The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased Bcell maturation and a greater dependence on B-cell activation signaling.

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.

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

Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Current available treatments include chemical castration and therapies targeting androgen receptor (AR) signaling pathways. Although initial responses are observed, 30% of patients have primary resistance to both forms of treatment and the majority of patients progress from androgen-dependent PCa (ADPC) to castration-resistant PCa (CRPC). Studies on mechanisms of resistance suggest that AR signaling remains a key driver in PCa even after treatment with AR-targeted therapies. Co-treatment with therapies that modulate alternative signaling pathways involved in oncogenesis in PCa including epigenetic modifiers, have been proposed as a modality to overcome resistance. Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of the polycomb repressive complex 2 and is one of the most highly upregulated genes in CRPC compared to localized PCa. Several reports suggest that EZH2 plays an oncogenic role both in ADPC and CRPC.Tazemetostat is a potent, selective, orally bioavailable, investigative first-in-class small molecule inhibitor of EZH2. Phase 2 clinical trials have demonstrated objective clinical responses in patients with B-cell lymphomas and molecularly defined solid tumors, including epithelioid sarcoma and other INI1-negative tumors. Given the implication of EZH2 in PCa, we explored the combination potential of tazemetostat with current second-line therapies, the androgen signaling inhibitors (ASIs) abiraterone and enzalutamide. Tazemetostat displayed both time- and dose-dependent antiproliferative activity in PCa cell lines dependent on AR signaling or those of the neuroendocrine subtype with IC50 values at or below 1 μM in 14-day assays. Synergistic activity was observed in vitro in a subset of cell lines when tazemetostast was combined with ASIs in a 7-day co-treatment model and this activity was further enhanced when cells were pretreated with tazemetostat prior to the cotreatment. To further validate these findings, we conducted in vivo studies in PCa cell line-derived xenograft models. Tazemetostat and enzalutamide showed single agent antitumor activity in a subcutaneous LNCaP xenograft mouse model. Co-administration of enzalutamide and tazemetostat markedly reduced tumor growth rate compared to either of the single agent(s). Similar results were observed with the combination of tazemetostat and abiraterone. Interestingly in the 22Rv1-derived in vivo xenograft model, a cell line that expresses AR-V7, a splice variant implicated in resistance to ASIs in CRPC, modest tumor growth inhibition was observed with tazemetostat and no significant antitumor activity was observed when given in combination with abiraterone or enzalutamide. A significant decrease in intratumoral H3K27me3 levels were observed in all treatment groups containing tazemetostat in both in vivo models, indicating an on-target activity of EZH2 inhibition. Current efforts are underway to investigate the mechanism of action and cellular inhibitory pathways for these combinations. In summary, our preclinical data demonstrates the importance of EZH2 inhibition in CRPC and provides a rationale for combination of tazemetostat with abiraterone and enzalutamide in the clinic. Citation Format: Vinny Motwani, Dorothy Brach, Chloe Pantano, Vania Estanek, Jeffrey A. Keats, Daniel T. Dransfield, Alejandra Raimondi. Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B011. doi:10.1158/1535-7163.TARG-19-B011

The EZH2 inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase 2 clinical trials for the treatment of non-Hodgkin lymphoma (NHL). EZH2 inhibitors have shown anti-proliferative effects in multiple preclinical models of NHL, mesothelioma, and molecularly defined solid tumors. Objective clinical responses have been reported in patients with B-cell lymphomas in phase 1 and in preliminary analysis of phase 2 studies of tazemetostat. Mounting evidence suggests that EZH2 regulates the maturation of both normal B-cells and B-cell lymphomas and may be an important mediator of cell fate in other B-cell malignancies. Consistent with this hypothesis, recent preclinical studies suggest that EZH2 may be a therapeutic target for treatment of multiple myeloma (MM), a disease arising from late stage B-cell lymphocyte plasmablasts. Inhibition of EZH2 has shown potent anti-proliferative effects in both in vitro and in vivo preclinical models of MM. Here, we show that synergistic anti-proliferative activity was observed when EZH2 inhibitors were combined with small molecule immune system modulators in both in vitro and in vivo multiple myeloma models and describe the mechanism of action of this enhanced effect. In particular, combination with IMiDs led to dramatic synergy across multiple MM cell lines. Like EZH2, Ikaros/Aiolos, the two important targets of IMiD response, are repressors of transcription. This led us to investigate the effects of the combination of tazemetostat and IMiDs on gene expression. Previous reports have indicated that both EZH2 and Ikaros/Aiolos play important roles in the regulation of interferon signaling via the IRF4 axis. We found that both agents singly elicited changes in interferon target genes, a phenomenon that could be enhanced when the two drugs were added in combination. Combination treatment also led to an increase in apoptosis as would be expected following downregulation of the IRF4 axis which is critical for MM cell survival. In this study, we provide evidence that EZH2 and Ikaros/Aiolos share a set of target genes, including IRF4, suggesting that these overlapping pathways may, in part, mediate the synergy between EZH2 inhibitors and IMiDs on cell viability. Based upon these in vitro findings, we conducted a xenograft study using the RPMI-8226 myeloma model and observed a notable enhancement in tumor growth inhibition with the combination EZH2 inhibitor and pomalidomide compared to the effect of either single agent. These data provide evidence of synergy between pomalidomide and EZH2 inhibitors in both in vitro and in vivo preclinical models, and provide a rationale for clinical development of this combination strategy for patients with multiple myeloma. Citation Format: Allison E. Drew, Vinny Motwani, Lindsey Eichinger, Jesse Smith, Alejandra Raimondi. Mechanism of action of synergistic activity of EZH2 inhibition and IMiDs in preclinical multiple myeloma 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 807.