Acute myeloid leukemia (AML) is a heterogeneous and devastating hematologic malignancy characterized by differentiation blockage and immature progenitor accumulation, positioning differentiation therapy as a promising therapeutic strategy. However, clinical success is largely confined to acute promyelocytic leukemia (APL) and isocitrate dehydrogenase (IDH)-mutated AML, leaving most AML subtypes with unmet needs. Herein, novel noscapine derivative ES428 is discovered that induces AML differentiation and exhibits potent anti-AML efficacy across diverse AML cell lines, primary patient samples, as well as cell line- and patient-derived xenograft models. Target deconvolution with combinatorial strategies identifies dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine synthesis, as the direct functional target. Integration of molecular dynamics simulations and comprehensive structure-activity relationship studies elucidates ES428’s unique mechanism via simultaneous engagement with DHODH and mitochondrial membrane lipids. This dual-engagement underpins ES428’s enhanced target engagement, efficacy, and selectivity in physiologically relevant mitochondrial membrane environment, potentially through stabilizing ES428-DHODH interaction in situ and facilitating ES428’s selective mitochondrial localization. Furthermore, ES428 triggers a mechanistic cascade linking decreased pyrimidine synthesis, reduced O-linked N-acetylglycosylation (O-GlcNAcylation), EP300/CREBBP catalytic inhibition, and transcriptional reprogramming. Our findings identify promising lead candidates, establish a novel DHODH-targeting strategy, and provide important mechanistic insights to advance differentiation therapies for myeloid malignancies.

Acute myeloid leukemia  /  Differentiation therapy  /  Noscapine derivative  /  Novel DHODH inhibitor  /  Protein‒lipid dual engagement  /  De novo pyrimidine synthesis  /  EP300/CREBBP  /  Transcriptional reprogramming