Abstract: Protein Arginine Methyltransferase-5 (PRMT5) has been reported to play a role in multiple diverse cellular processes including tumorigenesis. Overexpression of PRMT5 has been demonstrated in cell lines and primary patient samples derived from lymphomas, particularly Mantle Cell Lymphoma (MCL). Furthermore, knockdown of PRMT5 expression inhibits the proliferation of MCL cell lines. The mechanisms behind the oncogenic potential of PRMT5 are unclear, but the protein has been postulated to regulate processes such as cell death, cell cycle progression, and RNA processing through the dimethylation of arginine residues within a variety of cytoplasmic and nuclear target proteins. Epizyme developed small molecule inhibitors of PRMT5 enzyme activity in order to understand the role of PRMT5-mediated arginine methylation in tumorigenesis and to develop PRMT5-targeted cancer therapeutics. Here, we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with anti-proliferative effects in both in vivo and in vitro models of MCL. A diverse compound library was screened for inhibitors of arginine methylation by purified recombinant PRMT5:MEP50 complex and multiple hits were identified. The inhibitors are SAM uncompetitive, peptide competitive and bind with the PRMT5:MEP50 complex in a unique binding mode not previously observed. Further optimization yielded YQ36286, an orally available inhibitor of PRMT5 with enzymatic activity in biochemical assays with an IC50 in the low nM range and broad selectivity against a panel of other histone methyltransferases. YQ36286 demonstrated potent cellular activity as measured by its ability to inhibit symmetric dimethylation of SmD3, a cytoplasmic PRMT5 substrate in a time- and concentration-dependent manner. Treatment of MCL cell lines with YQ36286 led to inhibition of SmD3 methylation and cell killing, with IC50s in the nM range. Oral dosing of YQ36286 demonstrated dose-dependent anti-tumor activity in multiple MCL xenograft models. In xenograft studies with the Z138 MCL cell line, near 95% tumor growth inhibition was observed after 21 days of dosing with a corresponding decrease in symmetrically dimethylated levels of PRMT5 substrates. In summary, we have developed the first potent and selective small molecule inhibitor of PRMT5 that has cellular activity and in vivo efficacy. MCL cells are dependent on PRMT5 activity for their survival as demonstrated with YQ36286. This small molecule represents a starting point for the development of PRMT5 inhibitors as potential cancer therapeutics. Disclosures Penebre: Company stock options: Equity Ownership; Epizyme Inc.: Employment; GSK Research Funding: Research Funding. Kuplast:GSK research funding: Research Funding; Company Stock options: Equity Ownership; Epizyme Inc.: Employment. Majer:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Johnston:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Rioux:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Munchhof:Epizyme Inc.: Employment; GSK research funding: Research Funding. Jin:Epizyme Inc.: Employment; GSK research funding: Research Funding; Company stock options: Equity Ownership. Boriak-Sjodin:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Wigle:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Jacques:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. West:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Lingaraj:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Stickland:GSK research funding: Research Funding; Company Stock options: Equity Ownership; Epizyme Inc.: Employment. Ribich:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Raimondi:Epizyme: Employment, Equity Ownership; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Porter-Scott:Company stock options: Equity Ownership; GSK research funding: Research Funding; Epizyme Inc.: Employment. Waters:Epizyme, Inc: Employment, Equity Ownership; GSK research funding: Research Funding. Pollock:Epizyme: Employment, Equity Ownership; GSK research funding: Research Funding. Smith:GSK research funding: Research Funding; Epizyme: Employment, Equity Ownership. Barbash:GlaxoSmithKline Pharmaceuticals: Employment. Kruger:GlaxoSmithKline Pharmaceuticals: Employment, Equity Ownership. Copeland:Mersana: Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Employment, Equity Ownership; Celgene, Inc: Research Funding; Eisai Inc: Research Funding; Glaxo Smith Kline, Inc: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; New Enterprise Associates: Ad hoc consultant, Ad hoc consultant Other. Moyer:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Chesworth:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding. Duncan:Epizyme Inc.: Employment; Company Stock options: Equity Ownership; GSK research funding: Research Funding.

Abstract: CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC 50  = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC 50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.

Abstract: Tazemetostat is a small molecule inhibitor of the histone methyltransferase EZH2 and is currently in phase 2 clinical trials including relapsed refractory Non-Hodgkin Lymphomas (RR-NHL) Diffuse Large B Cell Lymphoma and Follicular Lymphoma. In the phase 1 clinical trial RR-NHL patients demonstrated positive clinical activity with a favorable safety profile. Acquired mutations in the D1 domain (I109K, Y111D, Y111L) and the SET domain (Y661D) of EZH2 have recently been reported as a mechanism of resistance to non-tazemetostat small molecule EZH2 inhibition. Given the clinical activity observed in the phase 1 tazemetostat clinical trial and these reports of pre-clinical EZH2 inhibitor induced resistance we embarked on investigations of the potential of tazemetostat to induce resistance in NHL cell lines. An EZH2 Y641F mutant DLBCL cell line WSU-DLCL2 was exposed to 1 μM tazemetostat, a dose which is 100 fold greater than the naïve line's 11 day growth IC50. After 8 weeks, growth of the tazemetostat treated cells matched that of the control cells. Subsequent increases in dose of tazemetostat up to 10 μM did not yield any changes in growth rate of the treated cells. EZH2 wild-type PMBCL cell line U2940 was exposed to a step wise increase in tazemetostat concentration for 7 weeks and finally maintained at the naïve cell line 11 day proliferation IC50 of 10 μM, with minimal effects on cell growth. Tazemetostat resistant cell lines were screened for acquired EZH2 mutations using a full coding next generation sequencing assay, with a mean depth of 17,126 across all positions. Sequencing results showed the resistant U2940 had gained mutations in EZH2 similar to those previously identified, a heterozygous Y661N mutation and a low frequency mutation of Y111H, consistent with a subclonal mutation. These acquired mutations have been reported to interfere with the binding of EZH2 inhibitors, which supports the minimal reduction of H3K27me3 as measured by ELISA in the resistant U2940 after treatment with tazemetostat. In contrast, after induction of tazemetostat resistance WSU-DLCL2 retained equipotent sensitivity to reduction of H3K27me3 by ELISA. Correspondingly, sequencing of EZH2 in the resistant WSU-DLCL2 line did not identify any additional mutations. These findings suggest continued target engagement with tazemetostat in the resistant WSU-DLCL2, and that a novel bypass mechanism may be engaged to confer resistance. In an attempt to identify the mechanism of resistance in the WSU-DLCL2 line, whole exome sequencing and RNA sequencing has been performed. Detailed mutational, gene expression and pathway analysis will be performed on these data to investigate mechanisms of treatment emergent resistance to tazemetostat. Understanding these mechanisms may guide hypotheses for rational combinations and provide direction for future preclinical and potentially clinical studies. Citation Format: Carly T. Campbell, Jeff S. Jasper, Scott R. Daigle, Scott A. Ribich, Heike Keilhack, Jesse S. Smith, Peter T. Ho, Stephen J. Blakemore. Evidence of EZH2 dependent and independent mechanisms of tazemetostat treatment emergent resistance in models of diffuse large B cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 312.

Abstract: 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.

Abstract: 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

Abstract: The deamination of adenosine to inosine (known as A-to-I editing) is one of the most prevalent RNA modifications that occurs in metazoans and is mediated by the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes. The enzyme ADAR/ADAR1 catalyzes the majority of A-to-I editing where it has been demonstrated to effect coding sequence, miRNA function and silencing of Alu repetitive elements1. A critical function of ADAR1 is to edit double stranded RNA (dsRNA) structures that can activate the cytoplasmic nucleoside sensors MDA5 and PKR, preventing aberrant activation of an innate immune type I interferon (IFN) response2,3. Consistent with this hypothesis, mutations in ADAR1 and other enzymes involved in nucleoside metabolism and/or sensing are found in Aicardi-Goutiéres Syndrome (AGS), an interferonopathy associated with spontaneous interferon production4. Through analysis of pooled CRISPR and shRNA screening data, we have identified that cancer cell lines with elevated expression of specific type I interferon stimulated genes (ISGs) are dependent on ADAR1 for their survival. Tumor cells can display elevated intrinsic type I interferon signaling and associated dsRNA burden due to multiple factors, including chronic cytoplasmic DNA activation of STING, oncovirus infection and other proinflammatory signals. We have identified a core subset of 26 ISGs that predict a cellular dependency on ADAR1 and demonstrate elevated expression of these ISGs in a diverse range of tumor types including head and neck squamous cell carcinoma, breast cancer, esophageal cancer, and lung cancer. Utilizing an isogenic cell line with low basal type I interferon signaling and knockout of ADAR1, we demonstrate that we can induce cell death through exogenous treatment with the dsRNA mimetic poly-(I:C) or type I interferons (IFNα or IFNβ) but not the type II interferon IFNγ. We believe that chronic ISG expression and dsRNA burden creates a dependency on ADAR1 to prevent activation of MDA5 and PKR. These data suggest an ADAR1 inhibitor could be beneficial for the treatment of tumors with elevated ISG expression. Eisenberg et al., Nature Review Genetics (2018) Ahmad et al., Cell (2018) Chung et al., Cell (2018) Crow et al., Nature Review Immunology (2015) Citation Format: Alexandra K. Gardino, Cindy Collins, Maureen S. Lynes, P. Ann Boriack-Sjodin, Robert A. Copeland, Scott A. Ribich. Elevated cancer-intrinsic type I interferon signaling confers a dependency on the RNA editor ADAR1 [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 B130. doi:10.1158/1535-7163.TARG-19-B130

Abstract: The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.