John E. Campbell

Loss of BAP1 function leads to EZH2-dependent transformation

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8—the H4K20me1 methyltransferase—reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

The Importance of Being Me: Magic Methyls, Methyltransferase Inhibitors, and the Discovery of Tazemetostat

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.

A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.

Abstract 5060: Activity of the EZH2 inhibitor tazemetostat as a monotherapy and in combination with multiple myeloma therapies in preclinical models

The EZH2 inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase 2 clinical trials for the treatment of non-Hodgkin’s Lymphoma (NHL). EZH2 inhibitors have shown anti-proliferative effects in multiple preclinical models of NHL and objective clinical responses have been reported in patients with B-cell lymphomas in phase 1 and phase 2 studies of tazemetostat. Mounting evidence suggests that EZH2 is an important regulator of B cell differentiation, both in normal B-cells and in B-cell lymphoma, and may be an important mediator of cell fate in B-cell malignancies in the clinic. Consistent with its essential role in regulating B cell differentiation, recent studies have also shown a dependence on EZH2 activity in multiple myeloma (MM), a disease arising from terminally-differentiated B-cell lymphocyte plasmablasts. Frequent genetic alterations of epigenetic modulators are observed in MM, pointing towards an important role in the initiation and maintenance of this disease. Dysregulation of the H3K27 methyltransferase EZH2, its corresponding histone demethylase UTX and the H3K36 methyltransferase WHSC1 in MM suggest that disruption of the balance of histone methylation may be fundamental to MM pathogenesis in a subset of cases. Indeed, inhibition of EZH2 alone has shown potent anti-proliferative effects both in in vitro and in vivo preclinical models of MM. Here, we describe the effects of small molecule EZH2 inhibitors as monotherapy and in combination with standard of care agents in preclinical models of MM. Tazemetostat selectively inhibits intracellular H3K27 methylation in MM cell lines and elicits a robust anti-proliferative effect in 14-day assays. Following demonstration of single agent activity, we then investigated potential for combinatorial activity of tazemetostat with first and second line therapies for multiple myeloma as well as other non-approved but emerging therapies. Synergistic anti-proliferative activity was observed when tazemetostat was combined with glucocorticoid receptor agonists (dexamethasone, prednisolone), small molecule immune system modulators (lenalidomide, pomalidomide) and proteasome inhibitors (bortezomib, ixazomib) when cells were primed with tazemetostat for seven days prior to the addition of the standard of care drugs. Combination activity was also observed with an alternate treatment schedule where cells were co-treated with tazemetostat along with the combination partner for seven days. Studies with selected therapeutic modalities were expanded into in vivo xenograft models to further evaluate monotherapy and combination activity of EZH2 inhibitors in MM. Citation Format: Allison E. Drew, Vinny Motwani, John E. Campbell, Cuyue Tang, Jesse J. Smith, Richard Chesworth, Robert A. Copeland, Alejandra Raimondi, Scott Ribich. Activity of the EZH2 inhibitor tazemetostat as a monotherapy and in combination with multiple myeloma therapies in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5060. doi:10.1158/1538-7445.AM2017-5060