Paired longitudinal single-cell profiling reveals clonal evolution and lymphoid lineage restriction during progression from chronic-phase CML to B-lymphoblastic blast crisis

Blast transformation remains the principal cause of mortality in chronic myeloid leukemia (CML), yet the cellular mechanisms underlying progression from chronic-phase CML (CML-CP) to B-lymphoblastic blast crisis (BLBC) remain poorly understood. We performed paired longitudinal single-cell transcriptomic profiling to characterize the cellular states, lineage trajectories, molecular programs, and clonal evolution associated with lymphoid blast transformation in CML. Single-cell RNA sequencing (scRNA-seq) was totally performed on five bone marrow samples, categorized into CML-CP ( n = 2) and CML-BLBC ( n = 3), including two longitudinally paired CP–BLBC sample sets from individual patients. Integrative analyses incorporating a publicly available scRNA-seq dataset from two patients with de novo Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph + B-ALL) were conducted to explore BLBC-specific cellular states. CD24 expression was further validated using a microarray dataset, bulk RNA sequencing (RNA-seq), quantitative real-time PCR (qRT‒PCR), and flow cytometry analysis. scRNA-seq analysis suggested substantial remodeling of the hematopoietic landscape during progression from CML-CP to BLBC. Two CML-CP samples exhibited heterogeneous erythroid- and myeloid-biased hematopoietic outputs associated with distinct transcriptional programs, whereas three BLBC samples acquired stem cell-like, relatively quiescent cellular states together with enhanced inflammatory, apoptotic, ERBB2, and epithelial–mesenchymal transition signaling. Blast transformation was associated with activation of a lymphoid-associated transcriptional program characterized by increased CD24 , DNTT , and RAG1 expression, consistent with the emergence of lymphoid-primed BCR::ABL1 -positive progenitor populations. Comparative analyses further suggested that CML-BLBC may exhibit a transcriptional landscape distinct from that of de novo Ph + B-ALL, with CD24 emerging as one of the most consistently upregulated genes enriched in ProB and PreB populations. Progressive CD24 upregulation during disease evolution was independently confirmed by using an external microarray dataset, an in-house bulk RNA-seq dataset, qRT‒PCR, and flow cytometric analysis. Moreover, elevated CD24 expression was associated with inferior response to imatinib in untreated CML patients. The paired longitudinal single-cell atlas delineates the evolutionary trajectory from CML-CP to BLBC and provides evidence for an association between the selective expansion of lymphoid-primed BCR::ABL1 -positive progenitors and lineage-restricted blast transformation. The upregulation of CD24 , DNTT , and RAG1 may constitute key molecular events underlying this process.