study, we have found that knocking down DOT1Lgene expression with siRNAs or doxycycline -inducible shRNAreduces the expression of well-knownMyc target genes, includingODC1 and E2F2 (30–32). Knocking down DOT1L gene expres-sion reduces H3K79 dimethylation at ODC1 and E2F2 genepromoter regions, and reduces N-Myc protein binding at theODC1 and E2F2 gene promoter regions containing Myc-respon-sive element E-Boxes. Protein coimmunoprecipitation and GSTpull-known assays demonstrate that DOT1L protein directlybinds to N-Myc protein. Taken together, our data indicate thatDOT1L and N-Myc form a protein complex at N-Myc target genepromoters, resulting inH3K79 dimethylation and transcriptionalactivation of N-Myc target genes including ODC1 and E2F2.

Upregulation of ODC1 contributes to N-Myc–mediated neu-roblastoma cell proliferation and tumorigenesis (31). E2F2 isinvolved in DNA synthesis (39), and loss of E2F2 results inreduced tumor incidence in a mouse model of c-Myc–inducedbreast cancer (40). In this study, we have demonstrated thatsuppression of E2F2 or DOT1L gene expression reduces neuro-blastoma cell proliferation. The data suggest that DOT1L isrequired for neuroblastoma cell proliferation through activatingthe transcription of N-Myc target genes, such as ODC1 and E2F2.

In this study, we have found that DOT1L expression correlateswith N-Myc, ODC1, and E2F2 expression in primary humanneuroblastoma tissues, and that a high level of DOT1L expressionin tumor tissues correlates with poor neuroblastoma patientsurvival, independent of disease stage, age at the timeof diagnosis,and MYCN amplification status, the current most importantprognostic markers for neuroblastoma patients (1). Importantly,

Table 1. Multivariable Cox regression analysis of DOT1L expression in tumor tissues as a factor prognostic for outcome in 476 neuroblastoma patientsa

Event-free survival Overall survivalFactors HR (95% CI) P HR (95% CI) P

High DOT1L expression (median level as cutoff) 1.90 (1.315–2.753) 0.0001 1.81 (1.076–3.045) 0.025MYCN amplification 2.03 (1.397–2.949) 0.0002 4.52 (2.885–7.080) 4.5E�11Age > 18 months 1.07 (0.749–1.529) 0.710 1.60 (1.000–2.563) 0.050Stages III & IVb 1.05 (1.032–1.072) 2.1E�7 1.07 (1.041–1.108) 8.0E�6High DOT1L expression (upper quartile as cutoff) 1.96 (1.329–2.903) 0.001 1.97 (1.180–3.281) 0.010MYCN amplification 1.69 (1.110–2.575) 0.015 3.70 (2.230–6.139) 4.1E�7Age > 18 months 1.02 (0.710–1.473) 0.904 1.54 (0.953–2.475) 0.078Stages III & IVb 1.05 (1.034–1.074) 5.5E�8 1.08 (1.043–1.111) 4.0E�6aDOT1L 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 theantilogs of the regression coefficients in the proportional hazards regression. Multivariable Cox regression analysis was carried out by including the above listed fourfactors 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 StagingSystem stages III and IV).





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knocking downDOT1L gene expression inmice xenografted withneuroblastoma cells reducesODC1 andE2F2 expression in tumortissues and reduces tumor growth. The data suggest that DOT1Lplays a critical role in N-Myc–mediated neuroblastoma byincreasing the expression of oncogenic genes, such as ODC1 andE2F2, and that DOT1L is a therapeutic target for neuroblastoma.

Several small-molecule DOT1L inhibitors, includingEPZ004777, EPZ5676, and SGC0946 have recently been reported(33–35). EPZ004777 displays specificity against DOT1L com-pared with a panel of eight other histone methyltransferases, andreducesMLL-rearranged leukaemogenesis (28, 34).Modificationsto 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 H3K79methylation and DOT1L target gene expression, and results ingrowth inhibition in MYCN-amplified, but not MYCN nonam-plified neuroblastoma cells. The data suggest that DOT1L inhi-bitors are potential anticancer agents against MYCN-amplifiedneuroblastoma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Wong, A.E.L.Tee, G. Milazzo, G. Perini,C.J. Scarlett, T. LiuDevelopment 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. H€uttelmaier, J. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Wong, A.E.L. Tee, J.L. Bell, R.C. Poulos, B. Atma-dibrata, Y. Sun, N. Ho, D. Ling, P.Y. Liu, X.D. Zhang, J.W.H. Wong, G. PeriniWriting, 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. LiuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.E.L.Tee, B. Atmadibrata, Y. Sun, D. LingStudy supervision: P. Polly, T. Liu

AcknowledgmentsWe thank Dr. Marco Herold at Walter and Eliza Hall Institute of Medical

Research, Melbourne, Australia, for providing the FH1tUTG construct. Chil-dren's Cancer Institute Australia is affiliated with University of New SouthWales, Australia and Sydney Children's Hospitals Network.

Grant SupportThis work was supported by National Health & Medical Research Council

Australia. G. Perini is supported by a grant of the Italian Association for CancerResearch (AIRC-IG15182) and T. Liu is the recipient of an Australian ResearchCouncil Future Fellowship.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 15, 2016; revised August 8, 2016; accepted January 17, 2017;published OnlineFirst February 16, 2017.

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2017;77:2522-2533. Published OnlineFirst February 16, 2017.Cancer Res Matthew Wong, Andrew E.L. Tee, Giorgio Milazzo, et al. Regulating Gene TranscriptionThe Histone Methyltransferase DOT1L Promotes Neuroblastoma by

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The Histone Methyltransferase DOT1L Promotes Neuroblastoma ... · Tumor and Stem Cell Biology The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription