Reawakening of Human Fetal Hemoglobin And

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

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 2017 1Epizyme Inc., Cambridge, MA; 2Cancer and Blood Disorders Center, Dana Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA

1 DISCLOSURES

I have the following financial relationships to disclose:

• Stockholder in and Employee of: Epizyme, Inc

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

• It is well-established that SCD patients exhibit fewer or no clinical symptoms if Post-birth EHMT1 they carry hereditary persistence of fetal hemoglobin (HPFH) mutations that lead EHMT2 Other epigenetic to increased blood levels of HbF machinery ‒ Re-expression of g-genes and increasing HbF is a powerful disease modifying treatment approach for SCD patients HBG locus • The gene responsible for HbF formation, HBG, is negatively regulated by EHMT1/2 b-globin locus post-birth Chrom 11 • EHMT2 (G9a) and EHMT1 (GLP) function in 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

HbE HbF HbA

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. 3 4. Bauer et al. Blood. 2012;120:2945-2953. 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

Prevention of EHMT1/2 repressive Re-expression of Anti-sickling Clinical benefit inhibition H3K9me2 mark HbF protein effects at HBG locus

EHMT1 EHMT1x HbF EHMT2 EHMT2x OFF ON

HBG locus H3K9me2

• HbF is a known modulator of SCD clinical symptoms

4 EPZ035544 IS A POTENT AND SELECTIVE EHMT1/2 INHIBITOR

• Potent, orally available EHMT1/2 inhibitor SAH • MOI: Peptide competitive, SAM non-competitive EPZ035544 • Highly selective vs. off-targets EHMT1/2 Ki: 4 nM, 9 nM ‒ HMT Panel: >2,000-fold by Ki Cellular H3K9 IC50: 67 nM LogP: 2.5, LogD: 0.5, TPSA: 73 ‒ Diversity Kinase Panel: >1,000-fold by Ki

10000 DOSE DEPENDENT MOUSE PK EPZ035544 IV -1 mg/kg 1000 PO -25 mg/kg PO -75 mg/kg Plasma exposure 100 required for IC50

1 Concentration (ng/mL) Concentration 0 6 12 18 24 30 36 42 48 Time (hr)

5 EHMT1/2-MEDIATED FETAL HEMOGLOBIN RE-EXPRESSION

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:

Decrease of Selective Increase of HbF EHMT1/2 inhibition H3K9me2 expression of HBG %HbF producing % H3K9me2 from genes cells Control mRNA Protein levels

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

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

H3K9me2 % HbF+ cells HBG mRNA HBG Protein H3K9me2 HBG Protein

mRNA (PCR) Protein (MS)

7 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

β-Locus H3K9me2 • Insignificant changes observed at Chr 16 HbA promoters • At β-Locus on Chr 11 – General H3K9me2 reduction across the locus upon compound treatment

H3K9Ac • Treatment causes an increase at HBG promoters only, HBA and other Hb gene loci are unaffected

H3K9me2 H3K9Ac RNA Pol II RNA Pol II • Treatment causes recruitment of RNA Pol II to HBG De-repression Activation Active promoters only, confirming active replication Transcription

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

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

Confirms RNA Pol II CHIP Seq Results

MOA for EHMT1/2-mediated HbF (P value) (P

10 induction is fully validated

log -

Expression of erythroid transcription

Downregulated Log2 Upregulated factors and known HBG repressors was genes (fold change) genes unchanged

9 TESTING THE RE-ACTIVATION OF DEVELOPMENTAL HEMOGLOBINS IN VIVO

Tool compound studies

Selective HbF protein Decrease of EHMT1/2 inhibition expression increase H3K9me2 of HBG genes (Hbeg in mouse)

1. In vitro: Human Erythroid Progenitors CD34+

2. In vivo: C57BL6 Mice

Robust correlation between Solid understanding of MOA target engagement and efficacy

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

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

√ √ Bodyweight Spleen weight

√ √ √ √ RBC WBC Platelets Neutrophils

11 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

H3K9me2 Hbεg mRNA % Hbεg /Total (Protein)

~50%

~100X

globin/Total

Protein by MS by Protein

Hb % %

Lost due technical issues with sample collection

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

Increase Selective of EHMT1/2 Decrease of expression of HbF protein inhibition H3K9me2 HBG genes (Hbeg in mouse)

In vitro In vivo Enables a rational clinical path

Solid understanding In vitro and in vivo induction of of MOA developmental hemoglobins

13 ACKNOWLEDGMENTS

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