The discovery and pre-clinical development of the first clinical stage EZH2-inhibitor, EPZ-6438 (E7438) Kevin W. Kuntz 1† , Roy M. Pollock 1,2 , Sarah K. Knutson 1 , Natalie Warholic 1 , Tim Wigle 1 , Chris Sneeringer 1,3 , Victoria Richon 1,4 , Richard Chesworth 1 , Margaret Porter Scott 1,3 , Robert A. Copeland 1 , Heike Keilhack 1 1 Epizyme Inc., 400 Technology Square, 4 th floor, Cambridge MA 02139, USA 2 Currently at Warp Drive Bio, 400 Technology Sq, Cambridge MA 02139, USA 3 Currently at Genentech, 1 DNA Way, South San Francisco CA 94080, USA 4 Currently at Sanofi, 270 Albany Street, Cambridge MA 02139, USA † corresponding author Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are 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 bioavailable small molecule inhibitor of EZH2 in preclinical models of NHL. Previously we have disclosed the properties of EPZ005687, a tool compound useful for exploring the in vitro biology of EZH2 inhibition. Multi-parametric optimization of the potency, pharmokinetics, oral bioavailability and tolerability of this series led to the discovery of the clinical compound, EPZ- 6438. Modulation of the log P was required to reach the optimal balance between clearance and bioavailability while maintaining the requisite potency. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 caused a dose- dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. EPZ-6438 recently entered clinical testing as E7438 in a dose escalation phase 1 trial in relapsed or refractory malignancies. Abstract Results Introduction Conclusions www.epizyme.com #277 P057 References 1. Knutson et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells, Nature Chemical Biology, 2012, 8:890-896. 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 lymphoma xenografts in mice, Blood (ASH Annual Meeting Abstracts) Nov 2012: 120: 3712. 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, 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. • 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. • Antitumor activity has been observed in several EZH2 mutant lymphoma xenograft models ranging from tumor growth inhibition to durable regressions (e.g. KARPAS422) at well tolerated doses and schedules. • EPZ-6438 (E7438) has transitioned into clinical development and results from the Phase I study are being presented in a separate oral presentation by V. Ribrag entitled “Phase 1 first-in-human study of the enhancer of zeste-homolog 2 (EZH2) histone methyl transferase inhibitor E7438 as a single agent in patients with advanced solid tumors or B cell lymphoma” in Plenary Session 5 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 Richon: Epizyme, Inc.: Equity Ownership, Patents , Stock options ; Sanofi.: Employment Methylation Demethylation Change of function mutation • Non-Hodgkin lymphoma PRC2 COMPLEX K27(me) 3 K27 Loss of function due to INI1 deficiency • Synovial sarcoma, MRT, others SWI/SNF COMPLEX INI1 At least 3 distinct genetically defined cancers • Non-Hodgkin lymphoma, germinal center (EZH2 point mutations) • Synovial sarcoma (SSX-SS18 fusion) • MRT (INI1-deletion) EZH2 Inhibition for Genetically Defined Cancers EZH2 Inhibition for Genetically Defined Cancers • EZH2 is the catalytic subunit of the multiprotein PRC2 (polycomb repressive complex 2) complex • PRC2 catalyzes mono-, di- and tri-methylation of H3K27 • H3K27 is the only significant substrate for PRC2 • H3K27me3 is a transcriptionally repressive histone mark • Hyper-trimethylation of H3K27 is tumorigenic in a broad spectrum of human cancers, including GC NHL Hit Expansion EPZ004851 EZH2 IC 50 2.5 mM EPZ004759 EZH2 IC 50 0.5 mM SAM competitive Nucleosome non-competitive Solubility <10 mM at pH 7 EZH2 HTS Yielded One Tractable Series Design Tenets 1.Improve solubility 2. Improve biochemical potency 3. Maintain good property space EPZ005030 EZH2 IC 50 0.4 mM 10 mM in cell Me assay Mouse PK F = 41 % Cl = 34 ml/min/kg Oral Bioavailability achieved: 1 st major hurdle overcome An HTS was performed under balanced conditions (Km for SAM and Km for Nucleosome) to identify inhibitors of EZH2, of which is EPZ004851 is an example. Additional analogs were purchased based on structural similarity to the original hit and EPZ004759 demonstrated improved potency. This compound was characterized for its mode of inhibition and was found to be SAM competitive. Aqueous solubility was identified as a key issue with EPZ004759, and therefore groups known to increase solubility were used to functionalize the template leading to EPZ005030 which showed improved solubility. EPZ005030 was assayed in mouse PK shown to have moderate clearance and bioavailability. Optimization of the 5,6 Ring Template Design Tenets Improve potency Maintain good PK EPZ005687 was identified as a tool compound to explore EZH2 biology EPZ005687 EZH2 IC 50 50 nM 1.1 mM in cell methylation assay Mouse PK Oral F ~40% Cl 11 ml/min/kg, Vdss 0.5 L/kg T 1/2 1.5 h ppb 99.2 (m) 99.7 (h) R1 = lipophilic groups preferred R2 = Me, Et & Pr preferred In order to increase potency, substitutions in a variety of vectors were explored. Equal-potent compounds could be made with different small groups in the R 2 position. Increasing the size and lipophilicity of groups in the R 1 position led to significant increases in potency. Combining a cyclopentyl group in the R 1 position with a change from a pyrrazolopyridine to an indazole led to EPZ005687 which showed increased cell potency while maintaining the mouse PK profile. Development of a new sub-series New sub-series expanded scope of SAR allowing optimization of potency and PK EPZ006093 33 nM 8 EPZ005991 EZH2 IC 50 200 nM EPZ006222 15 nM Cell Me IC 50 166 nM Mouse PK F = 2% Cl = 49 ml/min/kg ppb = 98% EPZ-6438 14 nM 94 nM 55% 13 ml/min/kg 93% 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 substitution on the aniline allowed an additional vector where polarity could be incorporated. When 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 with a benzyl-morpholine group, as in EPZ-6438, improved the solubility, decreased the clearance and improved the oral bioavailability without sacrificing potency. EPZ-6438 was selected for clinical development. EPZ-6438 is a Specific and SAM-Competitive Inhibitor of EZH2 EPZ-6438 is potent against both wild-type and mutant EZH2 EPZ-6438 is SAM competitive EPZ-6438 is similarly active against wild-type and SET domain mutants of EZH2. It is about 50-fold selective against EZH1. EPZ-6438 has shown no activity across a panel of protein methyltransferases up to the top concentration tested (10 or 50 μM), meaning it is greater than 1000-fold selective for EZH2. EPZ-6438 was shown to be a reversible inhibitor of EZH2 and is SAM competitive and nucleosome non-competitive. EPZ-6438 Specifically Inhibits Cellular H3K27 Methylation in a Time and Dose-Dependent Manner WSU-DLCL2 cells in vitro Methylation by ELISA WSU-DLCL2 cells in vitro Time Course at 1 μM WSU-DLCL2 cells in vitro 4-Day Treatment WSU-DLCL2 cells treated with EPZ-6438 for four days showed a concentration dependent decrease in H3K27 trimethylation. WSU-DLCL2 cells treated with 1 μM EPZ-6438 showed a time-dependent decrease of H3K27Me3 with greater than 50% reduction after 24 h and maximal decrease (within the limits of detection) at four days. Treatment of WSU-DLCL2 cells for four days with 2.7 μM EPZ-6438 showed a reduction of H3K27 mono-, di- and tri-methylation with no significant effects on other methyl marks. EPZ-6438 Selectively Kills EZH2 Mutant Cells Despite Similar Target Inhibition in Both Mutant and WT Cells EZH2 Y646F Mutant EZH2 Y646 WT H3K27me3 H3 Methylation IC 50 = 0.008 μM Methylation IC 50 = 0.0091 μM μM E7438 μM E7438 Day Day Treatment of WSU-DLCL2 cells (EZH2 Y646F mutant) with a range of concentrations of EPZ-6438 showed time and concentration dependent defects in proliferation. While at four days the number of cells were similar (within 10-fold), by seven days dramatic differences in cell numbers appeared. Treatment of OCI-LY19 cells (EZH2 wild-type) with a range of concentrations of EPZ-6438 showed no major proliferation defects even after 11 days of treatment. Both cell lines showed equal reduction of H3K27Me3 suggesting that the differences in phenotypic responses are due to differences in the cells’ dependency on EZH2 and not due to differences in drug exposure. KARPAS422 EZH2 Y646N Mutant Xenografts are Sensitive to Orally Dosed EPZ-6438 in a Dose Dependent Manner All doses were BID in efficacy study, no significant body weight loss during study Mice were kept alive and remain tumor free 63 days (top two dose groups) after cessation of dosing 28 day efficacy study 12 7 day PK/PD study Target inhibition in tumor (ELISA) 0 200 400 600 800 1000 1200 1400 1600 0 5 10 15 20 25 30 Tumor Volume (mm 3 ) Day Vehicle 80.5 mg/kg 161 mg/kg 322 mg/kg * * * Vehicle 75 mg/kg 150 mg/kg 301 mg/kg 602 mg/kg 0 50 100 Trimethylation Level of H3K27 (%) * * * * BID Mice implanted with KARPAS422 cells were randomized once tumors reached a median size of 150 mm 3 . The mice were treated orally with EPZ-6438 twice daily using the doses indicated. Tumor volumes were measured by calipers every 3-4 days. After 28 days, treatment was halted and the mice observed for an additional 63 days. The top two doses led to complete elimination of the tumor with no regrowth seen. In a separate study, the changes in the H3K27Me3 levels in tumors after seven days of BID PO dosing showed a dose dependent decrease. At the ~150 mg/kg dose level, complete elimination of the tumor is observed at 28 days while only ~60% decrease in H3K27Me3 is observed at 7 days. *statistically significant difference from vehicle