Identification and therapeutic targeting of leukemia stem cells at single-cell resolution in RUNX1::RUNX1T1 Leukemia

Abstract Acute myeloid leukemia with RUNX1::RUNX1T1 fusion (RUNX1::RUNX1T1 AML) is characterized by leukemic transformation originating at hematopoietic stem cell (HSC) stages, leading to aggressive leukemia with limited targeted therapies. Using single-cell transcriptomic analyses of CD34⁺CD56⁺ blast-enriched and CD34⁺CD56⁻ HSPC-like fractions from RUNX1::RUNX1T1 AML patients, we identified a substantial population of transcriptomically defined leukemia stem cells (LSCs) within the CD34⁺CD56⁻ compartment. These LSCs exhibited higher hierarchical potential, stronger HSC-like signatures, and pronounced enrichment for PRC2-related histone methylation programs compared with normal HSC populations. Further full-length single cell RNA-seq from sorted HSCs harboring RUNX1::RUNX1T1 mutations revealed substantial transcriptional alterations linked to EZH2 and PRC2 targets, reinforcing the crucial role of EZH2-mediated histone methylation in leukemic transformation. Supporting these observations, genetic ablation of Ezh2 in RUNX1::RUNX1T1 mice significantly accelerated leukemia progression, associated with myeloid progenitor expansion and differentiation blockade. Finally, pharmacological inhibition of H3K27 demethylation using GSK-J4 effectively inhibited RUNX1::RUNX1T1 patient-derived leukemic cells, underscoring therapeutic vulnerability. Collectively, these results emphasize EZH2-mediated histone modifications as critical in RUNX1::RUNX1T1 leukemogenesis and highlight epigenetic inhibition as a promising therapeutic strategy.