REAWAKENING OF HUMAN FETAL HEMOGLOBIN AND AN EPIGENETIC PATH TO THE CLINIC FOR SICKLE CELL DISEASE AND BETA-THALASSEMIA: IDENTIFICATION OF AN ORALLY-AVAILABLE, POTENT, AND SELECTIVE EUCHROMATIC HISTONE LYSINE METHYLTRANSFERASE 1 AND 2 (EHMT1/2) INHIBITOR

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Elayne Chan-Penebre,1 Veronica Gibaja,1 John Campbell,1 Oluwaseun Ogunbodede,1 Elizabeth Admirand,1 Dorothy Brach,1 Cuyue Tang,1 Alejandra Raimondi,1 Tami Hood,1 Alexis Cocozaki,1 Sule Karaman,1 Kimberly Stickland,1

Christopher Plescia,1 Michael Munchhof,1 Kelli Armstrong,1 Thomas Riera,1 Stuart Orkin,2 Ann Boriack-Sjodin,1 William Janzen,1 Stephen Blakemore,1 Scott A Ribich,1

Richard Chesworth,1 Jesse J Smith,1 and Kenneth W Duncan1

11 December 20171Epizyme Inc., Cambridge, MA; 2Cancer and Blood Disorders Center, Dana Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA

DISCLOSURES

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I have the following financial relationships to disclose:

• Stockholder in and Employee of: Epizyme, Inc

HISTONE METHYLTRANSFERASES EHMT1 AND EHMT2 ARE INDUCERS OF FETALHEMOGLOBIN: OFFERS A THERAPEUTIC APPROACH TO SICKLE CELL DISEASE

• It is well-established that SCD patients exhibit fewer or no clinical symptoms ifthey carry hereditary persistence of fetal hemoglobin (HPFH) mutations that leadto increased blood levels of HbF‒ Re-expression of g-genes and increasing

HbF is a powerful disease modifyingtreatment approach for SCD patients

• The gene responsible for HbF formation,HBG, is negatively regulated by EHMT1/2 post-birth

• EHMT2 (G9a) and EHMT1 (GLP) functionin a heterodimeric complex to di-methylate H3K9, a repressive mark of transcription

• EHMT1/2 play roles in multiple biological processes including hematopoiesis, imprinting, and immunity

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Post-birth

1. Krivega et al. Blood. 2015;126;665-672. 2. Renneville et al. Blood. 2015;126:1930-1939.

3. Cantu et al. Haematologica. 2014;99:1647-1649.4. Bauer et al. Blood. 2012;120:2945-2953.

b-globin locus

Chrom 11

HbE HbF HbA

HBG locus

Other epigenetic machinery

EHMT2

EHMT1

EHMT1/2 INHIBITION PREVENTS DI-METHYLATION OF H3K9 AT A SINGLE GENE LOCUS LEADING TO RE-EXPRESSION OF HbF THAT HAS DISEASE MODIFYING POTENTIAL FOR SCD

• HbF is a known modulator of SCD clinical symptoms

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H3K9me2

HbF

EHMT1/2 inhibition

Prevention of repressive

H3K9me2 mark at HBG locus

Re-expression of HbF protein

Anti-sickling effects

Clinical benefit

EHMT2

EHMT1xx

HBG locus

EHMT2

EHMT1

OFF ON

EPZ035544 IS A POTENT AND SELECTIVE EHMT1/2 INHIBITOR

• Potent, orally available EHMT1/2 inhibitor

• MOI: Peptide competitive,SAM non-competitive

• Highly selective vs. off-targets‒ HMT Panel: >2,000-fold by Ki

‒ Diversity Kinase Panel: >1,000-fold by Ki

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EPZ035544EHMT1/2 Ki: 4 nM, 9 nM

Cellular H3K9 IC50: 67 nMLogP: 2.5, LogD: 0.5, TPSA: 73

SAH

1

10

100

1000

10000

0 6 12 18 24 30 36 42 48

Co

nce

ntr

ation

(n

g/m

L)

Time (hr)

IV -1 mg/kg

PO -25 mg/kg

PO -75 mg/kg

DOSE DEPENDENT MOUSE PK

Plasma exposurerequired for IC50

EPZ035544

EHMT1/2-MEDIATED FETAL HEMOGLOBIN RE-EXPRESSION

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Human CD34+ erythroid progenitor cells were differentiated in the presence of

EPZ035544 and after 14 days, CD235+CD71+ cells were tested for the following

readouts

At Chr11 β-Locus:

Cantu et al. Haematologica. 2014; 99:1647-1649.

EHMT1/2 inhibition

Decrease of H3K9me2

% H3K9me2 from Control

Selective expression of HBG

genesmRNA

Increase of HbF%HbF producing

cellsProtein levels

% HBG/(HBG+HBB) = % HbF/(HbF + HbA)

ROBUST AND ON-TARGET IN VITRO ACTIVITY OF TOOL COMPOUND IN BLOOD PROGENITORS

• FACS analysis shows that EPZ035544 treatment of erythroid progenitors leads to dose-dependent decreases in H3K9me2 together with increases in % HbF+ cells

• 1:1 Correlation between the induction of HBG mRNA (PCR) and HBG protein (highly quantitative mass spec) is evident

• EPZ035544 shows no effects on in vitro erythroid differentiation as measured by CD235+ and CD71+ cell populations from differentiated CD34+ cells

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H3K9me2 % HbF+ cells H3K9me2HBG mRNA HBG Protein HBG Protein

mRNA (PCR)

Protein (MS)

EHMT1/2 INHIBITION IN HUMAN CD34+ CELLS LEADS TO THE DECREASE OF H3K9me2 TOGETHER WITH AN INCREASE OF H3K9Ac AND POL II OCCUPANCY AT THE HBG LOCUS

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• Insignificant changes observed at Chr 16 HbA

promoters

• At β-Locus on Chr 11

– General H3K9me2 reduction across the locus

upon compound treatment

• Treatment causes an increase at HBG promoters

only, HBA and other Hb gene loci are unaffected

• Treatment causes recruitment of RNA Pol II to HBG

promoters only, confirming active replication

H3K9Ac

H3K9me2

RNA Pol IIH3K9AcH3K9me2 RNA Pol II

De-repression Activation Active

Transcription

β-Locus

HBG GENES ARE THE MOST SIGNIFICANTLY UPREGULATED GENES UPON EPZ035544-TREATED HUMAN CD34+ CELLS

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EPZ035544 vs DMSO GENE EXPRESSION PROFILERNA Seq Analysis Selective up-regulation of HBG genes

Log2

(fold change)

-lo

g1

0(P

va

lue

)

Downregulated

genes

Upregulated

genes

MOA for EHMT1/2-mediated HbFinduction is fully validated

Confirms RNA Pol II CHIP Seq Results

Expression of erythroid transcription

factors and known HBG repressors was

unchanged

TESTING THE RE-ACTIVATION OF DEVELOPMENTAL HEMOGLOBINS IN VIVO

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1. In vitro: Human Erythroid Progenitors CD34+

Solid understanding of MOA

2. In vivo:

C57BL6 Mice

Tool compound studies

EHMT1/2 inhibitionDecrease of

H3K9me2

Selective expression

of HBG genes

HbF protein increase

(Hbeg in mouse)

Robust correlation between

target engagement and efficacy

LONG TERM DOSING OF EPZ035544 IN NAÏVE MICE IS WELL TOLERATED AND SHOWS NO SIDE EFFECTS

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Well tolerated at all dose group after 90 days of continuous dosing

√Bodyweight

√Spleen weight

√RBC

√WBC

√Platelets

√Neutrophils

EPZ035544 TREATMENT OF NAÏVE MICE INDUCES HBeg PROTEIN IN VIVO

• Solid reduction of H3K9me2 observed in PBMCs (peripheral blood mononuclear cells) from EPZ035544-treated naïve mice

• Dose-dependent induction of mouse embryonic Hbεγ mRNA with steady state achieved after 30 days

• Increase of Hbεγ protein over time in whole blood

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~50%

Lost due technical issues with sample collection

~100X

% H

bεγ

glo

bin

/To

tal

Pro

tein

by

MS

H3K9me2 Hbεg mRNA % Hbεg /Total (Protein)

SUMMARY: EPZ035544 CAN INDUCE DEVELOPMENTAL HEMOGLOBINS IN IN VITRO AND IN VIVO SYSTEMS

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Solid understanding

of MOA

In vitro and in vivo induction of

developmental hemoglobins

In vitro In vivo Enables

a rational

clinical

path

EHMT1/2inhibition

Decrease of H3K9me2

Selective expression of HBG genes

Increase of

HbF protein(Hbeg in mouse)

ACKNOWLEDGMENTS

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We would like to thank the employees of Epizyme, Inc.