Targeting EZH2 and PRC2 dependence as novel anticancer therapy

Abstract

Distinctive patterns of chromatin modification control gene expression and define cellular identity during development and cell differentiation. Polycomb repressive complex 2 (PRC2), the sole mammalian enzymatic complex capable of establishing gene-repressive high-degree methylation of histone H3 at lysine 27 (H3K27), plays crucial roles in regulation of normal and malignant hematopoiesis. Recently, increasing evidence has indicated that recurrent gain-of-function mutation and overexpression of EZH2, the catalytic subunit of PRC2, drive and promote malignant transformation such as B-cell lymphomagenesis, providing a rationale for PRC2 inhibition as a novel anticancer strategy. Here, we summarize the recently developed strategies for inhibition of PRC2, which include a series of highly specific, highly potent, small-molecule inhibitors of EZH2 and EZH1, an EZH2-related methyltransferase. PRC2 establishes functional crosstalk with numerous epigenetic machineries during dynamic regulation of gene transcription. Perturbation of such functional crosstalk caused by genetic events observed in various hematologic cancers, such as inactivation of SNF5 and somatic mutation of UTX, confers PRC2 dependence, thus rendering an increased sensitivity to PRC2 inhibition. We discuss our current understanding of EZH2 somatic mutations frequently found in B-cell lymphomas and recurrent mutations in various other epigenetic regulators as novel molecular predictors and determinants of PRC2 sensitivity. As recent advances have indicated a critical developmental or tumor-suppressive role for PRC2 and EZH2 in various tissue types, we discuss concerns over potentially toxic or even adverse effects associated with EZH2/1 inhibition in certain biological contexts or on cancer genetic background. Collectively, inhibition of PRC2 catalytic activity has emerged as a promising therapeutic intervention for the precise treatment of a range of genetically defined hematologic malignancies and can be potentially applied to a broader spectrum of human cancers that bear similar genetic and epigenetic characteristics.

Figure 1

Gain-of-function EZH2 mutations affect substrate specificity of the PRC2 complex. (A) Depiction of EZH2 and EZH1 domain structure with the site of gain-of-function mutations (either the hotspot Y641 mutation, A667G, or A687V) in the catalytic SET domain of EZH2 highlighted with a yellow asterisk. (B) Wild-type EZH2 is more efficient at catalyzing the turnover of H3K27me0 and H3K27me1 than H3K27me2 (shown in inset, black line). Y641N (blue bars) and A677G (green bars) exhibit the opposite trend. A687V (purple bars) is equipotent at catalyzing the turnover of H3K27me1 and H3K27me2, relative to WT EZH2. WT = wild-type. (Color version of figure is available online.)

Figure 2

Mechanistic insights into substrate selectivity of EZH2 mutants. (A) Wild-type EZH2 uses Y641 to control the methylation state of H3K27 by maintaining a hydrogen bond (dotted line, 2.9 Å) with the dimethylated substrate (green). SAH (purple), the methylation product of cofactor SAM, and the SAM-competitive EZH2 inhibitor UNC1999 (light blue) are also included in the model to illustrate their overlapped binding sites. (B) Mutation to Y641N enlarges the pocket and reduces the propensity for a hydrogen bond between the asparagine and H3K27me2 (dotted line, 5.6 Å), promoting trimethylation of H3K27. (C) Mutation of A677 to the smaller glycine residue (A677G) leads to a slight conformational change, enlarging the pocket and weakening the hydrogen bond between Y641 and H3K27me2 (dotted line, 4.2 Å), which enhances the ability of EZH2^A677G to trimethylate H3K27. (D) Mutation of A687 to valine (A687V) leads to a similar conformational change, which leads to a slightly larger pocket, and reduces the strength of the hydrogen bond between Y641 and H3K27me2 (dotted line, 3.2 Å). SAM = S-adenosylmethionine.

Figure 3

EZH2 gain-of-function mutations in driving B-cell lymphomagenesis. H3K27me3 and H3K4me3 often coexist at multiple genomic loci in germinal-center B cells, rendering these genes in a repressive but poised status. EZH2 gain-of-function mutants reinforce H3K27me3 occupancy and repression of the “bivalent genes” that are crucial for antiproliferation (such as CDKN2A) or terminal differentiation (such as PRDM1, IRF4, and BLIMP), promoting hyperplasia or cancerous transformation of germinal-center B cells.

Figure 4

Crosstalk between PRC2 and other epigenetic factors. PRC2 is the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. Various epigenetic machineries have crosstalk with PRC2 complex, either cooperatively or antagonistically. The KDM6 family of lysine demethylases such as UTX removes H3K27me3. Methylations of H3K4 and H3K36, as well as acetylation of H3K27, are prominent histone marks associated with transcriptional activation, which are established by the MLL complexes, the NSD family proteins, and the CBP/p300 acetyltransferases, respectively; preinstallation of H3K4me3, H3K36me2/3, and/or H3K27ac inhibits the activity of PRC2 and interferes with H3K27me3 installation. UTX physically interacts with the MLL complex linking H3K4 methylation with H3K27 demethylation. SWI/SNF is an ATP-dependent chromatin remodeling complex that can also antagonize PRC2. PRC2 was found to interact with other epigenetic factors such as histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) to further reinforce a repressive state of polycomb target genes. WT1, however, serves as a platform for recruiting DNA demethylases TET2 and TET3, thus facilitating conversion of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC) and attenuating the DNA methylation-mediated repression. Genetic alterations that affect such a myriad of epigenetic factors (red stars) are identified as the recurrent event in various hematopoietic malignancies, causing global or focal perturbation of PRC2 activity and H3K27me3 and thereby rendering cancer cells sensitive to PRC2 inhibition.

Figure 5

Genetic mutations found in hematopoietic malignancies confer on cancer cells sensitivity to PRC2 or EZH2 inhibition. (A) Although they do not affect the global H3K27me3 level, UTX inactivating mutations cause focal H3K27me3 enrichment in genes involved in antiproliferation and cell adhesion to promote cancerous transformation. (B) Biallelic inactivation of a core component of the SWI/SNF chromatin remodeling complex, SNF5 (also known as SMARCB1), enhances PRC2-mediated suppression of antiproliferation and lineage differentiation signature genes; other subunits of the SWI/SNF complex also function as tumor suppressors and their mutations in malignancies may confer a similar PRC2 dependence. Similarly, PRC2 is also required for acute leukemia caused by MLL rearrangements such as MLL-AF9. In addition to leukemia stem cell programs, MLL-AF9 oncoproteins directly promote expression of EZH2. PRC2 complexes assembled by both EZH2 and EZH1 repress myeloid differentiation and antiproliferation programs to promote acute leukemogenicity. (C) Because of the abnormal chromosomal translocation or gain-of-function somatic mutation (E1099K) found in multiple myeloma patients, hyperactivation of MMSET/NSD2 causes a global increase in H3K36me2 and decrease in H3K27me3. However, H3K27me3 is maintained and even enriched at certain critical regions protected by CTCF, an insulator factor. Focal enrichment of EZH2 leads to an enhanced suppression of lymphoid signatures and miR126, a negative regulator of c-MYC, thus enhancing tumorigenesis. (D) Loss-of-function mutation of WT1 induces a DNA hypermethylation phenotype in patients with acute myeloid leukemia (AML). Methylated genes found specific to WT1-mutated AMLs largely overlap with PRC2 gene targets such as myeloid maturation signatures, indicating cooperation between DNMTs and PRC2. WT1-mutated AMLs exhibit sensitivity to PRC2 inhibition. * = mutation of the labeled gene found in hematologic cancers.