A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.

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SET domain-containing protein 2 (SETD2), a histone methyltransferase, has been identified as a target of interest in certain hematological malignancies, including multiple myeloma. This account details the discovery of EPZ-719 , a novel and potent SETD2 inhibitor with a high selectivity over other histone methyltransferases. A screening campaign of the Epizyme proprietary histone methyltransferase-biased library identified potential leads based on a 2-amidoindole core. Structure-based drug design (SBDD) and drug metabolism/pharmacokinetics (DMPK) optimization resulted in EPZ-719 , an attractive tool compound for the interrogation of SETD2 biology that enables in vivo target validation studies.

SETD2, a lysine N -methyltransferase, is a histone methyltransferase that plays an important role in various cellular processes and was identified as a target of interest in multiple myeloma that features a t(4,14) translocation. We recently reported the discovery of a novel small-molecule SETD2 inhibitor tool compound that is suitable for preclinical studies. Herein we describe the conformational-design-driven evolution of the advanced chemistry lead, which resulted in compounds appropriate for clinical evaluation. Further optimization of this chemical series led to the discovery of EZM0414, which is a potent, selective, and orally bioavailable inhibitor of SETD2 with good pharmacokinetic properties and robust pharmacodynamic activity in a mouse xenograft model.

t(4;14) chromosome translocations are found in 15% of newly diagnosed multiple myeloma (MM) cases and are associated with high risk. MM cells with t(4;14) over-express the histone methyltransferase (HMT), WHSC1/MMSET/NSD2, which leads to deregulation of gene expression due to increased di-methylation of Histone H3 at lysine 36 (H3K36me2). This activity has been shown to be essential for the survival of t(4;14) MM cells. In addition to WHSC1, another HMT, SETD2, has been shown to methylate H3K36. SETD2 is the only known enzyme capable of tri-methylation of H3K36 and has been reported to play a role in transcriptional elongation and alternative splicing. CRISPR pooled screening has shown that SETD2 activity is required for viability of a variety of cancer cell lines. This led Epizyme to develop small molecule inhibitors of SETD2 enzyme activity in order to understand the role of SETD2 in tumorigenesis. Through our drug discovery efforts, we identified EPZ-040414, a potent and selective inhibitor of SETD2 with low nM cell biochemical activity and broad selectivity against a panel of other HMTs. The proposed role of SETD2 in H3K36 methylation led us to test a panel of MM cells, including 6 t(4;14) cell lines with EPZ-040414. Inhibition of SETD2 resulted in reduced global tri-methylation of H3K36 in t(4;14) bearing MM cell lines. In contrast, there was no effect on global di-methyl H3K36 levels, indicating that WHSC1 activity is not affected by SETD2 inhibition. Moreover, 5/6 t(4;14) MM cell lines showed a cytotoxic response to treatment with EPZ-040414 with IC50s ranging between 60 and 200 nM, while all non-t(4;14) MM cell lines showed limited responses between 1 and 8 μM. Moreover, screening of a 280 cancer cell line panel with a SETD2 inhibitor showed minimal anti-proliferative activity in most cell lines tested. These findings show that t(4;14) MM cell lines require SETD2 activity for survival, suggesting that SETD2 inhibitors are strong candidates for the treatment of this high risk subgroup of MM. Efforts to further understand the interaction between SETD2 and WHSC1 in the molecular pathogenesis of t(4;14) myeloma will be presented. The current chemical series represented by EPZ-040414 is potent, selective, orally available, and currently under further evaluation for its therapeutic potential. Figure. Figure. Disclosures Thomenius: Epizyme Inc.: Employment, Equity Ownership. Totman:Epizyme Inc.: Employment, Equity Ownership. Cosmopoulos:Epizyme Inc.: Employment, Equity Ownership. Brach:Epizyme Inc.: Employment, Equity Ownership. Ci:Epizyme Inc.: Employment, Equity Ownership. Farrow:Epizyme Inc.: Employment, Equity Ownership. Smith:Epizyme Inc.: Employment, Equity Ownership. Chesworth:Epizyme Inc.: Employment, Equity Ownership. Duncan:Epizyme Inc.: Employment, Equity Ownership. Tang:Epizyme Inc.: Employment, Equity Ownership. Riera:Epizyme Inc.: Employment, Equity Ownership. Lampe:Epizyme Inc.: Employment, Equity Ownership.

Tazemetostat (EPZ-6438) is a potent, selective, orally bioavailable small molecule inhibitor of EZH2, the enzymatic subunit of the polycomb repressive complex 2, which is currently being evaluated in multiple phase II clinical trials for the treatment of non-Hodgkin lymphoma, mesothelioma and molecularly defined solid tumors. Objective clinical responses have been observed in phase II studies of tazemetostat, including patients with B-cell lymphomas. EZH2 has been shown to play a key role in the maturation of B-cells and, consistent with this phenomenon, multiple B-cell malignancies are dependent on EZH2 for survival. In preclinical studies in mantle cell lymphoma (MCL), a disease arising from mantle zone cells surrounding germinal centers, treatment with EZH2 inhibitors, in in vitro and in vivo models demonstrated antiproliferative activity, suggesting EZH2 may be a promising therapeutic target for MCL. MCL is a distinct B-cell non-Hodgkin lymphoma characterized by the chromosomal translocation t(11;14) that leads to overexpression of cyclin D1. The disease most often presents at an advanced stage, and while initial responses occur, nearly all cases relapse or become resistant to frontline therapy and progress with poor prognosis. Novel targeted therapies are currently entering the clinic in combination with standard of care treatments, but heterogeneous response rates and durations of response show that there remains an unmet medical need in this population. The most widely used MCL treatment regimens include combinations of two or more Standard of Care (SOC) agents. In order to support potential future clinical investigations with tazemetostat we established in vitro assays to assess its combinatorial activity with 24 traditional and emerging therapies across seven MCL cell lines. All compounds were evaluated for their single agent activity in order to determine the appropriate starting doses to investigate synergy. We then looked at both a seven-day pretreatment with tazemetostat alone followed by a four-day cotreatment with combination partner as well as a seven-day cotreatment including both drugs simultaneously. Synergy status was determined via Loewe, BLISS and additional mathematical models. Robust synergy was observed with glucocorticoid receptor agonists, immunomodulatory drugs, venetoclax and a variety of B-cell receptor pathway modulators in both dosing regimens. In addition, we show that EPZ011989 (an EZH2 tool compound) induces tumor growth inhibition in a MCL xenograft model, and has combination benefit with ibrutinib. Preclinical data suggest that the sensitivity to tazemetostat as a single agent in MCL cell lines can be greatly enhanced in combination with new and emerging therapies. These combination approaches may be worthy of exploration in the clinic for the treatment of MCL. Citation Format: Tami L. Hood, Kat Cosmopoulos, Allison Drew, Kelli Armstrong, Jennifer Johnson, Smith Jesse, Alejandra Raimondi. Opportunity for therapeutic expansion in mantle cell lymphoma: Tazemetostat combination synergy status in preclinical MCL models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 808.

The NEDD8-activating enzyme (NAE) is an E1 involved in the activation of a large family of ubiquitin E3 ligases termed the cullin-RING ligases (CRLs) through conjugation of the cullin proteins with the ubiquitin-like modifier NEDD8. Polyubiquitination of CRL substrate proteins targets them for degradation by the proteasome. In this way, NAE regulates the stability of proteins required for cancer cell growth and survival. MLN4924 is an investigational small molecule that is a potent and selective inhibitor of NAE in Phase I clinical trials. In order to investigate the primary genetic determinants that confer sensitivity of cells to NAE inhibition, we have performed a genome-wide synthetic lethal RNAi screen using MLN4924 in the A375 melanoma cell line. We have also investigated the biological consequences of NAE inhibition by studying the regulation of protein and transcript levels in MLN4924-treated A375 cells using large-scale quantitative proteomics and gene expression profiling, respectively. The RNAi screen has identified 123 genes whose down-regulation modulates MLN4924-induced cell death, and approximately one-third of these interfere with components of the NEDD8 pathway itself, the cell cycle and apoptotic machinery, and DNA damage-response pathways. Of these genes, 99 were subsequently assessed using high throughput FACS analysis for their contribution to the major phenotype induced by MLN4924. The results emphasize replication, p53, BRCA1/BRCA2, and transcription-coupled repair as being particularly important for MLN4924-induced cell death. In addition, the interactions of 80 genes with roles in cell cycle and DNA damage repair were explored with proteasome inhibitors and 12 DNA damaging agents, demonstrating that MLN4924 induces DNA damage by a distinct mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3104. doi:1538-7445.AM2012-3104

Background: EZH2 is the functional histone methyltransferase component of the multiprotein complex known as Polycomb Repressive Complex 2 (PRC2). PRC2 catalyzes mono-, di- and tri-methylation of H3K27. Activating mutations in EZH2 produce altered substrate specificity, and mutant EZH2 acts in concert with wild-type EZH2 to produce increased H3K27 tri-methylation, abnormal repression of PRC2 targets, and tumorigenesis. Rhabdoid tumors are associated with loss of SMARCB1, a component of the SWI/SNF chromatin remodeling complex. Tumorigenesis in SMARCB1 deficient mice is blocked by inactivation of EZH2 (Cancer Cell 2010;18:316-28), and an EZH2 inhibitor induced regression in a rhabdoid tumor xenograft (PNAS 2013;110:7922-7). EPZ-6438 is a potent, specific and SAM-competitive EZH2 inhibitor that demonstrates favorable pharmacological properties and in vivo activity in multiple xenograft tumor models following oral dosing. It has entered clinical evaluation, and objective responses were noted in patients with NHL and rhabdoid tumor with twice daily oral dosing during phase 1 dose escalation studies. The aim of this preclinical study was to assess the antitumor activity of EPZ-6438 against pediatric solid tumor models with a focus on rhabdoid tumors of the kidney and CNS. Procedures: EPZ-6438 was evaluated using a dose of 400 mg/kg (mono-HBr salt, 350 mg/kg of active compound) administered twice-daily by oral gavage for 28 days. Standard PPTP measures of in vivo antitumor activity were utilized. Results: EPZ-6438 induced significant differences in event-free survival (EFS) distribution compared to control in 9 of 30 (30%) xenografts studied, with significant differences observed in 5 of 7 (71%) rhabdoid tumor xenograft lines compared to 4 of 23 (17%) non-rhabdoid lines (χ2 test p = 0.006). For the EFS T/C activity measure, intermediate activity requires an EFS T/C value > 2.0, and high activity additionally requires a reduction in final tumor volume compared to starting tumor volume. EPZ-6438 demonstrated intermediate or high EFS T/C activity in 2 of 26 (8%) and 1 of 26 (4%) xenografts evaluable for this measure, respectively. Intermediate/high activity for the EFS T/C metric was observed exclusively among rhabdoid tumor xenografts (3 of 5 rhabdoid tumor lines versus 0 of 21 non-rhabdoid tumor lines, χ2 test p<0.001). For the objective response metric, 1 of 7 rhabdoid tumor xenografts (G401) showed stable disease. PD2 (Progressive Disease 2) response indicates progressive disease with growth delay (EFS T/C >1.5) and was observed in an additional 5 of 7 rhabdoid tumor xenografts, but in only 3 of 23 non-rhabdoid tumor xenografts. For two rhabdoid tumors (G401 and KT-16), delayed reductions in tumor size to EPZ-6438 were noted following 1-2 weeks of tumor growth. Pharmacodynamic evaluation of H3K27 methylation status in treated and control tumors is ongoing. Conclusions: EPZ-6438 showed antitumor activity against rhabdoid tumor models, but showed no consistent activity against any other histology. The pattern of delayed tumor response observed for two rhabdoid tumor xenografts will need to be considered when developing clinical trials for EPZ-6438. Further preclinical testing using rhabdoid tumor lines and evaluating EPZ-6438 in combination with other anticancer agents may provide guidance for its pediatric clinical development. Citation Format: Raushan Kurmasheva, Kat Cosmopoulos, Melissa Sammons, Edward Favours, Jianwrong Wu, Peter Houghton, Malcolm Smith. Initial testing (Stage 1) of EPZ-6438 (tazemetostat), a novel EZH2 inhibitor, by the pediatric preclinical testing program (PPTP). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A137.