A covalently bound inhibitor triggers <scp>EZH</scp>2 degradation through <scp>CHIP</scp>‐mediated ubiquitination

<jats:title>Abstract</jats:title><jats:p>Enhancer of zeste homolog 2 (<jats:styled-content style="fixed-case">EZH</jats:styled-content>2) has been characterized as a critical oncogene and a promising drug target in human malignant tumors. The current <jats:styled-content style="fixed-case">EZH</jats:styled-content>2 inhibitors strongly suppress the enhanced enzymatic function of mutant <jats:styled-content style="fixed-case">EZH</jats:styled-content>2 in some lymphomas. However, the recent identification of a <jats:styled-content style="fixed-case">PRC</jats:styled-content>2‐ and methyltransferase‐independent role of <jats:styled-content style="fixed-case">EZH</jats:styled-content>2 indicates that a complete suppression of all oncogenic functions of <jats:styled-content style="fixed-case">EZH</jats:styled-content>2 is needed. Here, we report a unique <jats:styled-content style="fixed-case">EZH</jats:styled-content>2‐targeting strategy by identifying a gambogenic acid (<jats:styled-content style="fixed-case">GNA</jats:styled-content>) derivative as a novel agent that specifically and covalently bound to Cys668 within the <jats:styled-content style="fixed-case">EZH</jats:styled-content>2‐<jats:styled-content style="fixed-case">SET</jats:styled-content> domain, triggering <jats:styled-content style="fixed-case">EZH</jats:styled-content>2 degradation through <jats:styled-content style="fixed-case">COOH</jats:styled-content> terminus of Hsp70‐interacting protein (<jats:styled-content style="fixed-case">CHIP</jats:styled-content>)‐mediated ubiquitination. This class of inhibitors significantly suppressed H3K27Me3 and effectively reactivated polycomb repressor complex 2 (<jats:styled-content style="fixed-case">PRC</jats:styled-content>2)‐silenced tumor suppressor genes. Moreover, the novel inhibitors significantly suppressed tumor growth in an <jats:styled-content style="fixed-case">EZH</jats:styled-content>2‐dependent manner, and tumors bearing a non‐<jats:styled-content style="fixed-case">GNA</jats:styled-content>‐interacting C668S‐<jats:styled-content style="fixed-case">EZH</jats:styled-content>2 mutation exhibited resistance to the inhibitors. Together, our results identify the inhibition of the signaling pathway that governs <jats:styled-content style="fixed-case">GNA</jats:styled-content>‐mediated destruction of <jats:styled-content style="fixed-case">EZH</jats:styled-content>2 as a promising anti‐cancer strategy.</jats:p>