277 P057 the Discovery and Pre-Clinical Development of the First Clinical Stage EZH2-Inhibitor, EPZ-6438 (E7438)

#277 P057 The discovery and pre-clinical development of the first clinical stage EZH2-inhibitor, EPZ-6438 (E7438)

Kevin W. Kuntz1†, Roy M. Pollock1,2, Sarah K. Knutson1, Natalie Warholic1, Tim Wigle1, Chris Sneeringer1,3, Victoria Richon1,4, Richard Chesworth1, Margaret Porter Scott1,3, Robert A. Copeland1, Heike Keilhack1 1Epizyme Inc., 400 Technology Square, 4th floor, Cambridge MA 02139, USA 2Currently at Warp Drive Bio, 400 Technology Sq, Cambridge MA 02139, USA 3Currently at Genentech, 1 DNA Way, South San Francisco CA 94080, USA 4Currently at Sanofi, 270 Albany Street, Cambridge MA 02139, USA † corresponding author Abstract Results Mutations within the catalytic domain of the histone methyltransferase EZH2 have been EZH2 HTS Yielded One Tractable Series EPZ-6438 is a Specific and SAM-Competitive EPZ-6438 Selectively Kills EZH2 Mutant Cells Despite identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are Inhibitor of EZH2 Similar Target Inhibition in Both Mutant and WT Cells hypothesized to confer a cellular survival dependency on EZH2 enzymatic activity in these cancers. Here, we disclose the discovery of EPZ-6438 (E7438), as a potent, selective and orally EZH2 Y646F Mutant EZH2 Y646 WT bioavailable small molecule inhibitor of EZH2 in preclinical models of NHL. Previously we have Hit Expansion µM E7438 Design Tenets disclosed the properties of EPZ005687, a tool compound useful for exploring the in vitro biology 1.Improve solubility 2. Improve biochemical potency of EZH2 inhibition. Multi-parametric optimization of the potency, pharmokinetics, oral 3. Maintain good property space bioavailability and tolerability of this series led to the discovery of the clinical compound, EPZ- EPZ004851 EPZ004759 EPZ005030 m m EZH2 IC 0.4 mM 6438. Modulation of the log P was required to reach the optimal balance between clearance and EZH2 IC50 2.5 M EZH2 IC50 0.5 M 50 SAM competitive 10 mM in cell Me assay bioavailability while maintaining the requisite potency. EPZ-6438 selectively inhibits intracellular Nucleosome non-competitive Mouse PK Day Day m F = 41 % EPZ-6438 is potent against both lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in Solubility <10 M at pH 7 EPZ-6438 is SAM competitive Cl = 34 ml/min/kg wild-type and mutant EZH2 µM both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) E7438 Oral Bioavailability achieved: 1st major hurdle overcome H3K27me3 leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point H3 Methylation IC = 0.008 µM mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 caused a dose- Methylation IC50 = 0.0091 µM 50 An HTS was performed under balanced conditions (Km for SAM and Km for Nucleosome) to EPZ-6438 is similarly active against wild-type and SET domain mutants of EZH2. It is Treatment of WSU-DLCL2 cells (EZH2 Y646F mutant) with a range of concentrations of dependent tumor growth inhibition, including complete and sustained tumor regressions with identify inhibitors of EZH2, of which is EPZ004851 is an example. Additional analogs were about 50-fold selective against EZH1. EPZ-6438 has shown no activity across a panel EPZ-6438 showed time and concentration dependent defects in proliferation. While at correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. EPZ-6438 purchased based on structural similarity to the original hit and EPZ004759 demonstrated of protein methyltransferases up to the top concentration tested (10 or 50 µM), four days the number of cells were similar (within 10-fold), by seven days dramatic recently entered clinical testing as E7438 in a dose escalation phase 1 trial in relapsed or improved potency. This compound was characterized for its mode of inhibition and was found meaning it is greater than 1000-fold selective for EZH2. EPZ-6438 was shown to be a differences in cell numbers appeared. Treatment of OCI-LY19 cells (EZH2 wild-type) with to be SAM competitive. Aqueous solubility was identified as a key issue with EPZ004759, and reversible inhibitor of EZH2 and is SAM competitive and nucleosome non-competitive. a range of concentrations of EPZ-6438 showed no major proliferation defects even after refractory malignancies. therefore groups known to increase solubility were used to functionalize the template leading 11 days of treatment. Both cell lines showed equal reduction of H3K27Me3 suggesting to EPZ005030 which showed improved solubility. EPZ005030 was assayed in mouse PK shown that the differences in phenotypic responses are due to differences in the cells’ to have moderate clearance and bioavailability. dependency on EZH2 and not due to differences in drug exposure. KARPAS422 EZH2 Y646N Mutant Xenografts are Introduction Optimization of the 5,6 Ring Template EPZ-6438 Specifically Inhibits Cellular H3K27 Methylation in a Time and Dose-Dependent Manner Sensitive to Orally Dosed EPZ-6438 in a Dose Dependent Manner WSU-DLCL2 cells in vitro 7 day PK/PD study Methylation by ELISA 28 day efficacy study EZH2 Inhibition for Genetically Defined Cancers Target inhibition in tumor (ELISA) EZH2 Inhibition for Genetically Defined Cancers 1600 Design Tenets 1400

1200 100 100 ) 3 Vehicle Improve potency 1000 * 80.5 mg/kg * Maintain good PK 800 * 161 mg/kg SWI/SNF COMPLEX 50 50 600 322 mg/kg * * OMPLEX 400 * PRC2 C Change of function mutation EPZ005687 WSU-DLCL2 cells in vitro * * R1 = lipophilic groups preferred (mm Volume Tumor * 4-Day Treatment 200 WSU-DLCL2 cells in vitro 0 0 • Non-Hodgkin lymphoma R2 = Me, Et & Pr preferred EZH2 IC50 50 nM * Time Course at 1 µM 0 *

1.1 mM in cell methylation assay 0 5 10 15 20 25 30 TrimethylationLevel H3K27of (%) Vehicle

INI1 Day Vehicle

75 mg/kg 75

150 mg/kg 150 mg/kg 301 mg/kg 602

301 mg/kg 301 mg/kg 602

Mouse PK *statistically significant difference from vehicle mg/kg 150 1203 mg/kg 1203 Oral F ~40% BID QD Cl 11 ml/min/kg, Vdss 0.5 L/kg Loss of function due to INI1 deficiency .All doses were BID in efficacy study, no significant body weight loss during study T1/2 1.5 h • Synovial sarcoma, MRT, others .Mice were kept alive and remain tumor free 63 days (top two dose groups) after cessation ppb 99.2 (m) 99.7 (h) of dosing EPZ005687 was identified as a tool compound to explore EZH2 biology Mice implanted with KARPAS422 cells were randomized once tumors reached a median Methylation 12 3 In order to increase potency, substitutions in a variety of vectors were explored. Equal-potent WSU-DLCL2 cells treated with EPZ-6438 for four days showed a concentration size of 150 mm . The mice were treated orally with EPZ-6438 twice daily using the doses dependent decrease in H3K27 trimethylation. WSU-DLCL2 cells treated with 1 µM indicated. Tumor volumes were measured by calipers every 3-4 days. After 28 days, Demethylation compounds could be made with different small groups in the R2 position. Increasing the size and lipophilicity of groups in the R position led to significant increases in potency. Combining EPZ-6438 showed a time-dependent decrease of H3K27Me3 with greater than 50% treatment was halted and the mice observed for an additional 63 days. The top two 1 reduction after 24 h and maximal decrease (within the limits of detection) at four doses led to complete elimination of the tumor with no regrowth seen. In a separate a cyclopentyl group in the R1 position with a change from a pyrrazolopyridine to an indazole led to EPZ005687 which showed increased cell potency while maintaining the mouse PK profile. days. Treatment of WSU-DLCL2 cells for four days with 2.7 µM EPZ-6438 showed a study, the changes in the H3K27Me3 levels in tumors after seven days of BID PO dosing reduction of H3K27 mono-, di- and tri-methylation with no significant effects on showed a dose dependent decrease. At the ~150 mg/kg dose level, complete elimination K27 K27(me)3 other methyl marks. of the tumor is observed at 28 days while only ~60% decrease in H3K27Me3 is observed Development of a new sub-series at 7 days. At least 3 distinct genetically defined cancers • Non-Hodgkin lymphoma, germinal center (EZH2 point mutations) Conclusions • Synovial sarcoma (SSX-SS18 fusion) • MRT (INI1-deletion) • EPZ-6438 is a potent and selective small molecule inhibitor of EZH2 and EZH2 SET domain mutants. • EPZ-6438 inhibits cellular H3K27 methylation leading to killing of lymphoma cell lines expressing EZH2 SET domain mutants. EPZ005991 EPZ006093 EPZ006222 EPZ-6438 15 nM • EZH2 is the catalytic subunit of the multiprotein PRC2 (polycomb repressive EZH2 IC50 200 nM 33 nM 14 nM • Antitumor activity has been observed in several EZH2 mutant lymphoma xenograft models ranging from tumor Cell Me IC50 166 nM 94 nM complex 2) complex Mouse PK growth inhibition to durable regressions (e.g. KARPAS422) at well tolerated doses and schedules. F = 2% 55% • PRC2 catalyzes mono-, di- and tri-methylation of H3K27 Cl = 49 ml/min/kg 13 ml/min/kg • EPZ-6438 (E7438) has transitioned into clinical development and results from the Phase I study are being ppb = 98% 93% • H3K27 is the only significant substrate for PRC2 presented in a separate oral presentation by V. Ribrag entitled “Phase 1 first-in-human study of the enhancer of New sub-series expanded scope of SAR allowing optimization of potency and PK • H3K27me3 is a transcriptionally repressive histone mark zeste-homolog 2 (EZH2) histone methyl transferase inhibitor E7438 as a single agent in patients with advanced • Hyper-trimethylation of H3K27 is tumorigenic in a broad spectrum of human Disconnection of the five-membered ring led to an increase in potency as illustrated by the potency enhancement going from EPZ005991 to EPZ006093. Importantly a second solid tumors or B cell lymphoma” in Plenary Session 5 substitution on the aniline allowed an additional vector where polarity could be incorporated. References cancers, including GC NHL 1. Knutson et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells, Nature Chemical Biology, 2012, 8:890-896. When8 the aniline was alkylated with a tetrahydropyran and an ethyl group it furnished potent compound, EPZ006222, however the PK properties were not optimal. Replacing the chloro 2. Keilhack et al. Preclinical characterization of E7438, a potent, selective inhibitor of protein methyltransferase EZH2 with robust antitumor activity against EZH2 mutated non-Hodgkin with a benzyl-morpholine group, as in EPZ-6438, improved the solubility, decreased the lymphoma xenografts in mice, Blood (ASH Annual Meeting Abstracts) Nov 2012: 120: 3712. clearance and improved the oral bioavailability without sacrificing potency. EPZ-6438 was 3. Sneeringer et al. Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas, selected for clinical development. PNAS, 2010, 107(49): 20980-20985. 4. Knutson et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2, PNAS, 2013, 110(19): 7922-7927.

Disclosures: Kuntz, Knutson, Warholic, Wigle, Chesworth, Keilhack: Epizyme, Inc.: Employment, Equity Ownership, Patents , Stock options Copeland: Epizyme Inc. : Employment, Equity Ownership, Patents, Stock options ; Mersana: Membership on an entity’s Board of Directors or advisory committees. Pollock: Epizyme, Inc.: Equity Ownership, Patents , Stock options ;Warp Drive Bio.: Employment Sneeringer, Porter Scott: Epizyme, Inc.: Equity Ownership, Patents , Stock options ; Genetech.: Employment www.epizyme.com Richon: Epizyme, Inc.: Equity Ownership, Patents , Stock options ; Sanofi.: Employment