Hypoxia-Induced Extracellular Vesicles Regulate BCR::ABL1 Expression in Chronic Myeloid Leukemia

Introduction

Chronic Myeloid Leukemia (CML) is driven by the BCR::ABL1 oncogene, which encodes a constitutively active tyrosine kinase. Although tyrosine kinase inhibitors (TKis) are highly effective, they fail to eradicate leukemia stem cells (LSCs) that persist within the hypoxic bone marrow niche, sustaining minimal residual disease (MRD). In a subset of persisting LSCs, BCR::ABL1 protein expression or kinase activity can be markedly reduced or undetectable. Because hostile microenvironments can stimulate the biogenesis and release of extracellular vesicles (EVs), we investigated whether CML cells utilize EV secretion to clear BCR::ABL1 under hypoxic conditions.

Methods

CML cell lines were cultured under severe hypoxia (0.1 % O₂), and EVs were isolated from the conditioned media. EV cargo was analyzed for BCR::ABL1 mRNA and protein content. Functional assays were performed to evaluate cargo transfer efficiency, cell proliferation rates, and drug sensitivity. To block this mechanism, EV biogenesis and secretion were pharmacologically inhibited. Finally, the translational relevance of these findings was validated by isolating and analyzing EVs from CML patients’ plasma.

Results

Hypoxia significantly increased EV secretion in CML cells. Cargo analysis revealed that hypoxic EVs were loaded with high levels of BCR::ABL1 mRNA. Furthermore, these EVs successfully transferred their cargo to BCR::ABL1-negative recipient cells, which consequently exhibited increased proliferation rates and acquired sensitivity to TKis. Inhibiting EV secretion prevented BCR::ABL1 clearance, retaining high levels of the oncoprotein within CML cells and restoring their susceptibility to TKi therapy. Crucially, BCR::ABL1 mRNA was detected in EVs isolated from the CML patients' plasma, showing a significant inverse correlation with molecular remission status.

Conclusions

These findings demonstrate that EV secretion under hypoxic stress contributes to BCR::ABL1 clearance, supporting TKi therapy evasion. Targeting EV biogenesis might represent a promising therapeutic strategy to prevent oncogene clearance and potentially reduce MRD risk.