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

ABSTRACT 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.