Single-cell transcriptomic landscape reveals a relapse-associated, LAMP5⁺ B-cell state associated with PKNOX1 that confers chemoresistance in B-ALL

B-cell acute lymphoblastic leukemia (B-ALL) is a highly heterogeneous hematological malignancy which drives disease progression, therapeutic resistance and relapse. However, the biological characteristics and clinical significance of relapse-associated leukemic cell populations remain to be fully elucidated. Here we systematically characterized the transcriptional heterogeneity of malignant B-cell populations in B-ALL using single-cell RNA sequencing (scRNA-seq) datasets. Re-clustering analysis identified eight transcriptionally distinct B-cell subpopulations with diverse functional properties (S1-S8). Among them, a unique cluster, termed S6, was significantly enriched in samples from patients at relapse and exhibited a predominantly G1-phase distribution, indicative of a relatively slow-cycling cellular state. The S6 signature was largely driven by two relapse samples among five S6-positive patients. Functional enrichment analyses revealed activation of B-cell receptor signaling and adaptive cellular programs within this population. By constructing cluster-specific gene signatures, we assessed their prognostic significance in independent cohorts. The S6 signature exhibited the strongest association with poor overall survival, with high S6 scores serving as a robust predictor of poor clinical outcomes. Furthermore, we identified LAMP5 as a highly specific marker for the S6 population. Cross-dataset validation confirmed the existence of a corresponding cell population with similar transcriptional and functional characteristics. Finally, transcriptional regulatory network analysis identified PKNOX1 as a potential key regulator of the S6-associated cellular state. Functional validation in B-ALL cell lines (Nalm6 and RCHACV) demonstrated that PKNOX1 overexpression induces profound resistance to Cytarabine, Etoposide (VP16) and Idarubicin by suppressing chemotherapy-induced apoptosis. Collectively, our findings provide a comprehensive characterization of leukemic heterogeneity in B-ALL and identify a relapse-associated slow-cycling leukemic cell population linked to poor prognosis, offering novel insights into the cellular mechanisms underlying B-ALL and highlighting PKNOX1 and LAMP5 as potential biomarkers for risk stratification and therapeutic targets for intervention.