Single-cell RNA sequencing uncovers m6A regulator-defined T-cell subpopulations and YTHDF1–HSP90AB1–MYC therapeutic axis in T-cell acute lymphoblastic leukemia

BACKGROUND: N6-methyladenosine (m6A) drives T-cell acute lymphoblastic leukemia (T-ALL); however, its roles in leukemic heterogeneity and tumor microenvironment remodeling unclear. This study characterized N6-methyladenosine regulator-defined subpopulations and YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in leukemogenesis. METHODS: Integrated single-cell and bulk RNA sequencing from 17 pediatric T-cell acute lymphoblastic leukemia patients and 3 healthy donors were analyzed. Non-negative matrix factorization based on m6A regulator expression identified distinct T-cell subpopulations. CellChat, Monocle, and single-cell regulatory network inference and clustering analyses revealed intercellular communication patterns, developmental trajectories, and transcriptional regulatory programs. Functional validations included lentiviral-mediated YTHDF1 knockdown in Jurkat and Molt4 cells for proliferation, apoptosis, and migration assays, and a non-obese diabetic severe combined immunodeficiency mouse xenograft model. Methylated RNA immunoprecipitation quantitative polymerase chain reaction, RNA immunoprecipitation quantitative polymerase chain reaction, RNA stability assays, and Western blotting characterized YTHDF1 interactions with heat shock protein 90 alpha family class B member 1 (HSP90AB1). RESULTS: Single-cell transcriptomics identified m6A-defined T-cell subpopulations in T-ALL. YTHDF1-enriched cells exhibited hypoxia and cell-cycle programs, occupied early developmental states, and communicated with macrophages. Knockdown suppressed proliferation and migration, increased apoptosis, and reduced leukemic burden in vivo. The protein bound and stabilized m6A-modified HSP90AB1 to sustain MYC proto-oncogene signaling. CONCLUSION: This study identified a YTHDF1-high m6A-associated T-cell subpopulation in T-ALL. YTHDF1 promoted leukemic progression through regulation of the HSP90AB1-MYC axis and was associated with altered macrophage-related intercellular signaling.