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SETD2, a lysine

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 B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR.

Table S1: Conditions and seeding densities of all cell lines used in studies; Table S2. Single agent IC50 values for agents tested for 3 days in 96-well plates as described in Materials and Methods; Figure S1. Tazemetostat dosing reduced H3K27me3 in tumor xenografts and is tolerated by SCID mice; Figure S2: CD40L has little effect on growth of DLBCL cell lines; Figure S3. SU-DHL-5 cells were treated for 4 days with TAZ (0.1 to 1 µM) followed by addition of 500 ng/mL CD40L at the indicated time points (1-60 minutes; Figure S4. An ABC-DLBCL gene signature (1) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and ABC vs. GCB gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment.; Figure S5: CD40L, but not IL-21 induces PRDM1 in combination with tazemetostat (TAZ); Figure S6. A CD40 responsive gene set (2) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and Basso_CD40¬_SIGNALING_UP gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment; Figure S7. Model for tazametostat mechanism of action in DLBCL

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 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 B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2) is an ATP-dependent DNA helicase and a catalytic component of the SWI/SNF complex. SWI/SNF-mediated nucleosome remodeling is a critical regulator of chromatin accessibility resulting in transcriptional regulation of gene sets that determine and maintain cell state. SMARCA2's role as the catalytic driver of SWI/SNF activity is mutually exclusive with that of its close paralog, SMARCA4. Here, we show that the synthetic lethal relationship between SMARCA2 and SMARCA4 can be genetically engineered bi-directionally in SMARCA2/SMARCA4 wild type (WT) cell lines. While knockout (KO) of either protein individually minimally affects cell growth, a genetically engineered cell line with loss of either SMARCA2 or SMARCA4 becomes sensitive to loss of its paralog. While SMARCA2 is infrequently lost through genetic mutation, SMARCA4 mutations occur in a wide variety of cancer types. We present CRISPR-Cas9 pooled screening data showing that cell lines with SMARCA4 mutations have a selective sensitivity to loss of SMARCA2 across multiple cancer indications, including NSCLC and ovarian carcinoma. SMARCA2 and SMARCA4, though partially redundant, also play unique roles within the cell as is evidenced by the unique set of transcriptional changes that occur in the SMARCA2/4 isogeneic cell lines, including the differential modulation of genes involved in TGFB signaling, regulation of angiogenesis, and in determinants of epithelial or mesenchymal cell state. ChIP-seq studies show changes in SWI/SNF occupancy as well as in the chromatin landscape that are specific to the loss of either SMARCA2 or SMARCA4. These isogenic cell lines also respond differently to treatment with a novel SMARCA2/4 inhibitor, Compound 1, in transcriptional as well as functional effects, providing evidence that dual inhibition with a small molecule may not be equivalent to the complete genetic ablation of these two proteins The NCI-H358 NCSLC cell line expresses WT SMARCA2 and SMARCA4 and is weakly sensitive to treatment with Compound 1. CRISPR-Cas9 mediated KO of SMARCA4 confers several-fold greater sensitivity to treatment with the novel inhibitor compared to the parental cell line. Surprisingly, KO of SMARCA2 does not confer sensitivity to inhibitor treatment. This selectivity sensitivity to treatment with Compound 1 that we observe in the SMARCA4-KO cell line is also observed across a panel of SMARCA4-mutant NSCLC. The relationship between the transcriptional and phenotypic changes that occur in these isogenic knockout lines as well as in SMARCA4-mutated NSCLC cell lines in response to SMARCA2/4 inhibitor treatment have revealed novel mechanisms driven by specific SWI/SNF complexes. These data have allowed us to better understand the potential therapeutic utility of a SMARCA2/4 inhibitor in both a SMARCA4-mut and SMARCA4-wt NSCLC patient population. Citation Format: Lindsey W. Eichinger, Chloe Pantano, Vinny Motwani, Dorothy Brach, Selene Howe, Alejandra Raimondi, Daniel T. Dransfield, Kenneth W. Duncan, Kim Stickland, Cuyue Tang, Neil A. Farrow, John Lampe, Suzanne L. Jacques, Allison E. Drew. Aberrant SWI/SNF complexes lacking SMARCA2 or SMARCA4 differentially affect cell state and response to a novel SMARCA2/4 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2924.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2) is an ATP-dependent DNA helicase and a catalytic component of the SWI/SNF complex. SWI/SNF-mediated nucleosome remodeling is a critical regulator of chromatin accessibility resulting in transcriptional regulation of gene sets that determine and maintain cell state. The role of SMARCA2 as the catalytic driver of SWI/SNF activity is mutually exclusive with that of its close paralog, SMARCA4. Loss of functional SMARCA4, as is reported in a subset of NSCLC and other cancer types generally confers a dependency on SMARCA2. Cancers lacking functional SMARCA4 have been shown to be sensitive to loss of SMARCA2 function either through genetic ablation or the use of a small molecule SMARCA2/4 inhibitor. We describe the identification and characterization of a small molecule SMARCA2/4 inhibitor Compound 1 and its selective activity in the SMARCA4-mutant setting both in in vitro and in vivo model systems. The compound demonstrates inhibition of SMARCA2 and SMARCA4 enzymatic activity in biochemical assays and broad selectivity against other helicases. Selective sensitivity in anti-proliferative assays of 15-fold was observed in NSCLC cell lines with SMARCA4 protein loss compared to those harboring WT SMARCA4 protein. Oral dosing demonstrates dose-dependent in vivo SMARCA2 inhibition and anti-tumor activity in SMARCA4-mutant NSCLC xenograft models. This compound is suitable for further exploration of the role of SMARCA2/4 and SWI/SNF in vitro and in vivo in cancer and other indications. Citation Format: Allison E. Drew, Suzanne L. Jacques, Lindsey W. Eichinger, Chloe Pantano, Vinny Motwani, Cuyue Tang, Cuyue Tang, Neil Farrow, Dorothy Brach, Selene Howe, Alejandra Raimondi, Daniel T. Dransfield, Kenneth W. Duncan, Kim Stickland, Liyue Huang, John Lampe. Identification of a potent, orally-available SMARCA2/4 inhibitor with in vitro and in vivo activity in preclinical models of SMARCA4-mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1768.

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.

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

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.

The histone methyltransferase EZH2 is the enzymatic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of H3K27 thereby repressing target gene transcription. EZH2 is amplified, overexpressed, or mutated in multiple cancer types, most notably Follicular Lymphoma (FL) and germinal center Diffuse Large B-cell Lymphoma (GCB-DLBCL). We previously reported that preclinical models of malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SNF5, SMARCB1), are selectively killed by potent and selective inhibitors of EZH2. Here we report another class of SWI/SNF-altered cancers named small cell carcinoma of the ovary hypercalcemic type (SCCOHT) that is dependent on EZH2 activity. SCCOHT is a very aggressive form of cancer that responds poorly to conventional therapy with a one-year overall survival rate of only 50%. Very few novel agents have been approved for this indication; thus there is a need for targeted therapeutics in SCCOHT. SMARCA4 and SMARCA2 are co-inactivated in this tumor type that has many rhabdoid features. We demonstrate that tazemetostat, an EZH2 inhibitor currently in phase 2 clinical trials, induces potent and selective killing in SMARCA2 and SMARCA4-deficient ovarian cell lines. In addition to small molecule inhibitor data, we conducted functional genomics studies with CRISPR pooled screening, and confirmed that SCCOHT is also sensitive to CRISPR-mediated EZH2 gene ablation. Dose-dependent anti-tumor effects were observed upon tazemetostat treatment in SCCOHT xenografts deficient in both SMARCA2 and SMARCA4. We also report on additional non-ovarian tumor types with dual SMARCA2/SMARCA4 loss including NSCLC that exhibit EZH2 dependence representing additional potential therapeutic indications for tazemetostat treatment. Citation Format: Elayne Chan-Penebre, Kelli Armstrong, Allison Drew, Alexandra R. Grassian, Igor Feldman, Maria Roche, Peter Ho, Dorothy Brach, Alejandra Raimondi, Robert A. Copeland, Richard Chesworth, Jesse J. Smith, Scott A. Ribich. Selective killing of SMARCA2- and SMARCA4-deficient tumors by inhibition of EZH2: In vitro and in vivo 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 3345. doi:10.1158/1538-7445.AM2017-3345

The EZH2 inhibitor tazemetostat (EPZ-6438) is emerging as a promising therapeutic agent for the treatment of non-Hodgkin's Lymphoma (NHL). A significant body of work has now demonstrated in vitro and in vivo effects of EZH2 inhibition in preclinical models of lymphoma, in addition to objective clinical responses in early human trials. While EZH2 gain-of-function mutations clearly contribute to lymphomagenesis, patients with lymphomas harboring wild-type EZH2 also show responses to tazemetostat. This suggests a broad role of EZH2 in B-cell oncogenesis. Several recent mouse model studies have demonstrated the importance of wild-type EZH2 catalytic activity in the formation of germinal centers in non-diseased lymph nodes, suggesting a central role for EZH2 in B-lymphocyte maturation. These findings indicate that the importance of EZH2 to B-cell lymphoma likely lies in its ability to regulate B-cell differentiation. To understand the relationship between B-cell maturation and sensitivity to EZH2 inhibition, we evaluated changes in maturation markers and cellular proliferation following treatment of diffuse large B-cell lymphoma (DLBCL) cell lines with tazemetostat in combination with modulators of B-cell activation. Consistent with the importance of EZH2 in the regulation of B-cell differentiation, we observed increased expression of B-cell maturation markers in DLBCL cell lines treated with single agent tazemetostat in vitro. Furthermore, we demonstrate that tazemetostat pre-treatment of subsets of DLBCL cells lines (both EZH2 mutant and wild-type) can sensitize cells to inhibitors of B-cell activation pathways, which include glucocorticoids and BTK, MAPK and PI3K pathway inhibitors. Moreover, the anti-proliferative activity of single agent tazemetostat can be diminished or delayed by co-treatment with biological stimulators of B-cell activation including B-cell receptor ligation, CD40L, LPS and BAFF. Importantly, B-cell receptor ligation and co-stimulation agents have little proliferative effects on DLBCL cell lines on their own, suggesting that the protective function of these agents is directly related to the effects of EZH2 inhibition and not a generic stimulation of proliferation. Our findings suggest that EZH2 inhibition initiates a differentiation program that enables lymphoma cells to proceed through the normal processes of B-cell selection, growth regulation and maturation. Citation Format: Danielle Johnston, Dorothy Brach, Christopher Plescia, Alison Drew, Trupti Lingaraj, Natalie Warholic, Jesse J. Smith, Robert A. Copeland, Heike Keilhack, Elayne Penebre, Sarah K. Knutson, Scott Ribich, Michael J. Thomenius, Alejandra Raimondi. EZH2 plays a critical role in B-cell maturation and in non-Hodgkin's lymphoma: Interplay between EZH2 function and B-cell activation. [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 B85.