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Function of histone methyltransferase EZH2
and development of EZH2 inhibitors

2022/12/01 Literature Seminar
M1 Mayu Onoda

Contents▫Histone modification▫Function of histone methyltransferase EZH2
▫EZH2 inhibitors▫Summary

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Contents▫Histone modification▫Function of histone methyltransferase EZH2
▫EZH2 inhibitors▫Summary3

Histone modification

4▫Structure of nucleosome
H3 tailModification
H3Lys
H3 tailH4

H2A

H2B

DNAMüller, M. M.; Muir, T. W., Chem. Rev. 2015, 115, 2296.

Histone modification

5▫Modification (H3K27Me)▫Lysine residue methylation mechanism※※ HKMT
= Histone Lysine Methyltransferase

R. Fioravanti et al, Chem. Rec. 2018, 18, 1818.

Contents▫Histone modification▫Function of histone methyltransferase EZH2
▫EZH2 inhibitors▫Summary

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EZH2 (H3K27 Methyltransferase)▫EZH2 forms a PRC2 complex.

7▫The SET domain of EZH2 catalyzes methylation.

EZH2

H3K27 Methylation
An, R., Li, YQ., Lin, YL. et al, Cancer Gene Ther, 2022

EZH2 (H3K27 methyltransferase)

8▫ EZH2 (WT) and EZH2 (Mutant) prefer different methylation reactions.

H3K27 Methylation
EZH2 (WT)◎Me0→Me1〇Me1→Me2△Me2→Me3

EZH2 (A677 Mut)
〇Me0→Me1〇Me1→Me2〇Me2→Me3

EZH2 (Y641 Mut)
△Me0→Me1△Me1→Me2◎Me2→Me3

What makes this difference?
McCabe M.T.et al, Proc. Natl. Acad. Sci. USA, 2012, 109, 2989.
An, R., Li, YQ., Lin, YL. et al, Cancer Gene Ther, 2022

EZH2 (H3K27 methyltransferase)

9

What makes this difference?

CCNO

CNC

C

Me0→Me1: Hydrogen bond is important.
Me2→Me3: Wide space is important.

CONCC

・Loss of hydrogen bond.
・Hydrogen bondTyr (O atom) & K27 (N atom)

・Space is created.
・Hydrogen bondTyr (O atom) & K27 (N atom)

・Space is created.

CNC

Y641 and A677G EZH2 mutations alter the lysine binding pocket to affect H3K27 substrate specificity. A homology model of WT EZH2 was generated using the crystal
structure of GLP/EHMT1 bound to an H3K9me2 peptide substrate as described in SI Materials and Methods. Modeled structures of the active site region in WT (A),Y641N (B), Y641F (C), and A677G (D) EZH2 are depicted. For the Y641F and A677G mutant models, the lowest energy rotamer for the 641 residue (F or Y,respectively) was selected after rotating the dimethylated lysine into an orientation optimal for trimethylation. In C and D, the WT Y641 orientation is shown in a grayoutline to highlight the alternative low-energy conformations adopted by the 641 residue in these mutants. Key heavy atom distances (measurement unit = Å) areindicated with green lines. S-adenosyl-homocysteine (SAH) is colored with orange carbons, EZH2 residues are colored with magenta carbons, and the dimethylated
H3K27 is colored with cyan carbons.OPEN IN VIEWER

C

McCabe M.T.et al, Proc. Natl. Acad. Sci. USA, 2012, 109, 2989.

EZH2 (H3K27 methyltransferase)10▫ EZH2 (WT) and EZH2 (Mutant) prefer different methylation reactions.

Overexpression of WT➡ Me3 accumulation

Me3 accumulation

Gene silencing➡ Cancers

Cooperation with WT➡Me3 accumulation

McCabe M.T.et al, Proc. Natl. Acad. Sci. USA, 2012, 109, 2989.
An, R., Li, YQ., Lin, YL. et al, Cancer Gene Ther, 2022

EZH2 (H3K27 methyltransferase)

11▫H3K27Me3 is associated with disease.
・H3K27Me3 levels in cancer cell lines

・H3K27Me3 levels in WT and Mut EZH2 cell lines

EZH2 A677G Mut⇩Cell lines

EZH2 Y641 Mut➡ Lymphoma cells express mutant EZH2 and have high H3K27Me3 levels.
A subset of lymphoma cell lines exhibits elevated H3K27me3 levels. (A) Global H3K27me3 levels (normalized to total H3) were determined for 111 cancer cell lines from seven different cancer types using
H3K27me3 and total H3 ELISAs. Lymphoma cell lines harboring heterozygous Y641 mutations are indicated by black tick marks below the graph. MM, multiple myeloma. (B) Western blot analysis wasperformed with antibodies specific for H3K27me3, H3K27me2, H3K27me1, total histone H3, EZH2, and actin using protein lysates from a panel of lymphoma cell lines. Actin serves as a loading control.
EZH2 mutation status as determined from full-length Sanger sequencing is indicated.

McCabe M.T.et al, Proc. Natl. Acad. Sci. USA, 2012, 109, 2989.

Short summary12▫EZH2 methylates H3K27.
▫EZH2 mutants (A677, Y641) accumulate H3K27Me3.

This difference is due to hydrogen bonds and wide space.

▫H3K27Me3 is highly expressed in lymphoma cells.
➡ EZH2 Mutations (A677 or Y641) were identified.

EZH2 Inhibitors

Contents▫Introduction : Histone modification (methylation)▫EZH2 (H3K27 methyltransferase)▫EZH2 inhibitors ①▫Summary13

EZH2 inhibitors ①EPZ005687▫High-throughput screening of a 175,000-compound subset
against recombinant WT PRC2 Complex.

14

EZH2 Inhibitor

EPZ005687

Knutson, S et al, Nat Chem Biol, 2012, 8, 890.

EZH2 inhibitors ①EPZ00568715▫Biochemical characterization of EPZ005687
・Inhibition of EZH2 by EPZ005687 (In the presence of SAM)

EPZ005687 was competitive with SAM and noncompetitive with Nucleosome.

(a) Inhibition of EZH2 when activity is assessed under balanced conditions 24 for both SAM andpeptide substrates using a Flashplate assay to measure the transfer of a tritiated methyl groupfrom SAM to the peptide. The data are fit to a standard Langmuir isotherm for inhibition, and theIC50 of EPZ005687 was calculated to be 54 ± 5 nM with a Hill slope of 1. The data shown are
the average and s.d. of seven independent duplicate runs. (b) Plot of IC50 values of EPZ005687
as a function of SAM concentration relative to the Km of SAM ([SAM]/Km) measured using aFlashplate assay similar to the IC50 measurements described above. These values show a linear
relationship, as expected for SAM-competitive inhibition with a Ki of 24 ± 7 nM (± s.d. of threeexperiments). (c) Plot of IC50 values of EPZ005687 as a function of chicken erythrocyte
oligonucleosome concentration relative to the Km of nucleosome ([Nucleosome]/Km) measured
using a filter-binding microplate assay to measure the transfer of tritiated methyl groups fromSAM to the oligonucleosome. As expected for a noncompetitive inhibitor with respect to thissubstrate, the IC50 is unaffected as the concentration of oligonucleosome is increased. The
mean and standard error of three experiments are shown.

Knutson, S et al, Nat Chem Biol, 2012, 8, 890.
An, R., Li, YQ., Lin, YL. et al, Cancer Gene Ther, 2022

EZH2 inhibitor ①EPZ005687

16▫Biochemical characterization of EPZ005687
・Affinity map with methyltransferases
Figure 3: Ligand affinity maps of EPZ005687 across the family trees of humanlysine methyltransferases and arginine methyltransferase enzymes show
EPZ005687 is a selective and potent inhibitor of EZH2 and EZH1 enzymes.
The Ki of EPZ005687 was measured across a panel of recombinant lysine
methyltransferase (KMT; left) and arginine methyltransferase (RMT; right) enzymes atbalanced conditions24 of both the SAM and peptide or protein substrates. The EZH2
Tyr641 and Ala677 mutant enzymes are indicated above wild-type EZH2. The Ki values
were converted to pKi values and used to generate red circles of proportional sizes toindicate the extent of inhibition as shown in the legend. Larger circles correlate toincreased potency versus the enzymes, and gray circles indicate that inhibition was notmeasurable at concentrations up to 50 μM of EPZ005687.

➡ EPZ005687 can selectively inhibit WT and Mutant EZH2.
Knutson, S et al, Nat Chem Biol, 2012, 8, 890.

EZH2 inhibitor ①EPZ005687

17▫Ability of EPZ005687 in lymphoma cells(5.6 µM)(EZH2 WT lymphoma cell OCI-LY19 + EPZ005687)
overexpression(EZH2 Mutant lymphoma cell WSU-DLCL2 + EPZ005687)➡ EPZ005687 reduces H3K27Me3 expressed in WT and Mutant EZH2 cells.
(a) The wild-type EZH2 lymphoma cell line OCI-LY19 shows a dose-dependent decrease in H3K27me3 after treatment with EPZ005687 for 96 h. (b,c) A wild-type lymphoma cell line, OCI-LY19 (b), and a mutant lymphoma cell line, WSU-DLCL2 (c),show the specificity of H3K27 methylation inhibition by EPZ005687 across a broad panel of histone methylation marks. Quantification of methylation changes is represented in the bar graphs to the right of each panel of western blots.
Representative western blots (n = 1) were normalized to corresponding total H3 and expressed as percent change in EPZ005687-treated versus DMSO-treated cells.

Knutson, S et al, Nat Chem Biol, 2012, 8, 890.

EZH2 inhibitor ①EPZ005687

18▫Ability of EPZ005687 in lymphoma cells(Previous Slide) EPZ005687 reduces H3K27Me3 expressed in WT and Mutant EZH2 cells.

・Effect on cell proliferation

WTMutantMutant➡ EPZ005687 is effective against cell proliferation caused by Mutant EZH2.
Knutson, S et al, Nat Chem Biol, 2012, 8, 890.

Short summary

19▫ EZH2 inhibitor : EPZ005687
・Competes with SAM (=methyl donor).

EPZ005687

・Selectively inhibits EZH2 WT and Mutant.

EZH2 WT cell？
✖

〇

EZH2 Mutant cell〇

H3K27Me3 expression
is reduced.

〇

Cell proliferation
is inhibited.

(H3K27Me3 is overexpressed.)

EPZ005687？→ Hypothesis
EZH2 WT cell：H3K27Me3

Cell proliferation

EZH2 Mutant cell： H3K27Me3

Cell proliferation

Other factors

Knutson, S et al, Nat Chem Biol, 2012, 8, 890.

EZH2 inhibitor ①EPZ005687

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Effective in EZH2 mutation-positive
follicular lymphoma.
Approved in Japan in June 2021.

Research on diverse EZH2 inhibitors is ongoing.
(Next)New EZH2 inhibitors recently developed will be discussed.
S. K. Knutson et al, Proc. Natl. Acad. Sci. USA 2013,110,7922.
Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Contents▫Introduction : Histone modification (methylation)▫EZH2 (H3K27 methyltransferase)▫EZH2 inhibitors ②▫Summary21

EZH2 inhibitor ② IHMT-EZH2-115

22Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

EZH2 inhibitor ② IHMT-EZH2-115

23▫EPZ6438 was docked into the EZH2 crystal structure.

Van der Waals force

CentralIonic interaction

HeadTail
3 hydrogen bonds
Located in ligand-binding pocket

Van der Waals force

Figure 2. (A) Schematic illustration of the discovery of 29. (B) and (C) Binding modes of 1 with PRC2 (PDB: 5IJ7). 1 was labeled in color by atoms (carbon in yellow, nitrogen in blue, andoxygen in red). The hydrogen bonds were labeled as red dashed lines. The key amino acid residues for binding were labeled as follows: carbon in marine, nitrogen in blue, and oxygen in red.

Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

EZH2 inhibitor ③IHMT-EZH2-115

24▫Central Structure Optimization
EPZ6438

CentralHydrogen bonds could be formed.
Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

EZH2 inhibitor ③IHMT-EZH2-115

25▫Tail Structure Optimization
EPZ6438

Ionic interaction

Tail

Van der Waals force

Increased Van der Waals force.
Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

EZH2 inhibitor ③IHMT-EZH2-115

26▫Optimization complete➡ Pharmacokinetic characterization in rats.

Oral Bioavailability = 31%

EZH2 inhibitor ③IHMT-EZH2-115

27▫1(EPZ6438) vs 29(IHMT-EZH2-115)➡ 29 can strongly inhibit trimethylation.

➡ 29 can inhibit cell proliferation.
(EZH2 Mutant only)
Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Short summary (previous)

28▫ EZH2 inhibitor : EPZ005687
・Competes with SAM (=methyl donor).

EPZ005687

・Selectively inhibits EZH2 WT and Mutant.

EZH2 WT cell？
✖

〇

EZH2 Mutant cell〇

H3K27Me3 expression
is reduced.

〇

Cell proliferation
is inhibited.

(H3K27Me3 is overexpressed.)

EPZ005687？→ Hypothesis
EZH2 WT cell

H3K27Me3

Cell proliferation

EZH2 Mutant cell

H3K27Me3

Cell proliferation=EZH1?
(homolog)

Other factors

Knutson, S et al, Nat Chem Biol, 2012, 8, 890.

EZH2 inhibitor ③IHMT-EZH2-115▫1(EPZ6438) vs 29(IHMT-EZH2-115)

29

in vivo・Pfeiffer (EZH2 A677G)

・Karpas-422 (EZH2 Y641N)➡ 29 showed a stronger
anti-tumor effect than 1.

Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Summary

30▫Development of IHMT-EZH2-115
Hydrogen bondformation

Van der Waals force
enhancement

tazemetostat▫29 shows excellent antitumor effects in vivo.
29

1Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Thank you for your attention.

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Appendix

Appendix▫Histone methyltransferase (EZH2)

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Appendix

EZH2 is required

EZH2 is down-regulatedAppendix▫Differential methylation reactions in WT and Mutant EZH2
H3K27(Me0/Me1/Me2) peptide + EZH2 (WT/Mutant) + SAM
A677G EZH2 mutant exhibits a unique substrate specificity. Enzyme turnover number [kcat (min−1)], the substrateconcentration at which the reaction rate was half of Vmax [Km (μM)], and catalytic efficiency [kcat/Km (μM−1⋅min−1)] wereevaluated for recombinant five-member PRC2 complexes containing WT, A677G, or Y641 mutant EZH2 using peptides
from histone H3 AA21–44 with no methylation (K27me0), monomethylation (K27me1), or dimethylation (K27me2) at thelysine 27 position. Briefly, EZH2 (20 nM) was combined with varying concentrations of peptide and [3H]-SAM before
quenching with unlabeled SAM during the linear portion of their progress curves. Reactions were then captured using filter
plates, scintillation mixture was added, and signal was detected with a TopCount liquid scintillation counter (PerkinElmer).
Error bars represent the standard deviation from replicate experiments.

McCabe M.T.et al, Proc. Natl. Acad. Sci. USA, 2012, 109, 2989.

Appendix▫H3K27(Me0/Me1/Me2) peptide + EZH2 (WT/Mutant) + SAM → Activity assessment
・Reaction velocity

・Km Evaluation

・Kcat Evaluation

・Kcat/Km Evaluation

Me0Me1

Me2

Wild-type (○),Y641F (•), Y641H (□), Y641N (▪), Y641S (▴)

WT EZH2 prefers H3K27Me0→1 reaction
Mutant EZH2 prefers H3K27Me1→2 or Me2→3 reaction

C. J. Sneeringer et al,Proc. Natl. Acad. Sci. 2010,107,20980–510.

PRC2 complexes containing mutant EZH2 preferentially catalyze di- and trimethylation of histone H3K27. (A) Methyltransferase activity of mutant and WT complexes on unmethylated peptide (open bars), monomethylated peptide(hatched bars), and dimethylated peptide (closed bars). (B) Affinity for peptide substrates as defined by K1/2 is similar across all peptide methylation states for PRC2 complexes containing wild-type (○),Y641F (•), Y641H (□), Y641N (▪),
and Y641S (▴) EZH2. Note that the K1/2 values across all substrates and all enzyme forms varies less than 3.5-fold. For any particular methylation state of substrate, the variation in K1/2 value is less than 2-fold. (C) Enzyme turnover
number (kcat) varies with substrate methylation status in opposing ways for WT and Y641 mutants of EZH2. The kcat decreases with increasing K27 methylation states for wild-type (○), but increases for Y641F (•), Y641H (□), Y641N (▪),
and Y641S (▴) mutants of EZH2. (D) Catalytic efficiency (kcat/K1/2) decreases with increasing K27 methylation states for wild-type (○) but increases for Y641F (•), Y641H (□), Y641N (▪), and Y641S (▴) mutants of EZH2. (B–D) The lines
drawn to connect the data points are not intended to imply any mathematical relationship; rather, they are intended merely to serve as a visual aid to guide the eye of the reader.

Appendix

3▫Steps to generate EPZ005687

Improve solubility

Conversion toindazole

Introduction oflipophilic group

EPZ005687

Knutson, S et al, Nat Chem Biol, 2012, 8, 890.

Appendix

・Selectivity for H3K27
・WT cell・Y671F Mut cell

Appendix

Appendix

・Effect on cell proliferation (G1) (tazemetostat)Appendix

環アミドでの検討

Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

AppendixBin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Appendix

Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Appendix

Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Appendix

Bin Zhou et al, J. Med. Chem. 2021, 64, 15170.

Appendix
EZH1/2 両方阻害は有効？