Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor

Abstract

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B-cell lymphoma cells harboring gain-of-function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL-AF9, MLL-AF4, and AML1-ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.

Keywords: Dual inhibitor; EZH1; EZH2; H3K27me3; histone methyltransferase.

Figure 1

Enhancer of zeste homologs 1/2 (EZH1/2) dual inhibitors suppress trimethylation of histone H3 lysine 27 (H3K27me3) in cells more highly than do EZH2 selective inhibitors. (a) Plot of IC ₅₀ values of 77 compounds against polycomb repressive complex 2 (PRC2)‐EZH2 (x‐axis) and PRC2‐EZH1 (y‐axis). Cell‐based H3K27me3 reduction activities in HCT116 colorectal cancer cells are plotted as colored dots. (b) Structures of EZH2 selective inhibitor, OR‐S0, and EZH1/2 dual inhibitors, (R)‐OR‐S1 and (R)‐OR‐S2.

Figure 2

OR‐S1 and OR‐S2 are S‐adenosylmethionine (SAM)‐competitive and highly selective enhancer of zeste homologs 1/2 (EZH1/2) dual inhibitors. (a) Saturation transfer difference nuclear magnetic resonance (STD‐NMR) SAM/compound competition experiments for polycomb repressive complex 2 (PRC2). Upper spectrum shows an expansion of the aromatic region of the STD spectrum obtained for PRC2‐EZH2 in the presence of SAM. Middle and lower spectra show competition of SAM with (R)‐OR‐S1 and (R)‐OR‐S2. (b) Model of the PRC2/(R)‐OR‐S1 complex superimposed on the PRC2/S‐adenosylhomocysteine (SAH) complex (PDB ID: 5hyn44). EED, EZH2, (R)‐OR‐S1, and SAH are colored in gray, light brown, cyan, and pink, respectively. From PRC2/SAH complex, only SAH is shown. (c) In vitro inhibitory activities of OR‐S1 and (R)‐OR‐S2 against 24 histone lysine, six histone arginine, and four DNA methyltransferases. (d) In vitro inhibitory activities of (R)‐OR‐S1 and (R)‐OR‐S2 at 1 μM against 253 kinases.

Figure 3

Enhancer of zeste homologs 1/2 (EZH1/2) dual inhibitors show greater antitumor activity than an EZH2 selective inhibitor. (a) Dose‐dependent in vitro growth inhibition of KARPAS‐422 cells by OR‐S0, (R)‐OR‐S1, and (R)‐OR‐S2. (b) Summary of activities of OR‐S0, (R)‐OR‐S1, and (R)‐OR‐S2. Cell‐free polycomb repressive complex 2 (PRC2)‐EZH1 and PRC2‐EZH2 inhibition activities, cell‐based trimethylation of histone H3 lysine 27 (H3K27me3) inhibition activities in HCT116 cells, and in vitro growth inhibition activities against KARPAS‐422 cells of OR‐S0, (R)‐OR‐S1, and (R)‐OR‐S2 are shown. (c) Summary of pharmacokinetic parameters of single‐dose OR‐S0 and (R)‐OR‐S1 in BALB/c mice. (d–g) Antitumor activities and H3K27me3 reduction activities of (d,f) OR‐S0 and (e,g) (R)‐OR‐S1 in a KARPAS‐422 xenograft model. Mean estimated tumor volumes ± standard errors (n = 5, d,e), and mean relative H3K27me3/H3 values ± standard errors (n = 3, f,g) are plotted on the graphs.

Figure 4

In vitro growth inhibition activities of (R)‐OR‐S2 against various cancer cell lines. Fifty percent growth inhibitory activities against (a) hematological cancer cell lines and (b) solid cancer cell lines, with the cellular gene expression profiles of enhancer of zeste homolog 1 (EZH1) and EZH2. Gene expression profiles of EZH1 and EZH2 were obtained from the Cancer Cell Line Encyclopedia (CCLE).60 Information of the mutations of SWItch/Sucrose Non‐Fermentable (SWI/SNF) subunits in each cell line was obtained from CCLE, the Catalogue Of Somatic Mutations In Cancer (COSMIC),61 and the cBioPortal for Cancer Genomics.62, 63 ALL, acute lymphoblastic leukemia; APL, acute promyelocytic leukemia; BCL, B‐cell lymphoma; BURKITT, Burkitt lymphoma; DLBCL, diffuse large B‐cell lymphoma; HL, Hodgkin's lymphoma; MCL, mantle cell lymphoma; MM, multiple myeloma; PTCL, peripheral T‐cell lymphoma.

Figure 5

Antitumor activities of OR‐S1 against solid cancer cell lines. Antitumor activities in a xenograft model of OR‐S1 against (a) rhabdoid tumor cell line, G401 and (b) gastric cancer cell line, NCI‐N87. Mean estimated tumor volumes ± standard errors are plotted on the graphs (n = 5).