Allosteric Inhibition of Polycomb Repressive Complex 2 by an EZH2‐Selective Small Molecule Inhibitor

Polycomb Repressive Complex 2 (PRC2), containing homologous EZH1 or EZH2 as the catalytic subunit, is a conserved methyltransferase complex that represses gene transcription by transferring methyl group from SAM to H3K27. Gain-of-function mutations of EZH2 and aberrant H3K27 methylation have been linked to human cancers. SAM-competitive EZH1 and EZH2 dual inhibitors have been approved by the FDA for treating B-cell lymphoma and other cancers. Here, we characterized a small molecule C36, which potently inhibits EZH2/PRC2, but not EZH1/PRC2, with a novel SAM non-competitive mechanism. Cryo-EM structures revealed that C36 binds to a pocket at the interface of SET-Activation-Loop (SAL), stimulation-responsive motif (SRM), and I-SET domain of EZH2, and WD40 domain of EED. C36 binding induces conformational changes and disrupts allosteric communication between EZH2 and ligand-bound EED. C36 efficiently inhibits H3K27 trimethylation and PRC2 target gene expression in tumor cells and xenograft tumors with low hematotoxicity. Multi-omics analyses employing C36 uncovered the direct regulation of IFNB1 by EZH2/PRC2. The combined treatment of syngeneic LLC lung cancer with C36 and a PD-1 antibody significantly enhances anti-tumor efficacy. Our study identifies a new allosteric mechanism of PRC2 inhibition and paves the way for the development of highly selective EZH2/PRC2 inhibitors for combination therapy.