A687V EZH2 Is a Driver of Histone H3 Lysine 27 (H3K27) Hypertrimethylation

Published OnlineFirst September 24, 2014; DOI: 10.1158/1535-7163.MCT-13-0876

Molecular Cancer Cancer Biology and Signal Transduction Therapeutics

Heidi M. Ott1, Alan P. Graves2, Melissa B. Pappalardi1, Michael Huddleston2, Wendy S. Halsey2, Ashley M. Hughes2, Arthur Groy1, Edward Dul2, Yong Jiang2, Yuchen Bai3, Roland Annan2, Sharad K. Verma1, Steven D. Knight1, Ryan G. Kruger1, Dashyant Dhanak1, Benjamin Schwartz2, Peter J. Tummino1, Caretha L. Creasy1,3, and Michael T. McCabe1

Abstract The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell–derived cancer cell lines identiﬁed an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These ﬁndings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival. Mol Cancer Ther; 13(12); 3062–73. 2014 AACR.

Introduction lie EZH2 overexpression, including amplification (12, 13), EZH2 is the catalytically active methyltransferase com- E2F activation (12), and loss of repressive microRNAs ponent of the polycomb-repressive complex 2 (PRC2) and (miR101 and miR26a; refs. 14, 15). EZH2 is also recurrently functions to methylate histone H3 on lysine 27 (H3K27; mutated at specific residues in human non-Hodgkin lym- refs. 1, 2). Trimethylation of H3K27 (H3K27me3) is asso- phoma. EZH2 tyrosine 641 (Y641) is mutated in 14% to ciated with chromatin condensation and transcriptional 22% of germinal center B-cell (GCB) diffuse large B-cell repression of genes involved in development and differ- lymphomas (DLBCL) and 7% to 22% of follicular lym- entiation (3–5). EZH2 is overexpressed in many human phomas (FL; refs. 16–18). In addition, alanine 677 of EZH2 tumor types, including prostate, breast, neuroendocrine is mutated to a glycine (A677G) in roughly 1% to 2% of lung, renal, and others (6–11). Diverse mechanisms under- DLBCLs (19, 20). These recurrent EZH2 mutations affect residues located within the lysine binding pocket that are critical for the positioning of the K27 substrate during the methyl trans- 1 Cancer Epigenetics Discovery Performance Unit, Cancer Research, fer reaction. The lysine binding pocket of wild-type (WT) Oncology R&D, GlaxoSmithKline, Collegeville, Pennsylvania. 2Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania. EZH2 is designed to stabilize the K27me0 substrate, but is 3Molecular Medicine Unit, Cancer Research, Oncology R&D, Glaxo- also limited by steric hindrance with the larger K27me2 SmithKline, Collegeville, Pennsylvania. substrate. Mutations of Y641 to smaller residues (Y641N/ Note: Supplementary data for this article are available at Molecular Cancer F/S/H/C) relieve the steric hindrance with K27me2, Therapeutics Online (http://mct.aacrjournals.org/). thereby promoting the generation of H3K27me3, but this Corresponding Author: Michael T. McCabe, Cancer Epigenetics Discov- occurs at the expense of stabilizing the smaller H3K27me0 ery Performance Unit, Cancer Research, Oncology Research and Devel- opment, GlaxoSmithKline, 1250 South Collegeville Road, UP1450, Colle- and me1 substrates (19, 21, 22). Thus, although the Y641X geville, PA 19426. Phone: 610-917-6841; Fax: 610-917-4170; E-mail: EZH2 mutants have gained the ability to efficiently con- [email protected] vert H3K27me2 into H3K27me3, they require coordina- doi: 10.1158/1535-7163.MCT-13-0876 tion with the WT EZH2 as Y641X mutants possess little 2014 American Association for Cancer Research. activity with an H3K27me0 substrate (21).

3062 Mol Cancer Ther; 13(12) December 2014

Cellular Role of A687V EZH2 in ALL

The A677 residue of EZH2 is not located directly in the for expression of EED, SUZ12, RbAp48, AEBP2, and lysine binding pocket, but rather resides behind the Y641 FLAG–tev–EZH2 and PRC2 complexes were purified residue (19). The mutation of A677 to the smaller glycine using anti-FLAG M2 resin (Sigma) as previously residue increases activity with an H3K27me2 substrate by described (19). For mammalian expression studies, WT enlarging the lysine binding pocket. Importantly, howev- human EZH2 was subcloned into pIRES2-ZsGreen1 er, because this mutant retains the WT Y641 residue, it (Clontech) and site-directed mutagenesis was used as does not compromise the key interactions for positioning described above to obtain the A687V mutant. All com- K27me0 and me1. Therefore, the A677G mutant is an ponents and EZH2 mutations were confirmed by peptide efficient methyltransferase with any of the H3K27 sub- mapping analysis. strates (H3K27me0/1/2; ref. 19). These mutations provide a mechanism for lymphoma cells to dramatically increase Biochemical evaluation of methyltransferase activity global H3K27me3 and presumably permit aberrant tran- Unless otherwise stated, all reagents were obtained scriptional silencing of several genes involved in normal from Sigma and were at a minimum of reagent grade. cellular homeostasis. Peptides contained within the library (Supplementary Recently, recurrent mutation of EZH2 A687 (residue Table S2) were acquired from 21st Century Biochemicals, numbering based on NM_001203247/NP_001190176; AnaSpec, or Alta Bioscience. Library peptides all contain A692 in NM_004456/NP_004447) to a valine (A687V) a terminal biotin tag and range in purity from crude to has been reported in several large-scale sequencing 97%. Streptavidin SPA beads (RPNQ0261) and [3H]-S- studies of non-Hodgkin lymphoma (20, 23, 24). Bio- adenosyl-methionine (SAM) were purchased from chemical characterization of A687V EZH2 demonstrat- PerkinElmer. ed that this mutant exhibited a substrate preference All reactions were evaluated at ambient temperature in unique from that of WT, Y641X, and A677G EZH2. assay buffer containing 50 mmol/L Tris-HCl (pH 8.0), 2 A687V EZH2 exhibited greatly reduced activity with mmol/L MgCl2, 4 mmol/L DTT, and 0.001% Tween-20. an H3K27me0 substrate, 4-fold increased activity with The peptide library screen was run as 10 mL reactions in H3K27me1, and little change with H3K27me2 (25). Greiner 384-well plates that were prestamped with 100 nL Herein, we demonstrate that although A687V EZH2 peptide (1 mmol/L final) in 100% DMSO. [3H]-SAM (200 does not have increased activity with H3K27me2 sub- nmol/L, 0.016 mCi/mL final) was added to the plate strates, it promotes hypertrimethylation of H3K27 and followed by the addition of WT or mutant PRC2 (16 does so without dramatic depletion of H3K27me2 like nmol/L final). Reactions were quenched after 1 hour via other previously reported Y641 and A677 mutants. In the addition of unlabeled SAM and streptavidin-coated addition, we identify this mutation in a B-cell acute SPA imaging beads at 0.5 mmol/L and 1.5 mg/mL final lymphoblastic leukemia (ALL) cell line possessing ele- concentrations, respectively. Activity was analyzed by vated H3K27me3 levels and using GSK126 (26), a selec- reading the plates on a Wallac ViewLux CCD Imager tive inhibitor of EZH2 catalytic activity, we demonstrate (613/55 emission filter). that this A687V EZH2-mutant cell line is highly dependent on EZH2 function for survival. Cell culture The SUP-B8 ALL cell line that harbors a heterozygous Materials and Methods A687V EZH2 mutation was kindly provided by Dr. Ronald Levy (Stanford University, Stanford, CA) and was Structural modeling of EZH2 maintained in RPMI-1640 media (MediaTech) supple- As previously described (19), a homology model of mented with 10% FBS (Sigma-Aldrich). All other cell lines EZH2 was built using GLP/EHMT1 bound to an ¼ were obtained from either the ATCC or DSMZ, main- H3K9me2 peptide substrate (Protein Data Bank ID tained in the recommended cell culture media, and 2RFI) as a primary template and structurally compared authenticated via short tandem repeat analysis before with other related SET domain containing histone lysine storage in the GlaxoSmithKline cell repository. methyltransferases with determined crystal structures. Transient expression of WT and mutant EZH2 Cloning, expression, and purification of 5-member proteins in cells PRC2 complexes MCF-7 breast cancer cells (3 105) were seeded into Five-member PRC2 complexes were prepared as pre- 6-well tissue culture plates in RPMI-1640 media supple- viously described (19). For A687V EZH2, human EZH2 mented with 10% FBS the day before transfection. in pENTR/TEV/D–TOPO was mutagenized by site- Following the manufacturer’s recommendations, 2 mg directed mutagenesis (QuikChange II XL; Agilent Tech- plasmid DNA and 6 mL Lipofectamine 2000 (Invitrogen) nologies) using primers described in Supplementary were combined in 500 mLOpti-MEM(Invitrogen)and Table S1, the entire coding region of all mutants was incubated for 20 minutes at room temperature before confirmed by double-stranded DNA sequencing, and being added to cells. Cells were then incubated for 72 subcloned into pDEST8 with an N-terminal FLAG epi- hours at 37 Cwith5%CO2 and harvested for protein tope tag. Individual baculovirus stocks were generated lysates.

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3063

Ott et al.

Western blot analysis Sanger sequencing of EZH2 Cell lysate preparation and Western blotting were per- Isolation of genomic DNA and Sanger sequencing of formed as previously described (19). Antibodies used full-length EZH2, including exons 16 (Y641) and 18 (A677, included: EZH2 (BD Transduction Laboratories), histone A687), was performed as previously described (19). H3 (Abcam), H3K27me1 (ActiveMotif), H3K27me2 (Cell Signaling Technology), H3K27me3 (Cell Signaling Tech- Cell proliferation assay nology), EED (Santa Cruz Biotechnology), SUZ12 (Cell Cells were evaluated for sensitivity to GSK126 in a 6- Signaling Technology), or Actin (Sigma). Histone meth- day proliferation assay as described previously (26). Brief- ylation antibody specificity was previously confirmed ly, cells were seeded into 384-well plates at a density that using full-length recombinant methylated histones har- permitted proliferation for 6 days. Cells were then treated boring mono-, di-, or trimethylation at H3K4, H3K9, in duplicate with a 20-point 2-fold dilution series of H3K27, H3K36, and H3K79 (19). The abundance of GSK126 or 0.147% DMSO. After incubation with com- H3K27 methylation states was quantified using LI-COR pound for 6 days, cell proliferation was evaluated using Odyssey software and a standard curve of recombinant CellTiter-Glo (Promega) according to the manufacturer’s methylated histones, including H3K27me1 (Active Motif specifications. Data were fit with a 4-parameter equation #31214), H3K27me2 (Active Motif #31215), H3K27me3 to generate a concentration response curve and to deter- (Active Motif #31216), or total histone H3 (Active Motif mine the concentration of GSK126 required to inhibit 50% #31207; Supplementary Fig. S1). of growth (gIC50).

Analysis of H3K27 methylation by mass Caspase 3/7 assay spectrometry For detection of caspase 3/7 activity, Caspase-Glo 3/7 WT EZH2 NALM-6 cells were labeled with heavy (Promega) was used according to the manufacturer’s (isotopic) [U-13C6, 15N4]-L-Arginine and [U-13C6]-L- directions. Values were normalized to CellTiter-Glo (Pro- Lysine (Invitrogen) for six population doublings before mega) levels at each time point and expressed as a per- harvest and lysis. Acid-extracted cell lysates from the centage of vehicle-treated control. EZH2-mutant cell lines SUP-B8 and VAL were mixed 1:1 based on protein concentration with labeled NALM- Gene-expression profiling 6 lysate. Proteinase ArgC (Sigma) was added at a 10:1 SUP-B8 and NALM-6 cells (2 105/well) were seeded enzyme to the protein ratio and the samples were digested into 6-well tissue culture plates 24 hours before treat- for 2.5 hours at 37C. Digestion was stopped with formic ment with 0.1% DMSO or 500 nmol/L GSK126 for 72 acid. Peptide mixtures were separated on 100 mm inner hours. Cells were collected into TRizol reagent (Invitro- diameter 5-cm RP-PSDVB monolithic columns (Dionex) gen) and total RNA was isolated via phenol:chloroform using an acetonitrile:water:formic acid gradient at 500 extraction and the RNeasy Kit (Qiagen) according to the nL/min. The column was interfaced directly to an manufacturer’s instructions. Total RNA was labeled and LTQ-Orbitrap Velos Pro (ThermoFisher Scientific) mass hybridized to Affymetrix Human Genome U133 Plus 2.0 spectrometer. Peptide identifications and SILAC ratios oligonucleotide microarrays according to the manufac- were determined by data-dependent LC/MS-MS using a turer’s instructions (Affymetrix). CEL files were pro- resolution of 30,000 for the MS spectrum and MS/MS in cessed and differentially expressed probe sets were the ion trap. To quantify H3K27 methylation, targeted, determined as described previously (26). Briefly, signif- full-time LC/MS-MS spectra were acquired using HCD icant probe sets were identified after filtering for detec- with a resolution of 7,500 in the Orbitrap on precursor tion, an average fold change >2or<2, and P values ionsfortheH3peptide27to40containingonethrough adjusted for multiple testing correction by FDR (Benja- five methyl groups. No evidence was found for the mini Hochberg) < 0.1. Data are available through the peptide containing six methyl groups. Histones H3.1 Gene Expression Omnibus under accession GSE56954. and H3.2 have the same amino acid sequence through the 27 to 40 region, whereas H3.3 has an alanine to serine Gene ontology and gene set enrichment analysis switch at position 31. Given the rather low abundance of Functional analyses of differentially expressed probe H3.3 it was not included in the analysis. In addition, sets were performed using DAVID (27). Significantly given the very low abundance of acetylation on K27 and overrepresented GO Biological Process and Molecular K36, we also did not include peptides, which might Function terms (levels 3–5) were filtered for terms with contain acetylation in this region. Global methylation at least five significantly altered probes and EASE P < profiles for K27 were obtained by extracting the ion value of 0.01. For GSEA (28), log2-transformed micro- > intensity for the b3 N-terminal fragment ion containing array data were filtered (100% present calls and signal 6 0, 1, 2, and 3 methyl groups from each of the five methyl in at least one cohort). In total, 25,059 probesets were containing precursors and totaling the values. Because included. Gene set permutations were used to identify the b3 ion contains only the three N-terminal amino acids significantlyenrichedgenesetsfromthec2(curatedgene of the peptide, there is no contribution from methylation sets) and c3 (motif gene sets) MSigDB V3.0 databases on K36 in the intensities. (29).

3064 Mol Cancer Ther; 13(12) December 2014 Molecular Cancer Therapeutics

Cellular Role of A687V EZH2 in ALL

Results (31%) or isoleucine (35%) in other SET domain methyl- EZH2 A687 is mutated in human cancers and affects transferases (Fig. 1B and Supplementary Fig. S2). substrate specificity Recent biochemical studies (25) demonstrated that The A687V EZH2 mutation has been observed in two A687V EZH2 exhibits 4-fold increased catalytic efficiency independent studies of DLBCLs with incidences of 1 of 49 with a monomethylated K27 substrate. To further evalu- and 1 of 127 (20, 23). Although the incidence of this ate whether this was the only change in substrate spec- mutation appears to be quite low (1%–2%), it is similar ificity, we assessed PRC2 complexes containing WT or to what has been described for the A677G EZH2 mutation A687V-mutant EZH2 with a library of 602 peptides repre- (19, 20). A687 is located within the catalytic SET domain of senting sequences within histones H2A, H2B, H3, or H4 EZH2 (Fig. 1A) and lies adjacent to the highly conserved and possessing up to five posttranslational modifications, NHS motif found in most SET domain methyltransferases such as lysine and/or arginine methylation; lysine acet- (Fig. 1B). Interestingly, although the residues equivalent ylation; or phosphorylation of serine, tyrosine, and/or to EZH2 A687 in human EZH1 and Drosophila E(z) are threonine (Supplementary Table S2). Supporting the pre- both alanine, this residue is most frequently either valine vious report (25), this global analysis revealed that A687V EZH2 exhibited enhanced activity for three peptides that showed little activity with WT EZH2 (Fig. 1C and Sup- plementary Fig. S3). These three peptides derived from histone H3 all harbored monomethylation at either K27 EED A (n ¼ 2) or K9 (n ¼ 1). Methylation of H3K9-containing interaction Y641 A677A687 peptides is a known biochemical artifact for the PRC2 DNMT interaction Cys SET complex that only occurs in vitro (1, 2). Thus, the A687V 1 746 EZH2 complex is unique from both WT and previously reported Y641 and A677 mutants in that it gains activity B Y641 A677 A687 with a monomethylated, and not dimethylated, substrate. EZH2 ... EZH1 ... Dm E(z) ... G9A ... Transient expression of A687V EZH2 increases SETD8 ... NcDIM-5 ... global H3K27me3, while maintaining H3K27me2 SUV39H2 ... levels PRDM2 ... PRDM9 ... To explore the effect of the A687V EZH2 mutant on H3K27 methylation levels, WT and mutant versions of 100 C a, H3 AA14-34 K27me1 EZH2 were transiently expressed in MCF-7 cells. MCF-7 90 b, H3 AA20-40 K27me1 cells possess a WT EZH2 and have previously been shown a c, H3 AA1-21 K9me1 80 to exhibit increased H3K27me3 and decreased H3K27me2 70 b c in response to transient expression of Y641 and A677 EZH2 mutants (19). Interestingly, although A687V EZH2 60 exhibits increased efficiency in vitro for the dimethylation 50 reaction, but not the trimethylation reaction, exogenous 40 expression of A687V EZH2 reproducibly increased 30 H3K27me3 levels relative to the control (Fig. 2A and B). Histone H3 This represents approximately half of the effect observed 20 Histone H4 Histone H2b with the A677G, Y641F, or Y641N EZH2 mutants. How- A687V EZH2 (% of maximal activity) 10 Histone H2a ever, in contrast with these mutants that induced deple- 0 tion of global H3K27me2 in the process of generating 0102030405060 70 80 90 100 H3K27me3, A687V EZH2 maintained H3K27me2 at levels wt EZH2 (% of maximal activity) equivalent to that of WT EZH2. H3K27me1 levels were minimally affected by either WT or mutant EZH2. These

Figure 1. The A687V EZH2 mutation promotes increased activity with an observations suggest that the increased dimethylation H3K27me1 substrate. A, EZH2 domain architecture (UniProt Q15910). activity of A687V EZH2 may increase H3K27me3 through The A687 residue and other known sites of activating mutations are the law of mass action and over time equilibrium is highlighted within the SET methyltransferase domain. B, alignment of achieved with increased H3K27me3 and near WT levels fl human EZH2 with human EZH1, the y ortholog E(z), and six other related of H3K27me2. histone lysine methyltransferases showing that A687 is located immediately adjacent to the highly conserved NHS motif and is nearby the previously reported Y641 and A677 residues. Blue shading, identical Identification of a B-cell ALL cell line harboring residues; yellow shading, conserved residues. In order for a column to be heterozygous A687V EZH2 shaded, at least seven of nine residues must be conserved/identical. Dm, To identify a cell line possessing the A687V EZH2 Drosophila melanogaster. Nc, Neurospora crassa. C, comparison of biochemical activity for WT and A687V EZH2 across a library of 602 mutation, a panel of cancer cell lines of B-cell origin was peptides representing sequences within histones H2a (blue diamonds), sequenced for the A687 codon located in exon 18 of EZH2. H2b (maroon squares), H3 (green triangles), or H4 (purple circles). A single cell line, SUP-B8, was identified to harbor a

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3065

Ott et al.

Figure 2. Exogenous expression of A Recombinant histone A687V EZH2 stimulates trimethylation of H3K27 without diminishing H3K27me2. A, MCF-7 breast cancer cells were transiently No DNA Empty vector wt EZH2 A687V EZH2 A677G EZH2 Y641F EZH2 Y641N EZH2 transfected with mammalian H3K27me3 expression constructs encoding either WT or mutant forms of EZH2. H3K27me2 Transfection reagent alone (No DNA) and empty vector treatments H3K27me1 were included as controls. Cells were lysed 72 hours after Total H3 transfection, and whole-cell protein extracts were assessed for EZH2 levels of H3K27me3, H3K27me2, H3K27me1, total histone H3, Actin EZH2, and actin via Western blotting. Actin and total histone H3 were included as loading controls. To accurately quantify changes in B H3K27me1 H3K27me2 H3K27me3 protein levels, recombinant P = 0.01 P = 0.01 modified proteins (ActiveMotif) P < 0.01 P < 0.01 were titrated across a range of 150 P < 0.01 P < 0.01 concentrations on the same gel. B, P = 0.01 average values of H3K27me3, P < 0.01 200 P < 0.01 125 100 H3K27me2, or H3K27me1 normalized to total histone H3 from 100 80 150 at least three biologic replicates. Absolute concentrations of protein 75 60 were determined from a standard 100 curve based on the titrated 50 40 recombinant modified proteins. Values are presented as a 50 25 20 percentage of the empty vector control sample. Two-tailed t tests % of empty vector control 0 0 0 were performed comparing all transfected samples with the empty vector control. P values are shown for samples that were No DNA No DNA No DNA fi < wt EZH2 wt EZH2 wt EZH2 signi cantly different (P 0.05) from the empty vector control. Y641F EZH2 Y641F EZH2 Y641F EZH2 Empty vector Empty vector Empty vector A687V EZH2 A687V EZH2 A687V EZH2 Y641N EZH2 Y641N EZH2 Y641N EZH2 A677G EZH2 A677G EZH2 A677G EZH2

heterozygous A687V EZH2 mutation (Supplementary (Fig. 3A and B). Interestingly, although the A687V Fig. S4). The SUP-B8 cell line was established in 1988 from EZH2-mutant SUP-B8 cell line exhibited high H3K27me3, a 13-year-old female with B-cell ALL (30). As this was to its H3K27me2 levels were intermediate between those of the best of our knowledge the first observation of a EZH2 WT and Y641/A677-mutant cell lines. Thus, these potentially activating EZH2 mutation in B-cell ALL, we findings from a panel of B-cell ALL cell lines confirm the performed full-length sequencing of EZH2 in a panel of 11 transient overexpression studies and demonstrate that cell lines reported to derive from B-cell ALL. Although no A687V EZH2 promotes an imbalance in H3K27 methyl- other A687V EZH2 mutations were identified, a hetero- ation states different from Y641 or A677 EZH2 mutants. zygous Y641N EZH2 mutation was identified in VAL, a cell line derived from the bone marrow of a 50-year-old Cells harboring the A687V EZH2 mutation are female diagnosed with B-cell ALL (31). dependent upon EZH2 activity for growth and Because activating mutations at EZH2 Y641 and A677 in survival DLBCL cell lines lead to an imbalance of H3K27 methyl- To examine the extent to which A687V EZH2-mutant ation states with increased H3K27me3 and dramatically cells are dependent upon EZH2 activity for their survival, reduced H3K27me2 (19, 21), we examined the impact of we used a highly selective small-molecule inhibitor of the A687V and Y641N EZH2 mutations in the context of B- EZH2, GSK126. GSK126 has been shown to effectively cell ALL cell lines by Western blot and mass spectrometry inhibit the proliferation of many Y641 and A677 EZH2- approaches. Consistent with Y641 EZH2-mutant DLBCL mutant DLBCL cell lines (26). Among a panel of eight B- cell lines, the Y641N EZH2-mutant VAL cell line exhibited cell ALL cell lines, the A687V EZH2-mutant SUP-B8 cell elevated H3K27me3 and marked loss of H3K27me2 line exhibited the greatest sensitivity to GSK126 with a

3066 Mol Cancer Ther; 13(12) December 2014 Molecular Cancer Therapeutics

Cellular Role of A687V EZH2 in ALL

over a 6-day time course. Although little growth inhibition A ALL DLBCL and no activation of caspases 3 or 7 were observed through 6 days in NALM-6 cells (Fig. 5A and B), a

wt EZH2 A687VY641Nwt EZH2A677GY641N dose-dependent growth inhibition was observed in SUP-B8 cells after 4 days of treatment and was maximal on day 6 when cell number losses were evident for all doses above 100 nmol/L GSK126, suggesting induction of cell death mechanisms (Fig. 5C). Consistent with these

697 Tanoue KARPAS - 231 REH MN - 60 CCRF - SB SD1 RS411 NALM - 6 SUP - B8 VAL Toledo Pfeiffer KARPAS - 422 observations, caspase 3/7 activity was increased in a dose- H3K27me3 dependent fashion beginning on day 5 (Fig. 5D) demon- H3K27me2 strating that GSK126 potently induces apoptotic cell death H3K27me1 in A687V EZH2-mutant cells. Total H3 EZH2 EZH2 inhibition elicits transcriptional activation of EED silenced genes in the A687V EZH2-mutant cell line SUZ12 Previous studies have demonstrated that while both Actin sensitive and resistant lymphoma cell lines lose H3K27me3 with nearly equal potency in response to B 0.8 inhibition of EZH2, only sensitive cell lines exhibit a 0.7 NALM-6 robust program of transcriptional activation (26). When 0.6 SUP-B8 NALM-6 cells were treated with 500 nmol/L GSK126 for 0.5 VAL 72 hours, minimal transcriptional effects were observed 0.4 with only 35 upregulated probes and 10 downregulated 0.3 probes (Fig. 6A and B). SUP-B8, on the other hand, 0.2 exhibited marked transcriptional activation with 643 0.1 upregulated probes (Fig. 6A and B). Interestingly, the set

Fraction of total histone H3 0.0 me0 me1 me2 me3 of upregulated genes was enriched for H3K27me3 in their Histone H3K27 methylation promoter regions in GCBs (32), but acquired H3K27me3 throughout the gene body in EZH2-mutant DLBCL cell lines (26), suggesting that this gene set includes genes Figure 3. A B-cell ALL cell line harboring A687V EZH2 exhibits elevated targeted by both WT and mutant EZH2 (Supplementary H3K27me3 levels. A, Western blot analysis was performed with antibodies specific for H3K27me3, H3K27me2, H3K27me1, total histone Fig. S5). Analysis of gene ontology terms enriched among H3, EZH2, EED, and SUZ12, and actin using protein lysates from a panel the significantly changed probes from SUP-B8 cells sug- of B-cell ALL and DLBCL cell lines. Actin and total histone H3 serve as gests regulation of genes involved in several pathways, loading controls. EZH2 mutation status as determined from full-length including immune system development and function (e. Sanger sequencing is indicated. B, relative levels of histone H3K27me0, me1, me2, and me3 were determined using mass spectrometry and g., AICDA, CXCR5, IL7, IL15, LCK, LTA, LTB, STAT5A, heavy isotope amino acid labeling. Because contributions from the and TNF) and apoptosis (e.g., BCL2A1, BCL2L1, histone H3.3 variant and acetylation on the histone H3 AA27-40 peptide BCL2L11, CASP2, CFLAR, PDCD4, TNF, TNFRSF1A, were negligible based on analysis of the total histone H3 population, TNFSF10, TNFRSF21, and TRADD), whereas NALM-6 fi these are not included in this gure. only exhibited enrichment of two apoptosis-related terms (Fig. 6C; Supplementary Table S3). Gene set enrichment dose of 441 nmol/L being required to inhibit 50% of cell analysis (GSEA) additionally revealed an enrichment of proliferation (growth IC50; Fig. 4A). EZH2 WT cell lines, genes involved in lymphocyte and plasma cell differen- on the other hand, exhibited a range of sensitivity to tiation, interferon response, and apoptosis among the GSK126 with growth IC50 values ranging from 2.5 to 7.8 m genes that were significantly upregulated in the SUP-B8 mol/L. This increased sensitivity observed for SUP-B8 cell line (Supplementary Fig. S6). Thus, inhibition of EZH2 cells was comparable with that seen with A677G- and catalytic activity and loss of H3K27me3 in A687V EZH2 Y641N/F-mutant DLBCLs. In addition, sensitivity was cells induce a program of transcriptional activation for not correlated with proliferation rate (Fig. 4A) or mech- genes associated with lymphocyte activation and cell anistic potency as SUP-B8 exhibited comparable, or slight- death, suggesting a greater dependence on EZH2 in ly reduced, demethylation of H3K27me3 when compared A687V-mutant versus WT cells. with the most sensitive EZH2 WT cell line, NALM-6 (Fig. 4B). These data suggest that SUP-B8 sensitivity likely results from a greater dependence of these cells on EZH2 Discussion activity. Examination of Y641 and A677 EZH2 mutants suggests To evaluate the kinetics of growth inhibition and mech- that alterations to the lysine binding pocket architecture anism of cell death, SUP-B8 and NALM-6 cells were can affect substrate preferences leading to global hyper- examined for cell proliferation and caspase 3/7 activity trimethylation of H3K27 (19, 21, 22). In addition to these

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3067

Ott et al.

A 8,000 B wt EZH2 NALM-6 SUP-B8 mut EZH2 GSK126 GSK126

6,000

(nmol/L) 4,000 50 DMSO 25 nmol/L 125 nmol/L 500 nmol/L 2,000 nmol/L DMSO 25 nmol/L 125 nmol/L 500 nmol/L 2,000 nmol/L H3K27me3

Growth IC 2,000 H3K27me2

H3K27me1 0 Population Total H3 doublings 6.7 3.5 7.9 7.3 7.7 6.4 5.5 5.3 3.3 7.4 7.3 5.6 7.5 5.7 3.4 697 Reh MN60 Toledo Pfeiffer NALM-6SUP-B8 SUP-B15TANOUE SU-DHL-6 SU-DHL-8 SU-DHL-10 KARPAS-231 WSU-DLCL2 KARPAS-422 ALL Lymphoma

Figure 4. A687V EZH2-mutant ALL is highly dependent on EZH2 activity. A, sensitivity of ALL and DLBCL cell lines to EZH2 inhibition with GSK126. Cells were treated with a 20-point 2-fold dilution series of GSK126 (range, 36 mmol/L–70 pmol/L) for 6 days and cell growth was measured using CellTiter-Glo (Promega). Data are represented as the concentration of GSK126 required to inhibit 50% of growth (growth IC50). The growth of each cell line is indicated below the graph as the number of population doublings for vehicle-treated controls for each cell line. B, Western blot analysis of H3K27me3, H3K27me2, H3K27me1, and total histone H3 following treatment of NALM-6 and SUP-B8 cell lines with varying concentrations of GSK126 for 72 hours.

two mutated residues, numerous mutations have now cancers, however, appear to inactivate enzymatic activity been reported throughout the entire EZH2-coding likely reﬂecting a tumor-suppressive role for EZH2 and/ sequence. Most of these mutations observed in myeloid or PRC2 in myeloid cells (33). It is necessary, therefore, to

A Growth B Caspase 3/7

1,200 3125 nmol/L 600 3125 nmol/L 1563 nmol/L 1563 nmol/L ) ) 1,000 0 500 781 nmol/L 0 781 nmol/L 391 nmol/L 391 nmol/L 800 400 195 nmol/L NALM-6 195 nmol/L 98 nmol/L 98 nmol/L 49 nmol/L wild-type 600 49 nmol/L 300 24 nmol/L 24 nmol/L EZH2 DMSO 400 DMSO 200 Figure 5. A687V EZH2-mutant cells CTG signal (% of T CTG signal (% of Caspase 3/7 (% of T Caspase 3/7 (% of exhibit growth inhibition and 100 200 caspase activation in response to EZH2 inhibition. Temporal kinetics 0 0 012345 6 0123456 of GSK126-induced growth Days Days inhibition (A and C) and caspase C D 3/7 activation (B and D) in NALM-6 (A and B) and SUP-B8 (C and D) 12,000 3125 nmol/L 600 3125 nmol/L over a 6-day time course using 1562 nmol/L 1562 nmol/L CellTiter-Glo or Caspase-Glo 3/7, 10,000 ) 500

) 781 nmol/L 781 nmol/L 0 0 391 nmol/L 391 nmol/L respectively. SUP-B8 8,000 195 nmol/L 400 195 nmol/L A687V 98 nmol/L 98 nmol/L 6,000 300 EZH2 49 nmol/L 49 nmol/L 24 nmol/L 24 nmol/L 4,000 200 DMSO DMSO CTG signal (% of T CTG signal (% of Caspase 3/7 (% of T Caspase 3/7 (% of 2,000 100

0 0 0123456 0123456 Days Days

3068 Mol Cancer Ther; 13(12) December 2014 Molecular Cancer Therapeutics

Cellular Role of A687V EZH2 in ALL

A SUP-B8 NALM-6 B Upregulated Downregulated probes probes Figure 6. Activation of gene DMSO GSK126 DMSO GSK126 NALM-6 expression in A687V EZH2, but not NALM-6 SUP-B8 9 WT EZH2, cells. A, gene- 34 SUP-B8 1 expression proﬁling of SUP-B8 and 1 310 NALM-6 cells treated for 72 hours 642 with 0.1% DMSO or 500 nmol/L GSK126. Represented are 996 probe sets with signiﬁcantly altered C gene expression in either cell line SUP-B8 following GSK126 treatment. (n = 27) Immune system development Green, lower expression; red, and function higher expression. B, Venn Signal transduction diagrams depicting the number of up- and downregulated probe sets Apoptosis/cell death and their overlap across the cell lines. C, summary of gene ontology Cell communication terms enriched among the genes upregulated with GSK126 in SUP- Regulation of gene expression B8 or NALM-6 cells. NALM-6 Other (n = 2)

consider that EZH2 mutations may be activating or various SET domain mutants, different enzyme:substrate inactivating and a thorough understanding of the bio- interactions appear to be required for mono-, di-, and chemical and cellular consequences of each mutation trimethylation reactions. For the me0!me1 reaction, the may help clarify the oncogenic or tumor-suppressive key requirement appears to be proper positioning of the activities of EZH2 in different contexts. To this end, we lysine substrate’s e-amine at the methyl transfer pore. This have characterized the cellular effects of A687V EZH2 is accomplished in large part by the highly conserved and demonstrate that this mutant increases global tyrosine residue (Y641 in EZH2, Y245 in SET7/9; Supple- H3K27me3, but unlike Y641 and A687V EZH2 mutants, mentary Fig. S7) and the active site water molecule as retains near normal levels of H3K27me2. Importantly, evidenced by the loss of activity with unmethylated lysine these cells appear to be highly dependent on EZH2 substrates in the EZH2 Y641N/F/S/H/C and SET7/9 activity for their survival. Y245A mutants (19, 21, 22, 34). For the me1!me2 reaction, Because EZH2 and PRC2 crystal structures have the lysine substrate must be oriented with the Kme1 remained elusive, a homology model of the EZH2 SET methyl group rotated away from the methyl transfer pore domain was constructed to understand the location of so that a second methyl group may be transferred (Fig. A687 within the catalytic domain. This previously 7B). This requires that the conserved active site water described model is based on the protein sequence of EZH2 molecule be displaced from the lysine binding pocket or and a crystal structure of GLP/EHMT1 bound to a histone relocated within the enzyme to make room for the larger H3 peptide containing H3K9me2 (19). In this model, A687 substrate. Finally, the me2!me3 reaction is accomplished resides at the interface of the SAM- and peptide-binding when the Kme2 substrate is oriented with both methyl pockets (Fig. 7A). The A687 side chain is buried from groups positioned away from the methyl transfer pore. solvent and points away from the SAM pocket toward This step is limited by steric hindrance between the large I684 and F724, the phenylalanine–tyrosine switch residue Kme2 substrate and EZH2 Y641, SET7/9 Y245, or G9a of EZH2. The backbone carbonyl of A687 points into Y1067. Mutation of this restrictive tyrosine to a smaller the active site and forms H-bonds with both Y726 and residue converts these enzymes into efficient trimethyl- a conserved active site water molecule. While Y726 transferases (19, 21, 22, 34, 35). appears to contribute to efficient binding of SAM/SAH The active site water molecule in EZH2 appears to be (S-adenosyl homocysteine) as well as proper folding of the coordinated by A687, I684, and the lysine substrate (Fig. SET domain, the active site water molecule is stabilized by 7A). In SET domain enzymes in which the F/Y switch the carbonyl groups of A687 and I684 and likely helps residue is a tyrosine, the hydroxyl group of the tyrosine coordinate the lysine substrate. Thus, A687 appears to creates a fourth stabilizing interaction for this water (see occupy a highly conserved position within the SET SET8 and SET7/9 in Supplementary Fig. S7). On the basis domain and may function in multiple roles to regulate of the role of the active site water molecule in regulating binding of both SAM and lysine substrates. product specificity and the positioning of this water in Based upon study of our EZH2 active site model, part by EZH2 A687, we propose that mutation of A687 to related SET domain containing methyltransferases, and the larger valine residue results in expansion of the lysine

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3069

Ott et al.

A WT EZH2 + H3K27me0 B WT EZH2 + H3K27me1 I684 I684 A687 A687

F724 F724

Y726 Y726

2.8 Y641 Y641

K27 A677 K27 A677 me0 me1 SAM SAM

C A687V EZH2 + H3K27me0 D A687V EZH2 + H3K27me1 I684 I684 V687 V687 F724 F724

Y726 Y726 Y641 Y641

K27 A677 K27 A677 me0 me1 SAM SAM

Figure 7. EZH2 A687 coordinates an active site water molecule required for the stabilization of the H3K27me0 substrate. A homology model of EZH2 was generated using the crystal structure of GLP/EHMT1 bound to an H3K9me2 peptide substrate. SAM is colored with orange carbons, WT EZH2 residues are colored with gray carbons in A and B and red carbons in C and D, A687V EZH2 residues are colored with gray carbons in C and D, H3K27 is colored with cyan carbons, and water is indicated by a red sphere. Distances between atoms are indicated in angstroms. Represented are WT EZH2 with H3K27me0 (A), WT EZH2 with H3K27me1 (B), A687V EZH2 with H3K27me0 (C), and A687V EZH2 with H3K27me1 (D).

substrate pocket such that binding of the water is desta- group of a monomethyl lysine substrate. When a phe- bilized relative to the WT enzyme (Fig. 7C). In this sce- nylalanine occupies this position, the water molecule is nario, it would be easier for the methyl group of the boundlesstightlyandcanbedisplacedresultinginthe H3K27me1 substrate to be accommodated in the active increased ability to form Kme2 product. Thus, even site of A687V EZH2 through displacement of the less though EZH2 contains a phenylalanine in the switch tightly bound water, thereby enhancing turnover with an position and can accomplish dimethylation, the A687V H3K27me1 substrate (Fig. 7D). mutation appears to further weaken the binding of the Although there has not been much work focused on active site water and facilitates a further increase in the EZH2 A687, or the equivalent residue in other enzymes, rate of dimethylation. this proposal is supported by extensive work with SET Despite the fact that the predominant biochemical alter- domain F/Y switch residues that also coordinate the ation is a gain in activity with an H3K27me1 substrate (25), active site water molecule. In SET7/9, this residue is a when A687V EZH2 is transiently expressed in cells or tyrosine (Y305) and mutation to a phenylalanine found to occur naturally in an ALL cell line, the observed increases the amount of Kme2 product (34). Conversely, phenotype is increased trimethylation of H3K27, and not in G9A, this residue is a phenylalanine (F1152) and increased dimethylation (Figs. 2 and 3). The data pre- mutation to a tyrosine reduces the amount of Kme2 sented herein and in the literature suggest that the Y641, product (36). The mechanism by which the F/Y switch A677, and A687 EZH2 mutants all stimulate hypertri- residue affects product speciﬁcity is through the methylation of H3K27, but vary in the extent to which strength of hydrogen bonding to the active site water they induce hypodimethylation of H3K27. Unfortunately, molecule. When a tyrosine is in the F/Y switch position, the relevance of this difference is unknown because little is the phenolic OH group forms an H-bond to the water, understood about the distinct roles played by H3K27me2 resulting in the inability to accommodate the methyl and H3K27me3. Global studies of histone marks from

3070 Mol Cancer Ther; 13(12) December 2014 Molecular Cancer Therapeutics

Cellular Role of A687V EZH2 in ALL

þ CD4 T cells suggest that while H3K27me1 is enriched at in B-cell ALL, this observation raises the question whether actively transcribed genes, both H3K27me2 and EZH2 may be uniquely required for homeostasis in the H3K27me3 are present at genes that are silenced or lowly cell types from which these tumors arise. GCB DLBCL expressed (37). Work from Sarma and colleagues (38) and FL arise from centroblasts or centrocytes, respec- suggests that H3K27me2 is associated with lowly tively (41), and common B-cell ALL arises from pre–pro- expressed or poised genes, whereas the conversion to B cells or pro-B cells (42). EZH2 expression appears to be H3K27me3 may more completely silence gene expression. tightly regulated throughout B-cell differentiation. Interestingly, the transition from di- to trimethylation EZH2 levels are high in pro-B cells and then decrease appears to be stimulated by the presence of PHF1, a in pre-B cells and are nearly undetectable in immature Polycomblike (Pcl) family member, known to coimmu- na€ve B cells (43, 44). EZH2 is then upregulated during noprecipitate with a fraction of cellular PRC2 (38–40). The the germinal center reaction in centrocytes and centro- Drosophila Pcl protein has three human homologs, blasts before decreasing again in mature recirculating B including PHF1, MTF2, and PHF19, and all include at cells (43, 44). Thus, it appears that activating mutations least one PHD (Plant Homeo Domain) finger domain and of EZH2 are found in cancer cells arising from cell types a TUDOR domain. The existence of specific biological that highly express EZH2. This may suggest that these mechanisms for the regulation of H3K27me2 and tumors select for cells harboring preexisting activating H3K27me3 suggests that there may be distinct biological mutations in EZH2 because they provide an opportu- requirements for these two marks and perhaps study of nity for the mutant EZH2 to be expressed. Alternatively, these EZH2-mutant cell lines can provide some insight to it may be that silencing of EZH2 is critical for progres- their functions. sion to the next stage of differentiation and that acti- Considering that mutations of EZH2 Y641 are found vated EZH2 establishes an altered epigenetic state that inupto22%GCBDLBCLandFL,itissomewhat impedes further differentiation and promotes transfor- surprising that mutations of A677 and A687 appear to mation in the presence of additional oncogenic muta- be so rare (1%–2%) because they too increase tions. Further work with mouse models engineered to H3K27me3. We have previously proposed for the overexpress mutant EZH2 at various stages of B-cell A677GEZH2mutationthatthisisdueinparttothe differentiation may provide insight to these critical fact that glycine appears to be the only amino acid that questions. when substituted for A677 will promote hypertrimethy- This study has demonstrated that the A687V EZH2 lation and that when considering single-nucleotide mutation is capable of increasing global H3K27me3 in mutations of the A677 codon only a single mutation is cells and that a cancer cell line harboring this mutation capable of generating a glycine (19). The low incidence is dependent on EZH2 activity for its survival. Thus, of the A687V EZH2 mutation may be similarly there are now three EZH2 residues, that when mutated explained. At the nucleotide level, only one of nine in human cancers, alter the default substrate prefer- single-nucleotide mutations within the A687 codon will ences leading to aberrant methylation of H3K27. Our result in a valine residue. Other amino acids that can be studies with a specific EZH2 inhibitor demonstrate that generated by single-nucleotide mutations include glu- targeting of EZH2 in these mutant cell lines is an tamic acid, proline, threonine, serine, and glycine. We efficient strategy for inducing growth inhibition and hypothesize that glutamic acid and proline mutations cell killing. These studies and others (26, 45, 46) provide may disrupt proper enzyme folding due to their dra- encouraging data supporting the use of EZH2 inhibitors matically different properties when compared with the in patients harboring activating mutations of EZH2. WT alanine. Serine and threonine may be tolerated on the basis of their size alone; however, the polar side Disclosure of Potential Conflicts of Interest chains of these residues might also alter enzyme fold- No potential conflicts of interest were disclosed. ing. Finally, glycine, a slightly smaller residue, may be tolerated, but may not change the WT product speci- Authors' Contributions ficity as the active site water molecule would likely still Conception and design: A.P. Graves, Y. Jiang, R.G. Kruger, D. Dhanak, be sufficiently coordinated with this substitution. P.J. Tummino, C.L. Creasy, M.T. McCabe Therefore, it appears that the frequency of these muta- Development of methodology: A.P. Graves, A. Groy, Y. Jiang, R. Annan, S.K. Verma tions may relate to multiple independent factors that Acquisition of data (provided animals, acquired and managed patients, can be rationalized when placed within the context of provided facilities, etc.): H.M. Ott, A.P. Graves, M.B. Pappalardi, theenzyme’sactivesiteandthevariousinteractions M. Huddleston, W.S. Halsey, A.M. Hughes, E. Dul, Y. Jiang, B. Schwartz Analysis and interpretation of data (e.g., statistical analysis, biostatis- requiredforproperfunctioning. tics, computational analysis): H.M. Ott, A.P. Graves, M.B. Pappalardi, Although activating mutations in EZH2 (Y641F/N/S/ M. Huddleston, Y. Jiang, Y. Bai, R. Annan, S.D. Knight, B. Schwartz, C.L. Creasy, M.T. McCabe H/C, A677G, and A687V) have thus far been found Writing, review, and/or revision of the manuscript: H.M. Ott, primarily in GCB DLBCL and FL (16–20, 23), this study A.P. Graves, M.B. Pappalardi, R.G. Kruger, B. Schwartz, C.L. Creasy, identified cases of Y641N and A687V EZH2 mutations in M.T. McCabe Administrative, technical, or material support (i.e., reporting or orga- B-cell ALL cell lines. Although much additional screening nizing data, constructing databases): M.B. Pappalardi, S.K. Verma is required to determine the incidence of these mutations Study supervision: R.G. Kruger, D. Dhanak, P.J. Tummino, M.T. McCabe

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3071

Ott et al.

Other (produced the A687V mutation in the EZH2 gene that was used to DPU and Platform Technology and Science organization who provided express the protein described in this article): E. Dul guidance and support. Other (directed the design, synthesis, and analysis of the chemical The costs of publication of this article were defrayed in part by the compounds used for the generation of data in this article): S.K. Verma 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. Acknowledgments The authors wish to acknowledge Don Fisher for coordinating reagent Received October 16, 2013; revised September 8, 2014; accepted generation, and all members of GlaxoSmithKline’s Cancer Epigenetics September 9, 2014; published OnlineFirst September 24, 2014.

References 1. Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P, Reinberg 19. McCabe MT, Graves AP, Ganji G, Diaz E, Halsey WS, Jiang Y, et al. D. Histone methyltransferase activity associated with a human multi- Mutation of A677 in histone methyltransferase EZH2 in human B-cell protein complex containing the Enhancer of Zeste protein. Genes Dev lymphoma promotes hypertrimethylation of histone H3 on lysine 27 2002;16:2893–905. (H3K27). Proc Natl Acad Sci U S A 2012;109:2989–94. 2. Muller J, Hart CM, Francis NJ, Vargas ML, Sengupta A, Wild B, et al. 20. Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL, Corbett Histone methyltransferase activity of a Drosophila Polycomb group RD, et al. Frequent mutation of histone-modifying genes in non- repressor complex. Cell 2002;111:197–208. Hodgkin lymphoma. Nature 2011;476:298–303. 3. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K. Genome-wide 21. Sneeringer CJ, Scott MP, Kuntz KW, Knutson SK, Pollock RM, Richon mapping of Polycomb target genes unravels their roles in cell fate VM, et al. Coordinated activities of wild-type plus mutant EZH2 drive transitions. Genes Dev 2006;20:1123–36. tumor-associated hypertrimethylation of lysine 27 on histone H3 4. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, et al. (H3K27) in human B-cell lymphomas. Proc Natl Acad Sci U S A Role of histone H3 lysine 27 methylation in Polycomb-group silencing. 2010;107:20980–5. Science 2002;298:1039–43. 22. Yap DB, Chu J, Berg T, Schapira M, Cheng SW, Moradian A, et al. 5. Kirmizis A, Bartley SM, Kuzmichev A, Margueron R, Reinberg D, Green Somatic mutations at EZH2 Y641 act dominantly through a mecha- R, et al. Silencing of human polycomb target genes is associated with nism of selectively altered PRC2 catalytic activity, to increase H3K27 methylation of histone H3 Lys 27. Genes Dev 2004;18:1592–605. trimethylation. Blood 2011;117:2451–9. 6. Findeis-Hosey JJ, Huang J, Li F, Yang Q, McMahon LA, Xu H. High- 23. Lohr JG, Stojanov P, Lawrence MS, Auclair D, Chapuy B, Sougnez C, grade neuroendocrine carcinomas of the lung highly express enhancer et al. Discovery and prioritization of somatic mutations in diffuse large of zeste homolog 2, but carcinoids do not. Hum Pathol 2011;42: B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl 867–72. Acad Sci U S A 2012;109:3879–84. 7. Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, et al. EZH2 24. Bodor C, Grossmann V, Popov N, Okosun J, O'Riain C, Tan K, et al. is a marker of aggressive breast cancer and promotes neoplastic EZH2 mutations are frequent and represent an early event in follicular transformation of breast epithelial cells. Proc Natl Acad Sci U S A lymphoma. Blood 2013;122:3165–8. 2003;100:11606–11. 25. Majer CR, Jin L, Scott MP, Knutson SK, Kuntz KW, Keilhack H, et al. 8. Simon JA, Lange CA. Roles of the EZH2 histone methyltransferase in A687V EZH2 is a gain-of-function mutation found in lymphoma cancer epigenetics. Mutat Res 2008;647:21–9. patients. FEBS Lett 2012;586:3448–51. 9. Takawa M, Masuda K, Kunizaki M, Daigo Y, Takagi K, Iwai Y, et al. 26. McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller Validation of the histone methyltransferase EZH2 as a therapeutic GS, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with target for various types of human cancer and as a prognostic marker. EZH2-activating mutations. Nature 2012;492:108–12. Cancer Sci 2011;102:1298–305. 27. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative 10. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha analysis of large gene lists using DAVID bioinformatics resources. Nat C, Sanda MG, et al. The polycomb group protein EZH2 is involved in Protoc 2009;4:44–57. progression of prostate cancer. Nature 2002;419:624–9. 28. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette 11. Wagener N, Macher-Goeppinger S, Pritsch M, Husing J, Hoppe-Seyler MA, et al. Gene set enrichment analysis: a knowledge-based approach K, Schirmacher P, et al. Enhancer of zeste homolog 2 (EZH2) expres- for interpreting genome-wide expression profiles. Proc Natl Acad Sci sion is an independent prognostic factor in renal cell carcinoma. BMC U S A 2005;102:15545–50. Cancer 2010;10:524. 29. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo 12. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is P, Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioin- downstream of the pRB-E2F pathway, essential for proliferation, and formatics 2011;27:1739–40. amplified in cancer. EMBO J 2003;22:5323–35. 30. Carroll WL, Link MP, Cleary ML, Bologna S, Carswell C, Amylon MD, 13. Saramaki OR, Tammela TL, Martikainen PM, Vessella RL, Visakorpi T. et al. Idiotype as a tumor-specific marker in childhood B-cell acute The gene for polycomb group protein enhancer of zeste homolog 2 lymphoblastic leukemia. Blood 1988;71:1068–73. (EZH2) is amplified in late-stage prostate cancer. Genes Chromo- 31. Dyer MJ, Lillington DM, Bastard C, Tilly H, Lens D, Heward JM, et al. somes Cancer 2006;45:639–45. Concurrent activation of MYC and BCL2 in B-cell non-Hodgkin lym- 14. Sander S, Bullinger L, Klapproth K, Fiedler K, Kestler HA, Barth TF, et al. phoma cell lines by translocation of both oncogenes to the same MYC stimulates EZH2 expression by repression of its negative reg- immunoglobulin heavy chain locus. Leukemia 1996;10:1198–208. ulator miR-26a. Blood 2008;112:4202–12. 32. Beguelin W, Popovic R, Teater M, Jiang Y, Bunting KL, Rosen M, et al. 15. Varambally S, Cao Q, Mani RS, Shankar S, Wang X, Ateeq B, et al. EZH2 is required for germinal center formation and somatic EZH2 Genomic loss of microRNA-101 leads to overexpression of histone mutations promote lymphoid transformation. Cancer Cell 2013; methyltransferase EZH2 in cancer. Science 2008;322:1695–9. 23:677–92. 16. Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, et al. 33. Hock H. A complex Polycomb issue: the two faces of EZH2 in cancer. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large Genes Dev 2012;26:751–5. B-cell lymphomas of germinal-center origin. Nat Genet 2010;42:181–5. 34. Del Rizzo PA, Couture JF, Dirk LM, Strunk BS, Roiko MS, Brunzelle JS, 17. Bodor C, O'Riain C, Wrench D, Matthews J, Iyengar S, Tayyib H, et al. et al. SET7/9 catalytic mutants reveal the role of active site water mole- EZH2Y641mutationsinfollicularlymphoma.Leukemia2011;25:726–9. cules in lysine multiple methylation. J Biol Chem 2010;285:31849–58. 18. Ryan RJ, Nitta M, Borger D, Zukerberg LR, Ferry JA, Harris NL, et al. 35. Wu H, Min J, Lunin VV, Antoshenko T, Dombrovski L, Zeng H, et al. EZH2 codon 641 mutations are common in BCL2-rearranged germinal Structural biology of human H3K9 methyltransferases. PLoS ONE center B-cell lymphomas. PLoS ONE 2011;6:e28585. 2010;5:e8570.

3072 Mol Cancer Ther; 13(12) December 2014 Molecular Cancer Therapeutics

Cellular Role of A687V EZH2 in ALL

36. Collins RE, Tachibana M, Tamaru H, Smith KM, Jia D, Zhang X, et al. 41. Nogai H, Dorken B, Lenz G. Pathogenesis of non-Hodgkin's lympho- In vitro and in vivo analyses of a Phe/Tyr switch controlling product ma. J Clin Oncol 2011;29:1803–11. specificity of histone lysine methyltransferases. J Biol Chem 42. Cobaleda C, Sanchez-Garcia I. B-cell acute lymphoblastic leukaemia: 2005;280:5563–70. towards understanding its cellular origin. Bioessays 2009;31:600–9. 37. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, et al. High- 43. Su IH, Basavaraj A, Krutchinsky AN, Hobert O, Ullrich A, Chait BT, et al. resolution profiling of histone methylations in the human genome. Cell Ezh2 controls B cell development through histone H3 methylation and 2007;129:823–37. Igh rearrangement. Nat Immunol 2003;4:124–31. 38. Sarma K, Margueron R, Ivanov A, Pirrotta V, Reinberg D. Ezh2 requires 44. Velichutina I, Shaknovich R, Geng H, Johnson NA, Gascoyne RD, PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo. Mol Cell Melnick AM, et al. EZH2-mediated epigenetic silencing in germinal Biol 2008;28:2718–31. center B cells contributes to proliferation and lymphomagenesis. 39. Cao R, Wang H, He J, Erdjument-Bromage H, Tempst P, Zhang Y. Role Blood 2010;116:5247–55. of hPHF1 in H3K27 methylation and Hox gene silencing. Mol Cell Biol 45. Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus 2008;28:1862–72. CR, et al. A selective inhibitor of EZH2 blocks H3K27 methylation and 40. Nekrasov M, Klymenko T, Fraterman S, Papp B, Oktaba K, Kocher kills mutant lymphoma cells. Nat Chem Biol 2012;8:890–6. T, et al. Pcl-PRC2 is needed to generate high levels of H3-K27 46. Qi W, Chan H, Teng L, Li L, Chuai S, Zhang R, et al. Selective inhibition trimethylation at Polycomb target genes. EMBO J 2007;26: of Ezh2 by a small-molecule inhibitor blocks tumor cells proliferation. 4078–88. Proc Natl Acad Sci U S A 2012;109:21360–5.

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3073

A687V EZH2 Is a Driver of Histone H3 Lysine 27 (H3K27) Hypertrimethylation

Heidi M. Ott, Alan P. Graves, Melissa B. Pappalardi, et al.

Mol Cancer Ther 2014;13:3062-3073. Published OnlineFirst September 24, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-13-0876

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2014/09/26/1535-7163.MCT-13-0876.DC1

Cited articles This article cites 46 articles, 24 of which you can access for free at: http://mct.aacrjournals.org/content/13/12/3062.full#ref-list-1

Citing articles This article has been cited by 6 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/13/12/3062.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/13/12/3062. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.