1. Background

#5060: Activity of EZH2 inhibitors as monotherapy and in combination with multiple myeloma therapies in preclinical models A. Drew, V. Motwani, J. Campbell, C. Tang, J. Smith, R. Chesworth, R. A. Copeland, A. Raimondi, S. Ribich

2. EZH2 inhibition as a therapeutic approach in multiple myeloma

3. Tazemetostat combination potential in MM 5. EZH2 inhibitor synergizes with MM SOC resulting in durable tumor regressions

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4. EZH2 inhibitor elicits dose-dependent TGI in all tested MM xenograft models

7. Conclusions

Acknowledgements We would like to thank the employees of Epizyme, Inc. for helpful discussions in regards to this publication.

6. EZH2 regulates survival signaling pathways that impinge upon the Ikaros target gene IRF4

(A) Synergy was determined by 7-day pre-treatment with tazemetostat + 4-day co-treatment with SOCs + tazemetostat and 7-day co-treatment with SOCs + tazemetostat (B) Synergy status is calculated through Loewe Additivity and Bliss Independence in Chalice Viewer Software

11 Day Pre-treatment Assay

7 Day Co-treatment Assay

Legend

Antagonism Additive Synergy Not yet tested

Standard Risk Intermediate Risk High Risk

Class Compounds U266 MOLP-8 LP-1 KMS-28-BM L-363 MM1.S

t(11;14) t(11;14) t(4;14) t(4;14) t(20;22) t(14;16) Grags Dexamethasone

Prednisolone IMiDs Pomalidomide

Lenalidomide

Thalidomide Proteasome Inhibitors Ixazomib

Bortezomib Carfilzomib

Chemotherapeutics Melphalan Vincristine Mafosfamide Etoposide Doxorubicin Bendamustine

HDACis Vorinostat Panobinostat

Bcl-2 inhibitor Venetoclax

Standard Risk Intermediate Risk High Risk

Compounds U266 t(11;14)

Molp-8 t(11;14)

LP-1 t(4;14)

KMS-28-BM t(4;14)

L-363 t(20;22)

MM1.S t(14;16)

RPMI-8226 t(14;16)

Dexamethasone Pomalidomide

Velcade

Panobinostat

Venetoclax

• Combination potential evaluated across 8 cell lines representing major MM translocations • Synergy evaluated through both an 11-day (tazemetostat pre-treatment) and a 7-day (co-treatment) assay

with MM standard of care agents and emerging therapies • Most consistent combination benefit seen across cell lines with immune system modulators (IMiD) and

glucocorticoid receptor agonists (Grag) • Synergy with glucocorticoid receptor agonists consistent with DLBCL preclinical data

Dosing

Dosing

MM1.S – t(14;16)

Dosing

RPMI-8226 – t(14;16)

Dosing

KMS-28-BM – t(4;14)

MOLP8 – t(11;14)

Responses in vitro can be cytostatic or cytotoxic, depending on cell line

Anti-proliferative effects of EZH2 inhibition are observed across the major

recurrent translocation classes of MM

B-A

LL

MC

L

DLB

CL-

ABC

MM

DLB

CL-

GC

B

Hod

gkin

FL

Bur

kitt

Taze

met

osta

t Pro

lifer

atio

n IC

50 (µ

M)

Cell Line Translocations Tazemetostat Day 14

IC50 (µM) ARH77 2.93

JJN3 t(14;16) 0.441 KMS12BM t(11;14) 0.288

L363 t(20;22) 0.280 LP1 t(4;14) 2.1

MM1R t(14;16) 0.058 MM1S t(14;16) 0.340 MOLP2 t(4;14) >10 MOLP8 t(11;14) 0.322

NCIH929 t(4;14) >10 OPM2 t(4;14) .303

RPMI8226 t(14;16) 0.123 U266B1 t(11;14) 0.678

KMS28BM t(4;14) 0.043

Tazemetostat has in vitro activity across a range of B-cell malignancies

Tazemetostat potently inhibits in vitro proliferation of MM cell lines

• EZH2 inhibitor tazemetostat (EPZ-6438) is in phase 2 clinical trials in non-Hodgkin’s Lymphoma (NHL)

• Objective clinical responses have been reported in patients with B-cell lymphomas

• EZH2 is an important regulator of B-cell differentiation in both normal B-cells and B-cell lymphoma

• Consistent with this role, recent studies have shown a dependence on EZH2 activity in multiple myeloma (MM), a disease arising from terminally-differentiated B-cell lymphocyte plasmablasts

• EZH2 expression is increased and PRC target genes are repressed in multiple myeloma compared to normal bone marrow1

• Frequent genetic alterations in epigenetic modulators in MM suggest an important epigenetic role in initiation and maintenance of this disease

• Dysregulation of UTX and WHSC1 suggest that disruption of the balance of H3K27 and H3K36 methylation may be fundamental to MM pathogenesis

• Inhibition of EZH2 alone has shown potent anti-proliferative effects both in in vitro and in vivo preclinical models of MM2,3

EZH2 inhibitors show activity across a wide spectrum of B-cell malignancies

Example: Pomalidomide/tazemetostat 7 day co-treatment in RPMI-8226 cell line

EPZ011989 inhibits tumor growth in MM.1S xenograft model

EPZ011989 is well-tolerated in all tested xenograft models

Dose dependent decrease in tumor growth in MM1.S xenograft model

H3K27 methylation in MM.1S tumors is decreased by 14 day EPZ011989 treatment

Plasma levels of EPZ011989 are dose proportional

• EPZ011989 is a selective small molecule EZH2 inhibitor with comparable in vitro and in vivo characteristics to tazemetostat1

• EPZ011989 efficacy was

tested in 4 MM xenograft models in CB17 SCID mice (BID p.o. administration)

• Robust tumor growth inhibition was observed with the SOC pomalidomide/ dexamethasone

• EPZ011989 was well-tolerated up to 250mg/kg BID

• H3K27me3 was ablated at all doses, confirming EZH2 target engagement

• Dose-dependent TGI was observed in all models

• Single agent EZH2i potently inhibits proliferation in MM cell lines • Single agent EZH2i induces dose-dependent TGI in 4 out of 4 MM xenograft models • In vitro combination data with EZH2i and SOC agents show robust synergy with IMiDs and other classes of agents • In vivo combination studies support combination benefit of EPZ011989 with backbone SOC agents pomalidomide and

dexamethasone in 4 out of 4 models • EZH2i-IMiD synergy may be mediated through joint repression of Ikaros target genes, resulting in increased apoptosis • These data provide evidence of a dependency in MM on EZH2 activity and show that the single agent preclinical activity of

EZH2 inhibitors can be further enhanced and expanded through rational combination strategies

• In vivo combination studies support combination benefit of EPZ011989 with backbone SOC agents pomalidomide and dexamethasone in 4 out of 4 models

• Combinations induce tumor regressions in 3 out of 4 models, and are tolerated to 21 days in all models • Additional studies are ongoing/planned with bortezomib and daratumumab

Clinical response to tazemetostat in NHL (E7438-G000-001 – NCT01897571)

EPZ011989-Pom/Dex combination induces tumor regression in MM.1S xenograft model

EPZ011989 combinations are tolerated in MM.1S xenograft model

Plasma levels of EPZ011989 when dosed in combination with SOC agents

EPZ011989-Pom/Dex combination induces tumor regression in MM1.S xenograft model

(Day 18)

MM1.S proliferation is potently inhibited by EZH2 inhibitors (Day 14)

21% TGI 38% TGI

**61% TGI

***97% TGI Pom/Dex

RPMI-8226

**40% TGI

***65% TGI ***68% TGI Pom/Dex

**45% TGI

MM1.S

***26% TGI ***50% TGI ***52% TGI ***52% TGI Pom/Dex

KMS-28BM

13% TGI 26% TGI 26% TGI

***46% TGI Pom/Dex

MOLP-8

**40% TGI

**45% TGI

***65% TGI

***68% TGI

* p< 0.05 ** p< 0.01 *** p< 0.001

**40% TGI

***65% TGI ***68% TGI Pom/Dex

**45% TGI

Day 17 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

Comparison to EZH2i # p< 0.05 ## p< 0.01 ## p< 0.001

• Pomalidomide treatment results in Ikaros and Aiolos degradation with subsequent upregulation of Ikaros target gene IRF4 • Tazemetostat treatment also reduces IRF4 expression, possibly via modulation of regulatory miRNAs1

• Combined treatment with pomalidomide and tazemetostat results in further repression of IRF4 • The synergistic effect of tazemetostat treatment in combination with IMiDs may result from an increase in apoptosis related to dysregulation of the Ikaros-IRF4 axis, a key regulator of

malignancy-specific gene expression in MM

RPMI-8226 cells were treated for 3 days with 31, 125, or 500nM taz and/or 12.5, 50, or 200nM pomalidomide.

Tazemetostat/pomalidomide combination results in enhanced repression of IRF4 in RPMI-8226

Tazemetostat/pomalidomide combination results in enhanced apoptosis in RPMI-8226

ED EPD

E ED EPD

E ED EPD

E ED EPD

E E D V PD

ED

EPD

From Weigert + Weinstock Blood 2012

From Ribrag et al. ASH 2015

1 Kalushkova et al PlosOne 2010

2Argawal et al Oncotarget 2015 3 Internal Epizyme Data

1Alzrigat et al. 2016

RPMI-8226 cells were treated for 7days with 31, 125, or 500nM taz and/or 3, 12.5, or 50nM pomalidomide.

In vivo studies conducted in CB17 SCID mice. EZH2i = EPZ011989 125mg/kg BID p.o. Dexamethasone 1mg/kg QD i.p., Pomalidomide 10mg/kg QD p.o.

***EZH2i ***Dex ***Pom/Dex ***EZH2i/Dex ***EZH2i/Pom/Dex

P values derived from one-way ANOVA with Dunnett’s multiple comparisons of treatment compared to vehicle except where noted

P values derived from one-way ANOVA with Dunnett’s multiple comparisons of treatment compared to vehicle

***Dex ***EZH2i

***Pom/Dex ***EZH2i/Pom/Dex

Vehicle

***EZH2i/Dex

Day 17 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

***Pom

***EZH2i ***Dex

***EZH2i/Pom Vehicle

***EZH2i/Dex

Day 21 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

Day 18 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001 ***EZH2i

***Dex

***Pom/Dex

***EZH2i/Dex

***EZH2i/Pom/Dex

Vehicle

Vehicle

##98% TGI

#93% TGI

##103% TGI

Day 14 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

Day 21 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

Day 14 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

Day 21 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

59% TGI

58% TGI

Day 14 ANOVA * p< 0.05 ** p< 0.01 *** p< 0.001

* Response evaluable: Measureable disease ≥ 1 dose ≥ 1 post-baseline scan

Day 0 Day 7 End Point • ATP proliferation (CellTiter-Glo)

Co-treatment • Tazemetostat • Combination partner

Day 0 Day 4 Day 7 Day 11 Pre-treatment • Tazemetostat

Re-dose/Split • Tazemetostat

Co-treatment • Tazemetostat • Combination partner

End Point • ATP proliferation (CellTiter-Glo)

DMSO

Nai

ve

1 Campbell et al . 2015 ACS Med Chem Lett

Tazemetostat (EPZ-6438)

EPZ011989

EPZ011989 mg/kg

***14%

***2%

* p< 0.05 ** p< 0.01 *** p< 0.001

***2%

EPZ011989 mg/kg

Taz

Pom

Taz+Pom

DMSO

IKAROS 70kDa

AIOLOS 70kDa

50kDa

38kDa

GAPDH

IRF4

Nai

ve

8kDa

Histone H3

H3K27me3

15kDa

Perc

enta

ge ch

ange

from

bas

elin

e Patients (n=16)

DLBCL FL MZL

CR+PR 5/10 (50%) 3/5 (60%) 1/1

9/16 (56%)

Per Protocol: Response Evaluable *

Non- GCB

Non- GCB

Non- GCB

Non- GCB

Und.

Non- GCB

Non- GCB

GCB

GCB Und.