Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone – a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.
EPZ-5676 [(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo [d]imidazol-2-yl)ethyl)cyclobutyl) (isopropyl)amino)methyl)tetrahydrofuran-3,4-diol], a small-molecule inhibitor of the protein methyltransferase DOT1L, is currently under clinical investigation for acute leukemias bearing MLLrearrangements (MLL-r). In this study, we evaluated EPZ-5676 in combination with standard of care (SOC) agents for acute leukemias as well as other chromatin-modifying drugs in cellular assays with three human acute leukemia cell lines: MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), and SKM-1 (non-MLL-r). Studies were performed to evaluate the antiproliferative effects of EPZ-5676 combinations in a cotreatment model in which the second agent was added simultaneously with EPZ-5676 at the beginning of the assay, or in a pretreatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. EPZ-5676 was found to act synergistically with the acute myeloid leukemia (AML) SOC agents cytarabine or daunorubicin in MOLM-13 and MV4-11 MLL-r cell lines. EPZ-5676 is selective for MLL-r cell lines as demonstrated by its lack of effect either alone or in combination in the nonrearranged SKM-1 cell line. In MLL-r cells, the combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the chemotherapeutic agents, suggesting that EPZ-5676 sets up a durable, altered chromatin state that enhances the chemotherapeutic effects. Our evaluation of EPZ-5676 in conjunction with other chromatinmodifying drugs also revealed a consistent combination benefit, including synergy with DNA hypomethylating agents. These results indicate that EPZ-5676 is highly efficacious as a single agent and synergistically acts with other chemotherapeutics, including AML SOC drugs and DNA hypomethylating agents in MLL-r cells.
Elongation factor P (EF-P) is a highly conserved ribosomal initiation factor responsible for stimulating formation of the first peptide bond. Its essentiality has been debated and may differ depending on the organism. Here, we demonstrate that EF-P is dispensable in Escherichia coli and Pseudomonas aeruginosa under laboratory growth conditions. Although knockouts are viable, growth rates are diminished compared with wild-type strains. Despite this cost in fitness, these mutants are not more susceptible to a wide range of antibiotics; including ribosome targeting antibiotics, such as lincomycin, chloramphenicol, and streptomycin, which have been shown previously to disrupt EF-P function in vitro. In Pseudomonas, knockout of efp leads to an upregulation of mexX, a phenotype previously observed with other genetic lesions affecting ribosome function and that can be induced by the treatment with antibiotics affecting protein synthesis.
Preclinical data have suggested that small molecule inhibitors for the histone methyltransferase EZH2 represent potential new treatment modalities for Non-Hodgkin lymphomas (NHL) expressing EZH2 change of function mutations. Our group has previously reported that selective inhibition of EZH2 results in specific killing of lymphoma cells bearing EZH2 mutations in vitro and in vivo, with minimal effects on non-mutant lymphoma cells [Knutson et al. Nature Chemical Biology 2012; Keilhack et al. Blood (ASH Annual Meeting Abstracts) 2012, 120, Abstract 3712]. Since epigenetic changes have been suggested to be involved in resistance of cancer cells to many anticancer agents, we studied EPZ-6438 (or E7438), our clinical stage EZH2 inhibitor, in combination with standard of care agents for NHL, second line therapies or targeted therapies that are being explored in this indication. With continuous exposure to EPZ-6438, cell-based assays of two different EZH2 mutant cell lines demonstrated combination benefits with all components of the CHOP chemotherapy regime, second line therapies but also with several targeted therapies (for instance other epigenetic drugs, PI3K pathway or other inhibitors). These effects were not observed in an EZH2 wild type lymphoma cell line of the activated B cell type. Strong combination benefit with CHOP was also observed in two different EZH2 mutant xenograft models. For instance, in the SUDHL6 Y646N xenograft model neither EPZ-6438 nor CHOP chemotherapy alone induced a significant antitumor effect, yet their combination produced durable tumor regressions even after cessation of dosing (figure 1). Importantly, this effect was preserved when doxorubicin was omitted from the CHOP chemotherapy regime in a third study with another EZH2 mutant xenograft model. Subsequently we showed that glucocorticoid receptor agonism may be a key mechanism of the combination benefit observed with CHOP, as the antiproliferative effect of EPZ-6438 was enhanced by either prednisolone or dexamethasone alone, in several EZH2 mutant lymphoma cell lines (in vitro). Taken together these data suggest that the single agent activity of EPZ-6438 in EZH2 mutant NHL may be further enhanced and expanded through rational combination strategies. Disclosures: Johnston: Epizyme: Employment, Equity Ownership, stock options Other. Knutson:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Warholic:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Klaus:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Wigle:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Iwanowicz:Epizyme, Inc.: Employment, Equity Ownership, stock options Other. Littlefield:Eisai Inc.: Employment. Porter Scott:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Smith:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Moyer:Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Copeland:Epizyme Inc. : Employment, Equity Ownership, Patents & Royalties, stock options Other; Mersana: Membership on an entity’s Board of Directors or advisory committees. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Kuntz:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Keilhack:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Raimondi:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, stock options Other.
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