Chemistry & Biology Article - Constellation Pharmaceuticals...Chemistry & Biology Article EZH2 Inhibitor Efﬁcacy in Non-Hodgkin’s Lymphoma Does Not Require Suppression of H3K27

Please cite this article in press as: Bradley et al., EZH2 Inhibitor Efficacy in Non-Hodgkin’s Lymphoma Does Not Require Suppression of H3K27 Mono-methylation, Chemistry & Biology (2014), http://dx.doi.org/10.1016/j.chembiol.2014.09.017

Chemistry & Biology

Article

EZH2 Inhibitor Efficacy in Non-Hodgkin’sLymphoma Does Not Require Suppressionof H3K27 MonomethylationWilliam D. Bradley,1,3 Shilpi Arora,1,3 Jennifer Busby,1 Srividya Balasubramanian,1 Victor S. Gehling,1

Christopher G. Nasveschuk,1 Rishi G. Vaswani,1 Chih-Chi Yuan,1 Charlie Hatton,1 Feng Zhao,1 Kaylyn E. Williamson,1

Priyadarshini Iyer,1 Jacqui Mendez,2 Robert Campbell,1 Nico Cantone,1 Shivani Garapaty-Rao,1 James E. Audia,1

Andrew S. Cook,1 Les A. Dakin,1 Brian K. Albrecht,1 Jean-Christophe Harmange,1 Danette L. Daniels,2

Richard T. Cummings,1 Barbara M. Bryant,1 Emmanuel Normant,1 and Patrick Trojer1,*1Constellation Pharmaceuticals, Inc., 215 First Street, Cambridge, MA 02142, USA2Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA3Co-first author*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.chembiol.2014.09.017

SUMMARY

The histone lysine methyltransferase (MT) Enhancerof Zeste Homolog 2 (EZH2) is considered an onco-genic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL)and follicular lymphoma due to the presence ofrecurrent, monoallelic mutations in the EZH2 cata-lytic domain. These genomic data suggest that tar-geting the EZH2 MT activity is a valid therapeuticstrategy for the treatment of lymphoma patientswith EZH2 mutations. Here we report the identifica-tion of highly potent and selective EZH2 small mole-cule inhibitors, their validation by a cellular thermalshift assay, application across a large cell panel rep-resenting various non-Hodgkin’s lymphoma (NHL)subtypes, and their efficacy in EZH2mutant-contain-ing GCB-DLBCL xenograft models. Surprisingly, ourEZH2 inhibitors selectively affect the turnover oftrimethylated, but not monomethylated histone H3lysine 27 at pharmacologically relevant doses.Importantly, we find that these inhibitors are broadlyefficacious also in NHL models with wild-type EZH2.

INTRODUCTION

Chromatin modifier-encoding genes are now appreciated to be

frequently targeted by genomic aberrations in cancer (Garraway

and Lander, 2013; Gonzalez-Perez et al., 2013; Lawrence et al.,

2014; Zack et al., 2013). Both, the activation and inactivation of

chromatin modifiers by mutations support the concept that

cancer cells utilize manipulation of chromatin structure as a

means to gain growth advantage. Non-Hodgkin’s lymphoma

(NHL) is a common hematological cancer comprised of multiple

subtypes, most of which arise from various differentiation

stages of the B cell lineage. Recent genomic and transcriptomic

studies have allowed for a molecular categorization of NHL sub-

types based on recurrent mutations (Kiel et al., 2012; Lohr et al.,

Chemistry & Biology 21,

2012; Morin et al., 2011; Rossi et al., 2012; Schmitz et al., 2012)

and specific gene expression profiles (Alizadeh et al., 2000; Lenz

et al., 2008; Malumbres et al., 2009; Rosenwald et al., 2002) that

provide hints about potential key oncogenic drivers and driver

pathways. These sequencing efforts aim to identify novel thera-

peutic avenues as well as to provide adequate diagnostic infor-

mation to improve the choice of existing therapeutic options

across NHL subtypes.

Histone lysine methylation, specifically histone H3 lysine 27

(H3K27) methylation, has gained attention as a putative onco-

genic driver pathway after recurrent mutations in the H3K27-

specific methyltransferase (MT) Enhancer of Zeste Homolog 2

(EZH2) (Morin et al., 2010) and the corresponding lysine deme-

thylase KDM6A/UTX (van Haaften et al., 2009) were discovered.

Either mutation results in aberrantly high H3K27 trimethylation

levels. In the case of EZH2, the catalytic component of the poly-

comb repressive complex 2 (PRC2; reviewed in Margueron and

Reinberg, 2011), monoallelic mutations of single residues within

its catalytic domain were found in germinal center B cell-like

diffuse large B cell lymphoma (GCB-DLBCL), follicular lym-

phoma (Guo et al., 2014; Lohr et al., 2012; Morin et al., 2010,

2011), and melanoma (Hodis et al., 2012). These mutations alter

the substrate specificity of EZH2, promoting the conversion from

H3K27 dimethylated (me2) to trimethylated (me3) states (Majer

et al., 2012; McCabe et al., 2012a; Sneeringer et al., 2010; Wigle

et al., 2011; Yap et al., 2011). In addition, EZH2 is prevalently

overexpressed in many cancer types including breast and pros-

tate cancer, in which EZH2 level elevation correlates with the

stage of the disease and poor prognosis (Kleer et al., 2003;

Varambally et al., 2002). For years, EZH2 has received significant

interest from academia and the pharmaceutical industry as a

potential oncology target, and EZH2 small molecule inhibitors

have been identified (Diaz et al., 2012; Garapaty-Rao et al.,

2013; Knutson et al., 2012; Konze et al., 2013; McCabe et al.,

2012b; Nasveschuk et al., 2014; Qi et al., 2012). Several of these

compounds eliminated tumor growth in GCB-DLBCL models

with activating EZH2 mutations (Beguelin et al., 2013; Knutson

et al., 2014; McCabe et al., 2012b).

PRC2 likely controls bulk levels of H3K27 mono, di, and trime-

thylation (H3K27me1, me2, and me3), but the context-specific

functional distinction of the three H3K27 methylation states is

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http://dx.doi.org/10.1016/j.chembiol.2014.09.017

Chemistry & Biology

EZH2 Inhibitor Efficacy in Non-Hodgkin’s Lymphoma


just beginning to emerge (Ferrari et al., 2014). While H3K27me3

is well established to correlate with transcriptional repression,

H3K27me1 is rather present on active genes. PRC2 harbors

either EZH2 or its close homolog EZH1 as its catalytic compo-

nent. Both PRC2 variants exhibit H3K27-specific MT activity,

however it was suggested that EZH1-containing PRC2 also

functions by other mechanisms (Margueron et al., 2008; Son

et al., 2013). EZH2-containing PRC2 is responsible for maintain-

ing global H3K27me3 levels in embryonic stem cells; however,

EZH2 deletion did not affect global H3K27me1 levels. Instead,

deletion of EED (which would inactivate both EZH1- and EZH2-

containing PRC2 complexes) or EZH1 deletion in hematopoietic

stem cells reduced global H3K27me1 levels (Hidalgo et al., 2012;

Xie et al., 2014). These genetic data suggest that EZH1-contain-

ing PRC2 is involved in maintaining global H3K27me1 levels.

Here, we report the identification and characterization of an

additional EZH2 inhibitor chemotype that we utilized to broadly

investigate EZH2 dependencies in NHL. Moreover, we used a

number of approaches to determine the impact of EZH2 inhibi-

tion on global H3K27 methylation and gene expression pro-

grams in disease-relevant lymphoma cell models and provide

evidence that our inhibitors selectively modulate H3K27me3,

but not H3K27me1 levels. Our data suggest that NHL cells are

indeed dependent on the catalytic activity of EZH2, and that

EZH1 cannot substitute for the pharmacologically mediated

loss of EZH2 MT activity in lymphoma. Consequently, inhibition

of EZH1 and H3K27me1 reduction are not required for our

EZH2 inhibitors to achieve in vitro and in vivo efficacy in NHL.

RESULTS

Identification of Potent, Selective, and Cell-ActiveEnhancer of Zeste Homolog 2 InhibitorsWepreviously carried out a high throughput screening campaign

to identify small molecule inhibitors of EZH2 (Garapaty-Rao

et al., 2013; Nasveschuk et al., 2014). Among the hits from these

efforts was CPI-905, a weak inhibitor with a pyridone headgroup

(Supplemental Experimental Procedures and Figure S1A avail-

able online). Taking advantage of both the modular composition

of CPI-905 and our previously published chemotype, we gener-

ated a hybrid compound series with significantly improved prop-

erties (described elsewhere). These efforts eventually led to the

identification of compound CPI-360 (Figure 1A; compound 1).

In biochemical assays using reconstituted PRC2 containing

wild-type (WT) or Y641 mutant EZH2, or WT EZH1 and peptide

or oligonucleosome substrates, this compound was determined

as a subnanomolar inhibitor of the EZH2 MT activity (Figures 1B

and S1B). CPI-360 functions on the basis of S-adenosyl-L-

methionine (SAM)-competition (Figure 1C), inhibits EZH1 about

100-fold less and shows exquisite selectivity across a large

panel of histone lysine and arginine, and DNA methyltrans-

ferases (Figure 1D).

Next, we established a cellular thermal shift assay (CETSA) for

EZH2, a recently published technique that proved useful in

determining drug binding to its cognate target in a cellular setting

(Martinez Molina et al., 2013). This assay is based on the

biophysical principle of ligand-induced thermal stabilization of

target proteins, and thus allows for a direct measurement of

cellular target engagement in intact cells (Figure 1E, top). A se-

2 Chemistry & Biology 21, 1–13, November 20, 2014 ª2014 Elsevier

ries of CETSA at various temperatures with DMSO and CPI-

360-treated disease-relevant KARPAS-422 GCB-DLBCL cells

(which harbor a monoallelic Y641N EZH2 mutation) determined

that 52�C is a temperature at which EZH2 protein stability is

increased (Figure 1E, bottom). Increased EZH2 stability was

observed at 52�C, but not at 37�C in a CPI-360 dose-dependent

manner (Figure 1F), suggesting that the effects are indeed ther-

mally induced. CPI-360-induced EZH2 thermo-stabilization was

time-dependent, since the half-maximal effective dose (EC50)

was lower after 24 hr compared to 4 hr of treatment (Figure 1F,

lower panels). We obtained similar data for HTGCB-DLBCL cells

(which harborWT EZH2; Figure 1G), suggesting that thermo-sta-

bilization of EZH2 was achieved irrespective of EZH2 status.

However, the CPI-360 EC50 in HT cells was lower compared to

KARPAS-422 cells, perhaps indicating a higher affinity of CPI-

360 for WT compared to mutant EZH2. Compound-induced

EZH2 stability increase was quickly lost upon compound

removal (Figure 1H), suggesting that EZH2 inhibitor-induced ef-

fects are reversible.

As expected from the CETSA experiments, CPI-360 potently

reduced global H3K27me3 and H3K27me2 levels in a dose-

dependent manner (Figures 2A and S2A), while not affecting

theglobal levels of other tri anddimethylationmarksor theprotein

levels of PRC2subunits (Figures2BandS2A). Progressive reduc-

tion of H3K27me3 and me2 levels was observed over time in the

presence of CPI-360 and compound removal resulted in gradual

H3K27 methylation mark recovery, consistent with reversible

suppression of EZH2 catalytic activity (Figure S2B). To assess

PRC2 complex integrity in response to CPI-360 treatment in

intact cells, we established a bioluminescence resonance energy

transfer (nanoBRET) assay utilizing Nano Luciferase- (NanoLuc)

and Halo-tagged PRC2 components in KARPAS-422 cells.

CPI-360 did not affect the interactions between EZH2 and EED

(Figure 2C), and EZH2 and SUZ12 (Figure 2D) in a cellular setting,

thus being distinct from other PRC2-targeted modalities that are

aimed to prevent PRC2 complex formation (Kim et al., 2013). Em-

ploying a similar nanoBRET assay, we also determined that CPI-

360 did not affect global binding of EZH2 (Figure 2E) or EED (data

not shown) to chromatin.

To assess the impact of CPI-360 on histone modification

patterns in GCB-DLBCL, we used a stable isotope labeling

by amino acids in cell culture (SILAC) liquid chromatography-

tandem mass spectrometry (LC-MS/MS) approach. As ex-

pected, H3K27me3- andH3K27me2-containing peptide species

decreased in abundance, while the H3K27 unmodified peptide

species were significantly increased (Figure 2F; Table S1). Inter-

estingly, H3K27me1 peptide species increased in abundance,

with the exception of a peptide that carries H3K36me3 in addi-

tion to H3K27me1 (H3K27me1/K36me3). Most of the captured

histone peptide species did not show any change upon com-

pound treatment.

Slow Kinetics of H3 Lysine 27 Trimethylation Turnoverand Selective Suppression of H3 Lysine 27Trimethylation, But Not H3 Lysine 27 MonomethylationTurnover by CPI-360 in Germinal Center B Cell-likeDiffuse Large B Cell LymphomaUpon target engagement, the kinetics of functional conse-

quences of EZH2 inhibitor treatment will determine the timing

Ltd All rights reserved

Figure 1. Identification and Characterization of a Potent, Selective, Reversible, and Cell Active Enhancer of Zeste Homolog 2 Inhibitor

(A) Chemical structure of CPI-360.

(B) Determination of the CPI-360 potency employing a biochemical assay with reconstituted PRC2 harboring WT EZH2 or Y641N-mutated EZH2 as the catalytic

component and histone H3 peptides comprising residues 17–38with either amonomethyl (WT) or dimethyl (Y641N) group on residue K27. Indicated half-maximal

inhibitory concentration (IC50) values were calculated from the mean of three experiments ±SD. CPI-360 was found to be about 5-fold more potent on WT

compared to the Y641N mutant EZH2.

(C) Determination of the mechanism of inhibition of CPI-360. Enzymatic assays were carried out with either 20 or 200 pMPRC2 (Standard assay and 10X Enzyme,

respectively) and with 10X KMapp of H3K27me1 (10X Peptide) or SAM (10X SAM). The�5-fold shift of IC50 values at higher SAM is consistent with CPI-360 being

SAM-competitive. Data are represented as the mean of triplicate experiments ±SD.

(D) The selectivity of CPI-360 was tested across a panel of recombinant DNA and histone methyltransferases, comprising a total of 30 different enzymes

(indicated on the left). With the exception of EZH1 and EZH2, none of the tested enzymes was inhibited at concentrations of up to 10 mM. All enzyme assays were

carried out under balanced conditions and data represent the IC50 values obtained from a 10-point dose response curve with 3-fold serial dilutions starting from a

top concentration of 10 mM.

(E) CETSA was employed to monitor CPI-360 cellular target engagement. Cartoon illustrates the CETSA principle (top); different cell treatment groups are

separated from culture medium, intact cells exposed to varying temperatures, lysed with adequate buffers, soluble from insoluble proteins separated by

centrifugation, and soluble protein pool examined for protein of interest by western blotting. KARPAS-422 GCB-DLBCL cells were incubated with DMSO or CPI-

360 [10 mM] for 2 hr and subsequently exposed to temperature shifts (indicated on top) for 3 min. Cellular extracts after centrifugation were interrogated for EZH2

protein levels by western blotting. No EZH2 thermal stabilization of CPI-360 versus DMSO-treated cells was observed at room temperature (no temperature shift),

but at 52�C. Vinculin served as a control.

(F) CETSA in KARPAS-422 cells in the presence of DMSO or various CPI-360 concentrations [0.01–40 mM]. Increasing concentrations of CPI-360 gradually

increased EZH2 thermo-stability at 52�C (top panel, top blot), but not at 37�C (top panel, bottom blot). Densitometry-based quantification of western blot signals

(EZH2 intensities normalized to vinculin intensities for each data point, see Supplemental Experimental Procedures for details) are graphed for illustration

purposes (bottom graphs). Shown are the mean of three (52�C; red curve) and two (37�C; gray curve) biological replicates ±SD. KARPAS-422 CETSAwas carried

out after 24 (top) or 4 hr (bottom) incubation with CPI-360. Longer incubation times reduced the EC50 of EZH2 thermo-stabilization.

(G) CETSAwas carried out with HTGCB-DLBCL cells grown in the presence of DMSO or various CPI-360 concentrations [0.01–40 mM] for 24 hr. Quantification of

signals was carried out as described in (F) and data are shown as the mean of two biological replicates ±SD.

(H) CETSA was carried out with KARPAS-422 cells grown in the presence of DMSO or CPI-360 [10 mM]. After the 24 hr incubation period CPI-360 was removed

and cells continued to culture for 0.5, 1, 2 or 4 hr before exposure to 52�C. Nuclear extracts from temperature-exposed cells were resolved by SDS-PAGE and

immuno-blottedwith EZH2-specific antibodies. Vinculin served as a control. Quantifiedwestern blot data are shown as themean of two biological replicates ±SD.

Chemistry & Biology


Chemistry & Biology 21, 1–13, November 20, 2014 ª2014 Elsevier Ltd All rights reserved 3


Figure 2. Enhancer of Zeste Homolog 2 Inhibitor CPI-360 Modulates Histone Lysine Methylation States without Affecting Polycomb Repres-

sive Complex 2 Integrity or Chromatin-Binding

(A) Determination of the cellular potency of CPI-360. HeLa cells were incubated with various concentrations of CPI-360 over 4 days. Cells were lysed and

subjected to ELISA assays using H3K27me3-, H3K27me2-, and total H3-specific antibodies. CPI-360 treatment resulted in a dose-dependent reduction of

H3K27me3. Data are represented as the mean of duplicate experiments ±SEM.

(B) CPI-360 reduces H3K27 methylation levels in a dose-dependent manner without affecting EZH2, SUZ12, and EED protein levels. KARPAS-422 cells were

treated with DMSO or various concentrations of CPI-360 (indicated on top) for 4 days and whole cell extracts were analyzed by western blotting with antibodies

indicated on the right. GAPDH and H3 served as protein loading controls.

(C) Depiction of the PRC2 core complex. For the establishment of the nanoBRET assay, EZH2 and EED are expressed as fusion proteins with NanoLuc and Halo

tags, respectively. Tagged PRC2 complex components expressed in host cells form a complex. Close proximity between the NanoLuc and Halo tags is required

for BRET. Furimazine is added as substrate for NanoLuc that will upon excitation at 450 nm catalyze the transformation of substrate and function as the donor of

bioluminescence transferred to the Halo-tag with bound fluorescent ligand. The energy transfer fromNano-Luc to Halo will result in the emission of BRET that can

be detected at 610 nm. See Supplemental Experimental Procedures for details. The effect of CPI-360 on the EZH2-EED protein-protein interaction was explored.

Human embryonic kidney (HEK)293 cells were transfected with NanoLuc-EZH2 and Halo-EED, and 48 hr post transfection BRET signal was detected, indicating

a direct interaction between EZH2 and EED. The addition of CPI-360 [10 mM] over the 48 hr period did not affect the interaction. Data are represented as themean

of quadruplicate experiments ±SD.

(D) As in (C), but exploring the EZH2-SUZ12 protein-protein interaction. HEK293 cells were transfected with NanoLuc-EZH2 and Halo-SUZ12, and 48 hr post

transfection BRET signal was detected, indicating a direct interaction between EZH2 and SUZ12. The addition of CPI-360 [10 mM] over the 48 hr period did not

affect the interaction. Data are represented as the mean of quadruplicate experiments ±SD.

(E) CPI-360 does not globally affect PRC2-binding to chromatin. A cartoon describes a nanoBRET assay, which was developed tomonitor the interaction of Halo-

tagged H3.1 and H3.3 and Nano-Luc-tagged EZH2 in living cells (top). KARPAS-422 cells were treated with DMSO and CPI-360 [10 mM] for 2 days and BRET

signal was measured. Data are represented as the mean of quadruplicate experiments ±SD.

(F) EZH2 inhibition causes changes to the histone modification landscape. A SILAC labeling approach was employed in KARPAS-422 cells to determine changes

in histone modifications in an unbiasedmanner. KARPAS-422 cells were grown in the presence of DMSO and heavy medium or in the presence of CPI-360 [0.63,

2.5, and 10 mM] and light medium for 4 days. Cell populations were mixed, subjected to LC-MS/MS, and analyzed for changes in the abundance of histone

peptide species. Data are represented in form of a heatmap with increases and decreases in abundance shown in red and blue, respectively. Each data set

represents the average of duplicate experiments.

Chemistry & Biology



of relevant phenotypic responses. To measure H3K27 methyl-

ation turnover in GCB-DLBCL and to determine how CPI-360

affects incorporation of new methyl groups on H3K27 in

proliferating cells, we used an isotopic labeling approach (Fig-

ure 3A). Isotopically labeled methionine is added to the growth


medium, which over time results in the incorporation of ‘‘heavy’’

methyl groups onto histones, eventually turning over all histone

methylation from ‘‘light’’ to heavy. In KARPAS-422 cells,

approximately 60% of H3K27 peptides are trimethylated,

consistent with earlier studies suggesting that the presence of


Figure 3. Time-Dependent Suppression of H3 Lysine 27 Trimethylated, But Not H3 Lysine 27 Monomethylation, Turnover by CPI-360 in

Germinal Center B Cell-like Diffuse Large B Cell Lymphoma

(A) A methionine isotope labeling approach was used to determine histone methylation turnover. A schematic is shown illustrating that KARPAS-422 cells grown

in regular RPMI were shifted to RPMI medium containing methyl-13C, d3-L-Methionine and in the presence of DMSO or CPI-360 [3 mM] at time 0. Cells were

collected at 11 time-points, as early as 6 hr and up to 168 hr. Histone extraction, derivatization, desalting LC-MS/MSprocedure, and data acquisition were carried

out as described in the Experimental Procedures. This approach allows for the increased appearance of isotopically labeled (heavy) methyl-lysine-containing

histone peptide species over time.

(B) CPI-360 treatment [3 mM] reduces bulk H3K27me3 and H3K27me2 over time in KARPAS-422 cells. The abundance of isotopically labeled and unlabeled

histone peptide species harboring specific H3K27 methylation states (indicated on the right) were combined to determine the overall abundance of these

methylation states at various time points. While DMSO-treated cells have similar distribution of tri, di, and monomethylated H3K27-peptide abundance at each

time point, CPI-360-treated cells show a progressive reduction of tri and dimethylated, but increase in mono and unmethylated H3K27-peptides.

(C) CPI-360 suppresses the appearance of heavy H3K27me3, but not H3K27me1, peptide species. The turnover of individual H3K27 methyl-peptide species

(indicated on the right) was assessed in KARPAS-422 cells grown in the presence of DMSO (left) or CPI-360 [3 mM] (right).

(D) Thymidine, but not CPI-360-treated, cells show substantially delayed histone turnover. The total histone turnover was assessed in KARPAS-422 cells in the

presence of DMSO (blue), CPI-360 [3 mM] (red), or thymidine (green).

(E) Thymidine-treated KARPAS-422 cells show substantially delayed H3K27me3 turnover (shades of blue), while turnover of other histone peptide species

including that of H3K27me1, H3K36me1, and H3K36me2 (light green, green and red) is not affected.

(F) High CPI-360 concentrations partially affect H3K27me1 turnover. The turnover of H3K27me1 (top) or H3K36me1 (bottom) was assessed in KARPAS-422 cells

grown in the presence of DMSO (blue) or CPI-360 [30 mM] (red).

Chemistry & Biology



the Y641N-mutated EZH2 protein results in elevated H3K27me3

levels (Sneeringer et al., 2010). CPI-360 treatment dimin-

ished the abundance of ‘‘heavy and light’’ H3K27me3 and

H3K27me2 and substantially increased H3K27me1 and

H3K27 unmodified peptide species over time (Figure 3B), while

largely unaffected by DMSO treatment. Consistent with a previ-

ous study (Zee et al., 2010), we found H3K27me3 turnover to be

slow (t1/2 approximately 37–60 hr), while H3K27me1 turnover

was considerably faster (t1/2 = 6–25 hr) (Figure 3C, left panel).

Therefore, even complete target coverage at a given time point


will result in delayed H3K27me3 loss. As expected, CPI-360

effectively suppressed heavy H3K27me3 incorporation in

KARPAS-422 cells (Figure 3C) without affecting total histone

turnover (Figure 3D). Surprisingly, the turnover of H3K27me1

from light to heavy was not affected under these conditions.

Similar effects were observed with another EZH2 inhibitor

in additional GCB-DLBCL models (Figure S3), suggesting

that EZH2 generally controls global H3K27me3, but not

H3K27me1 levels in GCB-DLBCL. As with EZH2 inhibition,

H3K27me3, but not H3K27me1, turnover in lymphoma cells


Chemistry & Biology



was impaired when cells were arrested with thymidine (Fig-

ure 3E), suggesting that the mechanism for CPI-360-mediated

loss of bulk H3K27me3 is ‘‘dilution’’ of the mark due to histone

synthesis in S-phase. Histone methylation turnover was also

measured in the presence of very high CPI-360 concentrations.

Interestingly, H3K27me1 turnover was partially suppressed un-

der these conditions, while the turnover of other methylation

marks such as; for instance, H3K36me1 remained unaffected

(Figure 3F). Given the exquisite selectivity of the compound,

one might speculate that the impact on H3K27me1 turnover

at high compound concentrations stems from partial engage-

ment of EZH1. Regardless, such compound concentrations

are unlikely to be maintained in animals over time and are

thus considered pharmacologically irrelevant (see below).

CPI-360 Treatment Causes Time-DependentTranscriptional Changes in Germinal Center B Cell-likeDiffuse Large B Cell LymphomaIn order to detect potential transcriptional consequences of in-

hibition of the EZH2 catalytic activity, we treated KARPAS-422

cells with DMSO, CPI-360, or the published EZH2 inhibitors

GSK-126 (McCabe et al., 2012b) and EPZ-6438 (Knutson

et al., 2013) for 4 days and subjected the samples to RNA-

sequencing (RNA-seq). All three inhibitors reduced global

H3K27me3 levels to similar extent (Figure 4A, top) and signifi-

cantly altered the expression of 1,248 genes (Figure 4A, bot-

tom; Table S2). The remarkable similarity of the induced gene

expression changes within a given lymphoma model suggests

that these effects are caused by on-target activity, since it is

unlikely that three different EZH2 inhibitor scaffolds exhibit

identical off-target activity profiles. Genes induced by EZH2

inhibitors potentially indicate direct PRC2 target genes, since

PRC2 is well known to function in transcriptional repression.

We carried out chromatin immunoprecipitation and sequencing

(ChIP-seq) to determine H3K27me3 sites across the KARPAS-

422 genome. Interestingly, the majority of EZH2 inhibitor-

induced genes, but virtually none of the EZH2 inhibitor-down-

regulated genes, are marked with H3K27me3 (Figure 4A,

bottom right). We conclude that the latter group is comprised

of genes that are indirectly regulated by EZH2 inhibitors. These

genes include cell cycle regulators promoting proliferation,

similar to what has been shown previously (McCabe et al.,

2012b).

If H3K27me3 levels are ultimately governing transcriptional

repression, one would expect that H3K27me3 loss pre-

cedes transcriptional activation. Indeed, CPI-360-induced

gene expression changes were only apparent after prolonged

treatment (Figures 4B and 4E), and high doses of CPI-360

did not accelerate induction of gene expression at earlier

time points (Figure 4B; compare 1.5 and 20 mM CPI-360 treat-

ment for 2 days). Interestingly, restoration of gene silencing af-

ter compound removal was also time-dependent (Figure 4C).

Inspection of H3K27me3-positive genes chosen from our KAR-

PAS-422 ChIP-seq campaign showed that CPI-360 affected

promoter proximal H3K27me3 levels over time, but did not

substantially affect EZH2 and SUZ12 chromatin binding (Fig-

ure 4D). The delayed reduction in local H3K27me3 generally

correlated well with delayed induction of gene expression

(Figure 4E).


Enhancer of Zeste Homolog 2 Inhibitors Show Efficacyin Enhancer of Zeste Homolog 2 Mutant-ContainingLymphoma ModelsPreviously, EZH2 inhibitors have shown efficacy in EZH2mutant-

containing GCB-DLBCL models (Beguelin et al., 2013; Knutson

et al., 2014; McCabe et al., 2012b). Similarly, CPI-360 affected

the viability of Y641N mutant EZH2-containing KARPAS-422

(Figure 5A), but not of WT EZH2-containing OCI-LY19 cells (Fig-

ure 5B) in longer term growth assays. No phenotypic response

was observed in KARPAS-422 cells after 4 days of treatment

despite the fact that the target is already potently engaged as

measured by dose-dependent, global H3K27me3 reduction af-

ter 4 days (Figure 5A). This delayed phenotypic response stands

in contrast to the consequences observed upon treatment with

3-deazaneplanocin A (DZNep), an S-adenosyl homocysteine hy-

drolase inhibitor that has previously been suggested to function

as an anticancer agent by promoting PRC2 degradation. How-

ever, DZNep’s specificity has been questioned (Miranda et al.,

2009), and thus we wanted to compare DZNep to our EZH2 in-

hibitor in the context of lymphoma. Despite it being a nucleoside

analog, DZNep expectedly did not inhibit EZH1 or EZH2 catalytic

activity in biochemical assays (Figure S4). DZNep caused potent

cell viability defects in OCI-LY19 cells by day 4 of treatment,

while no effect on global H3K27me3 levels was observed at

this time point (Figure 5B). To the contrary, CPI-360 did not

cause any phenotypic changes in these lymphoma cells, but

effectively reduced H3K27me3 levels. Thus, DZNep causes

viability defects in lymphoma cell models that are independent

of inhibition of H3K27 methylation.

In order to define the phenotypic consequences upon EZH2

inhibitor treatment, we carried out a series of flow cytometry-

based experiments, measuring cell cycle stage distribution and

various markers of cell death. CPI-360 treatment gradually ar-

rested KARPAS-422 cells in the G1 cell cycle stage (Figure 5C

and data not shown), which was followed by the induction of

apoptosis starting after 13 days of treatment (Figure 5D). We

conclude that the kinetics of the induction of apoptosis is slow,

and the underlying mechanism is likely to be distinct from most

cytotoxic agents. Treatment with EZH2 inhibitors in lymphoma

causes a cascade of time-dependent events that include

H3K27me3 reduction and the induction of gene expression,

which are followed by the induction of a G1-arrest and finally re-

sults in apoptosis.

Twice daily, subcutaneous administration of 200 mg/kg of

CPI-360 reduced tumor growth (TGI 44%) of KARPAS-422 xeno-

grafts in mice (Figure 5E) without affecting body weight or

causing any overt adverse effects (Figure 5F). However, inade-

quate pharmacological properties of CPI-360 did not allow for

complete target coverage. Subsequently, we identified CPI-

169 as a more potent EZH2 inhibitor with improved microsomal

stability (Figures S5A–S5G). Interestingly, CETSA-based anal-

ysis suggested that CPI-169 significantly increased EZH2 ther-

mal stability and–compared to CPI-360–maintained stabilization

for a longer time period after compound removal (Figure S5B). Its

application in the KARPAS-422 xenograft model resulted in

tumor regression (Figures 5E and S5E). Tumors showed dose-

dependent reduction in global H3K27me3 levels (Figure 5G)

and induction of gene expression (Figure 5H) that correlated

with dose and impact on tumor growth. Also, the proliferative


Figure 4. Time-Dependent Induction of Polycomb Repressive Complex 2 Target Genes by Enhancer of Zeste Homolog 2 Inhibitor CPI-360 in

Germinal Center B Cell-like Diffuse Large B Cell Lymphoma Correlates with Loss of Local H3 Lysine 27 Trimethylated Levels

(A) Gene expression profiling fromKARPAS-422 cells treatedwith 1.5 mMof the EZH2 inhibitors CPI-360, GSK-126, and EPZ-6438. (Top)Whole cell extracts from

samples that were subsequently subjected to RNA-seqwere analyzed bywestern blotting. H3K27me3 levels were determined. Total H3 levels served as a loading

control. Two biological replicates (1 and 2) for each treatment groupwere analyzed. (Bottom) Shown is a heatmap representing 1,248 genes that were significantly

altered (log2 fold change (FC) > 1 and p < 0.05) in any two comparisons. Increases and decreases in gene expression are indicated by red and blue, respectively.

ChIP-seqwas carried out inKARPAS-422 cells to determine the presence ofH3K27me3across the genome. A heatmap illustratingH3K27me3 enrichment around

transcriptional start sites is shown on the right. Low and high H3K27me3 enrichment are indicated by blue and yellow, respectively.

(B) Induction of gene expression by EZH2 inhibition is time-dependent. ABAT, APOL3, and FBXO39 gene expression changes were assessed in KARPAS-422

cells upon treatment with DMSO and CPI-360 [1.5 and 20 mM] for 2, 3, 6, and 10 days. Gene expression changes are represented as the fold change over control.

Data are represented as the mean of triplicate experiments ±SD.

(C) Restoration of gene silencing after removal of EZH2 inhibitors is time-dependent. ABAT, APOL3, and FBXO39 gene expression changes were assessed in

KARPAS-422 cells upon treatment with DMSO and CPI-360 [1.5 mM] for 4 and 8 days or 8 days and subsequent compound removal and continued growth in the

absence of compound for up to 8 days (8-day washout period). Gene expression changes are represented as the fold change over control. Data are represented

as the mean of triplicate experiments ±SD.

(D) Reduction of promoter-proximal H3K27me3 levels is time-dependent. KARPAS-422 cells were treated with CPI-360 [1.5 mM] for 4 and 8 days and samples

were analyzed by ChIP using EZH2-, SUZ12-, and H3K27me3-specific antibodies. Gene names are indicated at the bottom. Data are represented in each graph

as the mean of duplicate experiments from two biological replicates ±SD.

(E) Induction of PRC2 target gene expression correlates with promoter-proximal reduction of H3K27me3 levels. The transcript levels were assessed in KARPAS-

422 cells upon treatment with DMSO and CPI-360 [1.5 mM] for 2, 3, and 6 days. Gene expression changes are represented as the fold change over control. Data

are represented the mean of triplicate experiments ±SD.

Chemistry & Biology



potential of tumor cells was substantially reduced upon treat-

ment (Figure S5I). CPI-169 achieved plasma concentrations

that covered the target for at least 12 hr (Figure S5F, plasma

concentration 12 hr post last dose = 2.9 mM) when compared

to the 90%-effective concentration (EC90) required to reduce

global H3K27me3 levels in KARPAS-422 at day 11 of treatment

(Figure S5C; EC90 = 0.388 mM). Importantly, the compound con-

centration that can be achieved andmaintained in animals effec-

tively suppresses H3K27me3 levels and results in lymphoma


tumor regression, while global H3K27me1 levels remain un-

changed (Figure 5G).

Enhancer of Zeste Homolog 2 Inhibitors Affect theGrowth of Wild-Type and Mutant Enhancer of ZesteHomolog 2-Containing NHL Cell Lines as Single Agentsor in Combination with ABT-199To assess the potential application of EZH2 inhibitors in NHL, we

carried out long term growth assays across a panel of 43


Figure 5. Enhancer of Zeste Homolog 2 Inhibitor CPI-360 Causes Selective Lymphoma Cell Killing In Vitro and Tumor Regression In Vivo

(A) Viability (upper) and H3K27me3 levels (lower) in response to CPI-360 treatment were assessed at 4, 7, and 10 days in KARPAS-422 cells. Data are represented

as the mean of triplicate experiments ±SD.

(B) Viability (upper) and H3K27me3 levels (lower) in response to CPI-360 (left) or DZNep (right) were assessed at 4, 7, and 10 or 11 days in OCI-LY19 cells. Data are

represented as the mean of triplicate experiments ±SD.

(C) Cell cycle stage distribution was assessed in KARPAS-422 cells treated with CPI-360 for 10 days. Cells were cultured in the presence of DMSO or various

concentrations of CPI-360, fixed with ethanol, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS). Shown is the per-

centage of cells in each cell cycle stage. Cell cycle stages are indicated.

(D) The type of cell death induced by CPI-360 treatment was assessed in KARPAS-422 cells after 13 days of treatment. Cells were cultured in the presence of

DMSO or various concentrations of CPI-360, fixed with ethanol, stained with annexin V, and subjected to FACS. Shown is the percentage of living, apoptotic, and

necrotic cells.

(E) In vivo efficacy experiment using a KARPAS-422 subcutaneous xenograft model (n = 10 per cohort) was carried out to assess the impact of CPI-360 and CPI-

169 (chemical structure shown on the top right) on tumor growth. Both compounds were dosed twice a day at 200mg/kg administered subcutaneously (200mpk

SC BID) for 28 days, and the tumor volume was plotted as a function of time. Data are represented as the mean tumor volume per cohort and time point ±SD.

(F) Bodyweight was assessed for vehicle and compound-treated animals for the experiment described in (E). No significant adverse effects were observed during

the course of the experiment. Data are represented as described in (E).

(G) Vehicle and compound-treated tumors were collected at 14 and 28 days respectively and analyzed for global H3K27me3 (n R 4; left graph) and H3K27me1

(n = 3; right graph) levels using an ELISA assay. Changes in H3K27me3, but not H3K27me1, levels upon compound treatment were significant (Student’s t test;

***p = 0.0001 and ****p < 0.0001).

(H) Total RNA was collected from tumors (n = 3), cDNA was generated and subjected to qPCR analysis measuring the steady-state expression levels of three

PRC2 target genes, ABAT, APOL3, and FBXO39. These three genes were initially identified in the RNA-seq campaign described in Figure 4A. All three genes are

substantially induced in tumors of the compound-treated compared to vehicle-treated animals. Data are represented as the mean gene expression change from

triplicate experiments ±SD.

Chemistry & Biology



lymphoma cell lines representing GCB- and ABC-DLBCL, BL,

and MCL. We observed selective cell killing in response to

CPI-360 or CPI-169 treatment across the panel, with GCB-

DLBCL being the most prevalently affected subtype (Figures

6A and S5H). A number of the mutant EZH2-containing cell lines


were effectively killed by EZH2 inhibitors in a time- and dose-

dependent manner (Figure S6A). In contrast to other sensitive

cell models, Pfeiffer and SUDHL10 cell viability was already

affected at 4 days of treatment. We cannot rule out that differ-

ences in seeding density, doubling time, or cell health impact


Figure 6. Enhancer of Zeste Homolog 2 Inhibitors Cause Cell Killing in Various NHL Subtypes

(A) Viability across a panel of 43 NHL cell lines treated with CPI-360 for 4, 7, and 11 days was assessed. Data are represented as themean GI50-value of duplicate

experiments.

(B) NHL cell lines that represent GCB-DLBCL, ABC-DLBCL, BL, and MCL subtypes were found sensitive to EZH2 inhibitors. Viability (upper) and global

H3K27me3 levels (lower) were determined at 4, 7, and 11 days of CPI-360 treatment. Data are represented as the mean of triplicate experiments ±SD.

(C) KARPAS-422 cells were pretreated with CPI-169 for 4 days followed by cotreatment with CPI-169 and BCL2-specific inhibitor ABT-199 for 2 days. Viability

was assessed using aCTGassay. Data are represented as the average percentage of viable drug-treated compared to DMSO-treated cells (indicated in the table)

from triplicate experiments. The Bliss independence method was used to determine combinatorial activity. The graph (bottom) shows a dose-dependent effect

on KARPAS-422 cell viability of CPI-169 alone or in combination with 20 nM ABT-199. The red dotted line shows the predicted additive effect according to the

calculations using the Bliss independence method. Data are represented as the mean of triplicate experiments ±SD.

Chemistry & Biology



the phenotypic response timing, however even the ‘‘fast re-

sponders’’ gained sensitivity over longer treatment periods,

supportive of a time-dependent mechanism of action of our

compounds. Interestingly, the potential applicability of EZH2 in-

hibitors is not restricted to this particular genotype, since GCB-

DLBCL cell lines with WT EZH2, such as HT, showed a viability

defect upon treatment (Figure 6B). Interestingly, EZH2 depen-

dency extended beyond GCB-DLBCL, since the viability of indi-

vidual BL, ABC-DLBCL, and MCL cell lines was also impaired

upon CPI-360 treatment (Figure 6B, upper panels). Phenotypic

response did not correlate with default EZH2 protein or global

H3K27me3 levels (Figure S6B), but in all cell lines we observed

potent suppression of H3K27me3 upon treatment (Figure 6B,

lower panels) that preceded any phenotypic response.


Given that our EZH2 inhibitors caused phenotypic responses

in NHL cell models beyond cases of EZH2-containing GCB-

DLBCL models, we focused on the identification of agents that

synergize with EZH2 inhibitors, and thus may further expand

the applicability of EZH2 inhibitors in NHL. The high incidence

of oncogenic genomic aberrations of the antiapoptotic factor

BCL2 in DLBCL (De Paepe and De Wolf-Peeters, 2007), the

fact that EZH2 mutations frequently cooccur with BCL2 muta-

tions and structural aberrations in NHL (Morin et al., 2011), and

the recent finding showing combinatorial effects of pan-inhibi-

tors of the BCL-family and EZH2 inhibitors in several lymphoma

models (Beguelin et al., 2013) provided a sufficient rationale to

explore potential codependency on EZH2 and BCL2. We used

the BCL2-selective inhibitor ABT-199 that is currently in clinical


NHL cell line CPI-169sensitive1

ABT-199 sensitive2 Synergy3 EZH2

mutationBCL2 transcript

level41 SUDHL16 yes yes 488 no2 WSU-DLCL2 yes yes 295 yes3 DB yes* yes 285 yes4 KARPAS-422 yes yes 224 yes5 SUDHL5 no no 178 no6 SUDHL6 yes yes 157 yes7 Ramos yes no 130 no8 TMD8 yes no 120 no9 WSU-NHL no yes 97 no10 OCI-LY19 no yes 91 no11 SUDHL4 yes* yes 85 yes12 Farage yes no 79 no13 DOHH-2 no yes 71 no14 Toledo no yes 62 no15 WSU-FSCCL no yes 53 no16 SUDHL10 yes no 49 no17 U2932 no yes 47 no18 NUDHL1 yes yes 33 no19 MC116 yes no 30 no20 OCI-LY7 yes no 27 no21 RL no yes 14 yes22 Raji no no 6 no23 Pfeiffer yes no 6 yes24 OCI-LY1 yes yes 2 yes25 HT yes no 0 no

1 GI50 < 5 µM in 11 day viability assay2 GI50 < 1 µM in 2 day viability assay3 Synergy is determined by calculating the Bliss independence volume4 Transcript level represented as BCL2 transcripts per 1000 TBP transcripts * Only sensitive (GI50 < 5 µM) in 14+ day growth assay

Figure 7. Synergy of Enhancer of Zeste Ho-

molog 2 and B Cell Lymphoma 2 Inhibitors

in Suppressing the Growth of NHL Cell Lines

The potential synergy of EZH2 and BCL2 inhibitors

was assessed across a panel of 25 NHL cell lines.

The table summarizes the response to EZH2 and

BCL2 inhibitors (treatment as described in Fig-

ure 6C) used as single agents or in combination.

Synergy is preferentially observed in cell lines that

respond to both single agents. Response to EZH2

inhibitors does not correlate with BCL2 expression.

The BCL2 transcript levels were determined by

qPCR across the panel of NHL cell lines (column on

the right) and normalized to TBP transcript levels.

Data are represented as the mean of triplicate

experiments ±SD.

Chemistry & Biology



trials for lymphoma (Souers et al., 2013) rather than pan-BCL

family inhibitors. We first tested the EZH2-BCL2 inhibitor combi-

nation in the EZH2 inhibitor sensitive KARPAS-422 cell line.

These agents showed synergy in suppressing growth of KAR-

PAS-422 cells at multiple dose-combinations (Figure 6C). The

Bliss independence criterion was employed to investigate syn-

ergy. As illustrated in Figure 6C (lower panel), the calculated

combinatorial activity of CPI-169 and ABT-199 was greater

than a predicted additive effect. Subsequent exploration of 25

NHL cell lines with an EZH2-BCL2 inhibitor combination is sum-

marized in Figure 7 (see also Table S3 for calculation of Bliss in-

dependence volumes). The cell lines in which the EZH2-BCL2 in-

hibitor combination showed synergy did not correlate with EZH2

mutation status, nor with BCL2 genomic aberrations. However,

as previously suggested (Souers et al., 2013) there was a corre-

lation of response to ABT-199 and BCL2 levels (Figures 7 and

S6B). Overall, synergy of EZH2 and BCL2 inhibitors was not

broadly observed across the NHL cell line panel, but there was

a trend toward synergy in cell models that phenotypically re-

sponded to both single agents.

DISCUSSION

We identified an EZH2 inhibitor chemotype, examples of which

exhibit adequate pharmacokinetic properties to allow for the

execution of in vivo studies. Consistent with previous data, our

compounds achieved in vivo efficacy in a mutant EZH2-contain-

ing GCB-DLBCL xenograft model, whereby the impact on tumor

growth correlatedwell with the administered dose, the exposure,

and the level of target engagement. We have used these com-

pounds for an in depth exploration of EZH2 inhibitor time depen-

dency using mass spectrometry. As expected, H3K27me3 turn-

over in GCB-DLBCL cells was relatively slow and dependent on

10 Chemistry & Biology 21, 1–13, November 20, 2014 ª2014 Elsevier Ltd All rights reserved

cell cycle progression. Hence, demethy-

lases unlikely play a major role in altering

global H3K27me3 levels upon EZH2 inhi-

bition. Rather, the synthesis of new his-

tones during replication leads to a dilution

of H3K27me3. The fact that cell cycle ar-

rest impedes the turnover of H3K27me3,

but not of other histone methylation

marks, (Figure 3E) supports this claim.

Interestingly, our compounds selectively suppressed

H3K27me3, but not H3K27me1, turnover at pharmacologically

relevant doses. This is surprising given that PRC2 likely controls

bulk levels of all three H3K27 methylation states. Recent genetic

data suggest that EZH2 controls H3K27me3 levels, but it is

rather EZH1 that controls bulk H3K27me1 levels (Hidalgo et al.,

2012; Xie et al., 2014). Our data suggest that CPI-360 completely

inhibits EZH2 catalytic activity, since we entirely suppress

H3K27me3 turnover over time. Since our compounds do not

impact H3K27me1 turnover under the used experimental condi-

tions (Figure 3C; only very high CPI-360 doses partially affected

H3K27me1 turnover, Figure 3F), we do not achieve inhibition of

EZH1, consistent with the biochemical data estimating at least

a 100-fold potency difference of these compounds between

EZH2 and EZH1. Alternatively, one may consider the existence

of a hitherto unknown H3K27 monomethylase that is only poorly

inhibited by current EZH2 inhibitors.

EZH2 small molecule inhibitors are important tools to study the

contribution of the catalytic activity to overall PRC2 function. It is

now clear that EZH2MT activity is required for the selective sup-

pression of gene expression, and that certain cancer cell con-

texts are indeed dependent on this activity. It is still unclear

which of the EZH2 inhibitor-mediated transcriptional changes

drive apoptosis of lymphoma cells. Since three different EZH2

inhibitor scaffolds elicit similar transcriptional changes, it is likely

that these effects are caused by compound on-target activity.

It was previously shown that EZH2 inhibition largely caused

different gene expression changes in different DLBCL cell

lines (McCabe et al., 2012b). We have shown here that also

ABC-DLBCL, BL, and MCL cell lines phenotypically respond

to EZH2 inhibitors. However, the underlying transcriptional

changes that precede the phenotypic effects are yet to be iden-

tified. It will be interesting to determine in future studies if an

Chemistry & Biology



EZH2-controlled gene signature can be elucidated across NHL

subtypes. Such a signature, if it exists, may have utility as a pre-

clinical and clinical biomarker.

The slow H3K27me3 turnover is a plausible explanation for the

delayed induction of gene expression and time-dependent

phenotypic responses upon compound treatment. However, dif-

ferences in H3K27me3 turnover rates and proliferation indices

cannot fully explain the range of phenotypic response times

among the investigated NHL cell lines. The molecular events

thatgovern thephenotypicEZH2 inhibitor response timing remain

to be identified. In adequate models, the mechanism is powerful

and leads to complete and durable tumor regression. The selec-

tive suppression of H3K27me3 at efficacious doses, and thus a

selective suppression of the PRC2 functional repertoire, (inhibit-

ing EZH2, but not necessarily EZH1 activity) is sufficient to elicit

growth defects in NHL. However, it remains to be investigated if

simultaneous interference with EZH1-containing PRC2 function

improves the therapeutic potential of our inhibitors.

Here we have characterized an EZH2 inhibitor that can be

used for in vitro and in vivo experiments and will aid the research

community to explore EZH2 biology. Our compounds are

currently being optimized with the goal to create a therapeutic

agent for clinical applications.

SIGNIFICANCE

For over a decade the histone lysine methyltransferase

EZH2 is considered an oncogenic driver due to its prevalent

overexpression in many cancer types. The discovery of

recurrent somatic mutations in the EZH2 catalytic domain

in diffuse large B cell lymphoma, follicular lymphoma, and

melanoma has greatly increased the relevance of EZH2 as

a candidate oncology target. EZH2 small molecule inhibitors

have been recently discovered and demonstrated efficacy

in various mutant-EZH2-containing lymphoma xenograft

models. These data suggest that pharmacological inhibition

of EZH2may be a promising lymphoma therapeutic strategy.

We have identified an additional EZH2 inhibitor chemotype,

examples of which exhibit adequate pharmacokinetic prop-

erties to be used for in vivo studies. We have employed a

recently discovered method, cellular thermal shift assays,

to demonstrate direct cellular target engagement for any

EZH2 inhibitor. Moreover, we have used an isotopic labeling

and mass spectrometry approach to demonstrate that our

EZH2 inhibitors selectively suppress H3K27me3, but not

H3K27me1, turnover at pharmacologically relevant doses.

These findings suggest efficacy of current EZH2 inhibitors

in lymphoma depends on comprehensive EZH2 inhibition,

but does not require inhibition of EZH1. Interestingly, the ef-

ficacy of EZH2 inhibitors was not limited to cases of mutant

EZH2-containing lymphomas, but included various NHL cell

models with WT EZH2. Thus, the application of EZH2 inhibi-

tors in lymphoma may be broader than initially anticipated.

EXPERIMENTAL PROCEDURES

Biochemical Assays

PRC2 and oligonucleosome reconstitution and radiometric biochemical as-

says using oligonucleosomal substrates were essentially carried out as

Chemistry & Biology 21, 1

described previously (Garapaty-Rao et al., 2013). Methyltransferase selectivity

panel profiling with CPI-360 and CPI-169 was carried out using the services of

Reaction Biology Corp.

Cell Culture and Viability Assays

The lymphoma cell lineswere obtained fromATCCor DSMZandwere grown in

media recommended by the vendor. All media contained 10% fetal bovine

serum and 1% penicillin/streptomycin (Invitrogen). For long-term assays, cells

were plated onto compound containing 96-well plates. Cells were passaged

every 4 days (for seeding densities and split ratios see Table S4) to plates con-

taining fresh EZH2 inhibitors. Relative cell numbers were assessed by Cell

Titer-Glo (CTG) luminescent cell viability assay (Promega) using an Envision in-

strument (Perkin Elmer). Prism 6.0 (Graphpad Software, 2013) was used for

curve fitting. See Supplemental Experimental Procedures for more details.

Cell Cycle and Apoptosis

KARPAS-422 cells were plated in 96-well plates and treated with various

doses of CPI-360 for a total of 14 days. The cells were split on day 4, day 8,

and day 11. Cells were collected on day 4, day 6, day 8, day 10, and day 14

and fixed in 70% ice-cold ethanol overnight at �20�C. The fixed cells were

stained with 20 mg/ml propidium iodide (PI) and the celluar DNA content was

assessed onGuava Easycyte. The percentage of cells in the different cell cycle

phases were obtained by analysis with Guava Cytosoft program. Apoptosis

was assessed at day 10, day 12, day 13, and day 14. The cells were stained

with PI and Annexin-FITC using Annexin V:FITC Apoptosis Detection Kit I

(BD Biosciences; #556547) according to the provided instructions. Data was

acquired on Guava Easycyte, and the percentage of cells undergoing

apoptosis was calculated using Guava Cytosoft program.

Cellular Thermal Shift Assay

Experiments were carried out as described previously (Martinez Molina et al.,

2013).

DNA and RNA Sequencing Data

The ChIP-seq and RNA-seq data are available in the Gene Expression

Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds) under the acces-

sion number, GSE62058. See Supplemental Experimental Procedures for

more details.

ELISA and Western Blot Experiments

The Meso Scale Discovery ELISA assay was performed as described previ-

ously (Garapaty-Rao et al., 2013). For antibodies used in this study see Table

S7.

Histone Mass Spectrometry

For all cell pellets to be analyzed by mass spectrometry, the histone isolation

was performed using EpiQuik kit (Epigentek) per instructions. Resultant solu-

ble histone protein was precipitated using 20% trichloroacetic acid for 3 hr on

ice. After isolation, the histones were propionylated (Sigma) as previously

described (Garcia et al., 2007) to block free amines on the histone proteins

and then treated with trypsin (Promega) for overnight digestion. Peptides

were then subjected to another round of propionylation to block the termini

created from the digestion. Each sample was desalted via Stage Tip prior to

analysis by Orbitrap MS.

Peptides were resuspended in 0.1% formic acid and analyzed by direct on-

line injection into an LTQ-Orbitrap XL electron-transfer dissociation mass

spectrometer (Thermo Scientific) using an Agilent 1260 high-pressure liquid

chromatography system. Chromatographic separation was performed over

a gradient of 0%–55% acetonitrile containing 0.1% formic acid over 55 min

on a 100 mm 3 15 cm analytical column packed with HALO C18 packing ma-

terial (NewObjective) with a flow rate of 300 nl/min. Peptides were sprayed us-

ing 20 mm emitter tip, using spray voltage of 2.0 kV, and capillary temperature

of 200�C. Survey scans were acquired in the Orbitrap from a range of mass/

charge ratio [m/z] 300–2,000 at 30,000 resolution with poly dimethylcyclosilox-

ane from ambient air (m/z 445.120025) as a lock mass (Olsen et al., 2005). For

each cycle, the eight most intense ions were fragmented by collision induced

dissociation in the ion-trap at a target of 20,000 ions or 100 ms maximum

–13, November 20, 2014 ª2014 Elsevier Ltd All rights reserved 11

http

Chemistry & Biology



injection time before being excluded for 30 s. For sample prep and data anal-

ysis, see Supplemental Experimental Procedures.

Quantitative PCR Experiments

Cell lines were pelleted by centrifugation at 500 3 g for 2 min, and imme-

diately lysed in buffer RLT (QIAGEN). RNA was purified from lysed cells us-

ing RNeasy columns (QIAGEN) using the manufacturer’s protocol. Two hun-

dred nanograms of RNA were converted to cDNA using SuperScript III

Reverse Transcriptase (Invitrogen) and 250 ng random primers (Invitrogen).

Quantitative (q)PCR was performed using resultant cDNA, Roche FastStart

Universal Probe Master Mix (Roche Diagnostics), and appropriate TaqMan

probes (Invitrogen) on a Stratagene MX3005P qPCR instrument (Strata-

gene). Ct and delta Ct values were generated and analyzed using standard

protocols. For primer sets and probes, see Supplemental Experimental

Procedures.

Xenograft Studies

Each mouse was inoculated subcutaneously in the right flank with the

KARPAS-422 tumor cells (1 3 107) in 0.2 ml of PBS with matrigel (1:1) for

tumor development. Treatments commenced when the average tumor

size reached approximately 100–200 mm3. Each group consisted of ten

randomly assigned tumor-bearing mice. The mice were dosed with vehicle

(10% DMSO + 60% polytheylene glycol 400 + 30% ddH2O), CPI-360

(200 mg/kg, subcutaneously [SC], twice daily [BID]) or CPI-169 (200 mg/

kg, SC, BID) as per Institutional Animal Care and Use Committee guidelines.

Tumor size was measured three times a week using a caliper, and the tumor

volume (V) was expressed in mm3 using the formula, V = 0.5a 3 b2, where a

and b were the long and short diameters of the tumor, respectively. The mice

were weighed every day. Tumor growth inhibition (TGI) was calculated as

TGI (%) = (1� (T1�T0) / (C1�C0)) 3 100, where C1-mean tumor volume

of control mice at time t; T1-mean tumor volume of treated mice at time t;

C0-mean tumor volume of control mice at time 0; and T0-mean tumor vol-

ume of treated mice at time 0. For processing of tumor samples, see Supple-

mental Experimental Procedures.

ACCESSION NUMBERS

The ChIP-seq and RNA-seq data are available in the Gene Expression

Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds) under the acces-

sion number GSE62058.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

six figures, and seven tables and can be found with this article online at

http://dx.doi.org/10.1016/j.chembiol.2014.09.017.

AUTHOR CONTRIBUTIONS

W.D.B. carried out CETSA and ABT-199 combo experiments; S.A. carried out

cell panel viability screening and analyzed tumor samples; J.B. performed all

mass spectrometry-related experiments; S.B. and E.N. executed the in vivo

studies and performed apoptosis assays; V.S.G., C.G.N., R.G.V., J.E.A.,

A.S.C., L.A.D., B.K.A., and J.-C.H. contributed to the generation of the com-

pounds; C.-C.Y. performed the ChIP experiments; J.M. and D.L.D. performed

nanoBRET assays; C.H. and B.M.B. provided bioinformatics support; F.Z.,

K.E.W., P.I., and S.G.-R. helped with experiments and provided technical sup-

port; R.C., N.C., and R.T.C. carried out biochemical assays; and P.T. designed

and oversaw all experiments and wrote the manuscript.

ACKNOWLEDGMENTS

W.D.B., S.A., J.B., S.B., V.S.G., C.G.N., R.G.V., C.-C.Y., C.H., F.Z., K.E.W.,

P.I., R.C., N.C., J.E.A., B.K.A., J.-C.H., R.T.C., B.M.B., E.N., and P.T. are em-

ployees and shareholders of Constellation Pharmaceutical, Inc. Promega Cor-

poration is the commercial owner by assignment of patents of the HaloTag and

NanoLuc technology and its applications.


Received: July 23, 2014

Revised: September 16, 2014

Accepted: September 30, 2014

Published: November 6, 2014

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