P204 | DECODING INTERMODEL HETEROGENEITY IN CHRONIC MYELOID LEUKEMIA: INSIGHTS FROM IN VITRO EXTRACELLULAR VESICLES’ RELEASE

Chronic Myeloid Leukemia (CML) is a blood disease driven by the BCR::ABL1 fusion gene, causing aberrant proliferation. Tyrosine Kinase Inhibitors (TKIs) revolutionized treatment, improving survival. Challenges like resistance and disease persistence remain, thus, accurate preclinical models are essential to better translate laboratory findings to the clinic. In this context, we recently uncovered cellular heterogeneity in CML cell lines (K562, LAMA84, KCL22) under TKI treatment, impacting viability, metabolism, and molecular marker expression. Given the growing interest in extracellular vesicles (EVs), naturally released nanoparticles involved in immune modulation, angiogenesis, and disease progression, we subsequently investigated EV release and BCR::ABL1 trafficking in K562 and KCL22 cells exposed to imatinib, nilotinib, and dasatinib. Cells were treated with TKI for 24 and 48 hours. EV were isolated via commercial kit from the culture media and characterized according to international guidelines (MISEV 2023 ). Vesicular BCR::ABL1 transcript was detected via digital PCR and both values were normalized to the total cell count determined via CCK8 assay. Cellular proliferation was evaluated by Ki67 expression and normalized to GAPDH expression. Untreated K562 and KCL22 cells exhibited similar Ki67 expression. 24h post TKI, KCL22 showed a marked reduction, while K562 required 48h to display comparable effects, suggesting differences in drug sensitivity and adaptive response. Vesicular BCR::ABL1 content was similar between untreated K562- and KCL22-derived EV at both timepoints (Fig 1A). However, TKI exposure increased BCR::ABL1 cargo in K562-EVs, especially after 48 hours (Fig 1C-E). EV quantification by Nanoparticle Tracking Analysis showed comparable EV release from untreated K562 and KCL22 cells (Fig. 1B). At 48 hours, imatinib and dasatinib significantly increased EV secretion in K562 (Fig. 1F-H), highlighting model-specific vesicular responses to TKI that may affect intercellular communication . Our findings support the need for adequate preclinical models to study CML progression and therapy response. Differences in EV release and BCR::ABL1 trafficking between CML models reflect the complexity of vesicular signaling in leukemia. Further studies are needed to clarify the functional impact of these EV-mediated interactions. While not directly clinically translatable, these results provide insights for future preclinical research.