Covalent targeting of PSMD14 by Eupalinolide B induces oncoprotein degradation and apoptosis in acute promyelocytic leukemia cells

Treatment of acute promyelocytic leukemia (APL) remains challenged by toxicities associated with current regimens, highlighting the need for novel and safer therapeutic agents. Here, we identify Eupalinolide B (EB), a natural sesquiterpene lactone isolated from Eupatorium lindleyanum DC., as a potent anti-leukemic compound targeting the human APL-derived HL-60 cell line. Through integrated chemoproteomic profiling and functional validation, we demonstrate that EB covalently binds and inhibits 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), a deubiquitinase enzyme (DUB) within the 19S proteasome regulatory particle. This inhibition disrupts PSMD14-mediated stabilization of key oncoproteins RAC-alpha serine/threonine-protein kinase 1 (AKT1) and cyclin-dependent kinase 4 (CDK4), promoting their proteasomal degradation. As a result, EB induces G2/M cell cycle arrest and apoptosis in leukemia cells. Both genetic knockdown and pharmacological inhibition of PSMD14 recapitulate EB's effects, confirming its essential role in leukemia cell survival and proliferation. Collectively, these findings uncover a previously unrecognized PSMD14–AKT1/CDK4 regulatory axis in leukemia and position EB as a promising chemical probe and lead compound for the development of targeted covalent inhibitors against oncogenic DUBs.