SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2

Abstract

Human cancer genome sequencing has recently revealed that genes that encode subunits of SWI/SNF chromatin remodeling complexes are frequently mutated across a wide variety of cancers, and several subunits of the complex have been shown to have bona fide tumor suppressor activity. However, whether mutations in SWI/SNF subunits result in shared dependencies is unknown. Here we show that EZH2, a catalytic subunit of the polycomb repressive complex 2 (PRC2), is essential in all tested cancer cell lines and xenografts harboring mutations of the SWI/SNF subunits ARID1A, PBRM1, and SMARCA4, which are several of the most frequently mutated SWI/SNF subunits in human cancer, but that co-occurrence of a Ras pathway mutation is correlated with abrogation of this dependence. Notably, we demonstrate that SWI/SNF-mutant cancer cells are primarily dependent on a non-catalytic role of EZH2 in the stabilization of the PRC2 complex, and that they are only partially dependent on EZH2 histone methyltransferase activity. These results not only reveal a shared dependency of cancers with genetic alterations in SWI/SNF subunits, but also suggest that EZH2 enzymatic inhibitors now in clinical development may not fully suppress the oncogenic activity of EZH2.

Figure 1. SWI/SNF mutant cancer cells require EZH2

SWI/SNF mutant and wild–type cell lines were transduced with either control or EZH2–targeting shRNAs. Proliferation was assessed by MTT assay. (a) Proliferation curves of SWI/SNF wild–type cell lines (ES2, OCI–LY–19, and SKM–1) and mutant cancer cell lines including SMARCA4 mutant (A549, H1299, and SW13), ARID1A mutant (TOV21G, HEC59, and OVISE), and PBRM1 mutant (RCC4, A704, and SK–RC–20). Error bars indicate means ± s.d. (n = 3). (Ordinary one–way ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) (b) Colony formation assays. Cells transduced with either control or EZH2 shRNAs were seeded at low density in standard 6–well plates. Colonies were visualized by crystal violet staining. The assays are representative of replicates of three independent experiments. (c) Western blot analysis of HCT116 isogenic cells (ARID1A wild-type parental, +/+; ARID1A heterozygous. +/−; or ARID1A homozygous deficient, −/−) after inducing with control or EZH2 shRNA. Actin was used as a loading control. (d). MTT proliferation curves of ARID1A isogenic cell lines. Error bars indicate means ± s.d. (Ordinary one–way ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (e) Distribution of EZH2 shRNA dependency based on SWI/SNF mutational status. Each bar represents one cell line; cell lines with homozygous inactivating SWI/SNF mutations are indicated in blue. Lower DEMETER scores indicates more dependency on EZH2. The statistical difference of the EZH2 dependency between SWI/SNF mutant and wild–type cells was calculated using an unpaired, two–sample Welch’s t–test.

Figure 2. The catalytic activity is only partially responsible for EZH2 dependence

(a) Treatment of A549 and SW13 (SMARCA4/SMARCA2 mutant), TOV21G and HEC59 (ARID1A mutant), and A704 (PBRM1 mutant), with GSK126 for 7 d impaired colony formation whereas ES2 (SWI/SNF wild–type), RCC4 (PBRM1 mutant), H1299 (SMARCA4 mutant), and OVISE (ARID1A mutant) cells were relatively resistant. (b) GSK126–sensitive (TOV21G, G401) and resistant (RCC4) cell lines were treated with increasing doses of GSK126 for 7 d. Immunoblots show levels of H3K27 tri–methylation and total H3. Proliferation curves are for cells treated with GSK126 10 µM ± s.d. (n = 3). (c) Effects of EZH2 shRNA knockdown and rescue with either full–length EZH2 or catalytically–dead EZH2–ΔSET (n=3). (d–g) Immunoblot analysis of EZH2 and H3K27me3 expression in GSK126–resistant (H1299) and sensitive (A549) cell lines and MTT proliferation assays before or after replacement with control (vector–only), wild–type, or point mutant EZH2. H3K27 tri–methylation and H3 blots are quantified. Actin and H3 were used as loading controls. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by 2–way Anova). Error bars indicate means ± s.d. (n = 3). (h) Immunoblot of EZH2 and H3K27 tri–methylation in G401 cells treated with SAH–EZH2 (42–68)_AB compared to negative control (dSAH–EZH2_Neg1). (i) Colony formation of SWI/SNF mutant and wild–type cells in response to dSAH–EZH2 stapled peptides (n=2).

Figure 3. Disruption of PRC2 stability occurs in sensitive cells following enzymatic inhibitor treatment

(a) Evaluation of H3K27 methylation and acetylation, total EZH1 and EZH2, and EZH2 phosphorylation in GSK126–sensitive (G401) and resistant (RCC4) cell lines following treatment with GSK126 for 7 d. EZH2 p–T487 and EZH2 blots are quantified and the numbers for quantifications are written below each blot. (b) Schematic of the location of Thr487 within EZH2. EED–, SUZ12–, and DNA methyltransferase (DNMT)–binding domains and the SET domain are indicated. (c–f) The integrity of PRC2 complex was evaluated by immunoblot using EZH2 or SUZ12 antibodies in both GSK126–sensitive (G401) and resistant (RCC4, H1299, and OVISE) cell lines. The assays are representative of replicates of three independent experiments.

Figure 4

In vivo inhibition of H3K27me3 via GSK126 caused regression of tumor growth of GSK126–sensitive cancers, but not GSK126–resistant cancers. Recipient mice received xenografts of A549 (a) a GSK126–sensitive line or H1299 (d) a GSK126–resistant cell line and once tumors reached 200 mm³, mice were randomized to treatment with either GSK126 or vehicle control (n = 4), ****P < 0.0001 (ordinary one–way ANOVA). (b and e) Mass of the dissected tumors (n = 4), *P = 0.0237 (unpaired t–test). (c and f) Immunoblot using the indicated antibodies for tumors isolated from mice treated with vehicle control or GSK126; Actin and H3 were used as loading controls.