Homoharringtonine suppresses acute myeloid leukemia progression by orchestrating EWSR1 phase separation in an m 6 A‐YTHDF2‐dependent mechanism

Homoharringtonine (HHT) is widely used in combination regimens for acute myeloid leukemia (AML), yet its direct cellular targets remain undefined, limiting precision application. Here, we identified EWS RNA-binding protein 1 (EWSR1) as the primary target of HHT through chemical proteomics and biophysical validation. HHT bound the RNA recognition motif of EWSR1 with micromolar affinity, inducing an allosteric conformational switch that promoted oligomerization and liquid-liquid phase separation (LLPS). EWSR1 condensates selectively recruited the N⁶-methyladenosine (m⁶A) reader YTHDF2, forming cytoplasmic hubs where HHT disrupted YTHDF2-mRNA interactions. This sequestration attenuated m⁶A-mediated RNA decay, stabilizing key transcripts such as TNFRSF1B and HMOX1, and thereby impairing AML cell proliferation. Integrated transcriptomics and single-cell RNA-seq analyses revealed that EWSR1 was markedly upregulated in AML, particularly in hematopoietic progenitor and myeloid subpopulations, and high EWSR1 expression correlated with poor prognosis and enhanced HHT sensitivity. In vivo, the anti-leukemic efficacy of HHT was significantly diminished upon EWSR1 knockdown, demonstrating that EWSR1 was required for therapeutic response. Collectively, these findings uncover a phase separation-centric mechanism by which HHT exerts anti-AML activity, establish the EWSR1-YTHDF2-m⁶A axis as a critical regulator of leukemia progression, and position EWSR1 as both a functional target and a predictive biomarker for optimizing HHT-based therapies.