Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2

Abstract

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.

Keywords: EZH2 inhibitor; epigenetic cancer therapy.

Fig. 1.

Effects of EPZ-6438 on cellular global histone methylation and cell viability. (A) Chemical structure of EPZ-6438. (B) Concentration-dependent inhibition of cellular H3K27Me3 levels in G401 and RD cells. (C) Selective inhibition of proliferation of SMARCB1-deleted G401 cells by EPZ-6438 in vitro (measured by ATP content). G401 and RD cells were replated at the original seeding densities on day 7. Each point represents the mean for each concentration (n = 3). (D) Proliferation IC₅₀ values (day 14) for SMARCB1 wild-type and mutant cells. Compound incubations for each experiment were performed in triplicate, and symbols represent the mean of two experiments for all cell lines. The horizontal lines represent the median. Mean calculation of duplicate experiments was not possible for RD and SJCRH30 cells; individual values are shown (Table S2).

Fig. 2.

EPZ-6438 induces G₁ arrest and apoptosis in SMARCB1-deleted MRT cells. Cell cycle analysis (by flow cytometry) and determination of apoptosis (by TUNEL assay) in RD (A) or G401 cells (B) during incubation with either vehicle or 1 µM EPZ-6438 for up to 14 d. G₁ arrest was observed as of day 7, and apoptosis was induced as of day 11. Data are represented as mean values ± SEM (n = 2). The DMSO control values shown are the average ± SEM from each time point. Cells were split and replated on days 4, 7, and 11 at the original seeding density.

Fig. 3.

EPZ-6438 induces changes in expression of SMARCB1-regulated genes and cell morphology. (A) Basal expression of SMARCB1-regulated genes in G401 SMARCB1-deleted cells, relative to RD control cells [measured by quantitative PCR (qPCR); n = 2]. (B) G401 and RD cells were incubated with either DMSO or 1 µM EPZ-6438 for 2, 4, and 7 d. Gene expression was determined by qPCR (n = 2) and is expressed relative to the DMSO control of each time point. (C) G402 cells were incubated with either DMSO (Left) or 1 µM EPZ-6438 (Right) for 14 d. Cells were split and replated to the original seeding density on day 7.

Fig. 4.

EPZ-6438 eradicates SMARCB1-deleted MRT xenografts in SCID mice. (A) Tumor regressions induced by twice daily (BID) administration of EPZ-6438 for 28 d at the indicated doses. Compound administration was stopped on day 28, and tumors were allowed to regrow until they reached 2,000 mm³ (data shown as mean values ± SEM; n = 8). (B) EZH2 target inhibition in G401 xenograft tumor tissue collected from mice euthanized on day 21. Each point shows the ratio of H3K27Me3 to total H3, measured by ELISA. The horizontal lines represent group mean values; gray symbols are values outside of the ELISA standard curve. (C) Change in gene expression in G401 xenograft tumor tissue collected from mice treated with EPZ-6438 for 21 d. Data are presented as fold change compared with vehicle ± SEM (n = 6, n = 4 for 500 mg/kg group). *P < 0.05, **P < 0.01, ****P < 0.0001, vs. vehicle, Fisher’s exact test.