Abstract
Introduction Resistance to tyrosine kinase inhibitors (TKIs) remains a major therapeutic challenge in chronic myeloid leukemia (CML). Although tumor-intrinsic mechanisms of resistance have been extensively studied, the role of the bone marrow microenvironment, particularly endothelial progenitor cells (EPCs) mediating TKI resistance is poorly understood. This study aims to elucidate the molecular, phenotypic, and functional alterations in EPCs derived from TKI-resistant CML patients, and to uncover the mechanisms by EPCs mediated TKI resistance through intercellular crosstalk.
Methods EPCs were isolated from TKI-resistant and TKI-sensitive CML patients and healthy donors. Their angiogenic capacity, migratory ability, intracellular reactive oxygen species (ROS) levels, and leukemic cell adhesion were assessed using standard in vitro assays. To explore the role of cell–cell contact, CML cells were co-cultured with EPCs either directly or in transwell systems. In vivo, co-injection xenograft models were used to evaluate TKI response. CRISPR-Cas9 screening targeting membrane proteins in K562 cells under EPC co-culture was used to identify critical mediators. Mechanistic studies included CRISPR-Cas9–mediated SEMA4D knockout and overexpression in CML cells, Plexin B1 knockdown in EPCs, and treatment with SEMA4D-neutralizing antibodies. Immunoprecipitation-mass spectrometry (IP-MS) was employed to identify SEMA4D-interacting proteins, followed by co-immunoprecipitation (Co-IP) for validation. Seahorse XF extracellular flux analysis was used to measure mitochondrial oxygen consumption and oxidative phosphorylation (OXPHOS) activity in CML cells. Results EPCs derived from TKI-resistant CML patients (resistant EPCs, n = 10) exhibited markedly reduced angiogenic (tube length: 17005 vs. 41084 vs. 45141, p < 0.001) and migratory capacities (migrate cells: 103 vs. 202 vs. 183, p < 0.001), along with elevated intracellular ROS levels (mean fluorescence intensity: 2683 vs. 1862 vs. 1812, p < 0.001) compared to EPCs from TKI-sensitive patients (sensitive EPCs, n = 10) and healthy donors (n = 10). Notably, the resistant EPCs demonstrated significantly enhanced adhesion to CML cells (adhered cells: 390 vs. 265 vs. 228, p < 0.001). Direct contact with resistant EPCs conferred TKI resistance to CML cells in vitro and in vivo. The CRISPR screen identified SEMA4D as a key mediator. SEMA4D knockout sensitized CML cells to TKI-induced apoptosis and reduced endothelial adhesion, while its overexpression promoted TKI resistance and adhesion. SEMA4D bonded to its receptor Plexin B1 which was upregulated in resistant EPCs. Disruption of the SEMA4D–Plexin B1 axis via neutralizing antibodies or Plexin B1 knockdown restored TKI sensitivity. Transcriptomic analysis of SEMA4D-overexpressing CML cells revealed significant enrichment of metabolic pathways including the tricarboxylic acid (TCA) cycle, pyruvate metabolism, and gluconeogenesis, suggesting a potential role of SEMA4D in regulating cellular metabolism. To explore the underlying molecular mechanisms, immunoprecipitation followed by mass spectrometry was performed, which identified pyruvate carboxylase (PC), a key anaplerotic enzyme, as a potential binding partner of SEMA4D. This interaction was further validated by co-immunoprecipitation assays. Functional metabolic analysis using Seahorse XF extracellular flux analysis demonstrated that SEMA4D overexpression enhanced mitochondrial oxygen consumption rate (OCR), while SEMA4D knockdown impaired OXPHOS, indicating that SEMA4D supports mitochondrial metabolic activity and contributes to the metabolic adaptation of CML cells during TKI treatment.ConclusionOur study demonstrates that bone marrow–derived endothelial progenitor cells from TKI-resistant CML patients promote leukemic cell survival through direct cell–cell contact. This microenvironmental protection is mediated by the SEMA4D–Plexin B1 axis, which facilitates CML cells–EPCs adhesion and confers TKI resistance. Beyond its adhesive role, SEMA4D interacts with pyruvate carboxylase to enhance mitochondrial OXPHOS and metabolic adaptation, further supporting leukemic persistence.
