Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.
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.
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.
PRMT5 is responsible for symmetric dimethylation of arginine residues in glycine and arginine rich (GAR) motifs on a variety of cytosolic and nuclear proteins including histones, spliceosome components, regulators of translation, transcription factors, kinases and others. PRMT5 driven methylation of some of these proteins has been implicated in tumorigenesis. For example, PRMT5 deposits repressive marks on histones and silences a subset of tumor suppressor genes, such as RB and ST7. PRMT5 methylation of non-histone substrates (such as E2F1 and p53) also contributes to cancer cell growth and death. PRMT5 driven methylation of spliceosome subunits and components of translational machinery has been well described but its connection to PRMT5's role in cancer has not been established. We have identified first-in-class small molecules that are highly potent, selective, reversible inhibitors of PRMT5. Cellular mechanistic studies revealed that PRMT5 inhibition decreases symmetric arginine dimethylation on a variety of cellular proteins including spliceosome components, histones and transcription factors. PRMT5 inhibition leads to gene expression and splicing changes ultimately resulting in the induction of p53 in lymphoma cell lines. In addition to impacting the p53 pathway, PRMT5 inhibition leads to attenuation of the expression of cell cycle related genes, genes involved in ribosome and spliceosome homeostasis, as well as genes important for cellular metabolism. PRMT5 inhibitor attenuates proliferation and induces cell death in a subset of mantle cell and diffuse large B-cell lymphoma cell lines and inhibits tumor growth in xenograft models of mantle cell lymphoma. These data underline the potential of PRMT5 inhibitors as a therapeutic strategy in mantle cell and diffuse large B-cell lymphoma. Citation Format: Olena Barbash, Sarah Gerhart, David Soong, Christine Thompson, Rocio Montes de Oca, Ping Zhang, Charles McHugh, Kristy Kuplast, Christina Majer, Richard Chesworth, Jesse Smith, Robert Copeland, Elayne Penebre, Kenneth Duncan, Neil Johnson, Chris Carpenter, Ryan Kruger. Protein arginine methyltransferase 5 (PRMT5) inhibition as a therapeutic strategy in B-cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-248. doi:10.1158/1538-7445.AM2015-LB-248
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