Aim: The aims are to verify if: (i) EVs released by CML cells carry BCR-ABL in their cargo and if that BCR-ABL is captured by recipient cells; (ii) EVs released by a CML drug resistant cell line, with mutant BCR-ABL, may transfer mutant BCR-ABL and a resistant phenotype to sensitive cells.
Introduction: BCR-ABL, the fusion gene originated by the t(9;22) translocation, is responsible for Chronic Myeloid Leukemia (CML). BCR-ABL codes for a constitutively active tyrosine kinase (TK), deregulating downstream pathways and promoting cell survival. Imatinib mesylate (Gleevec), a TK inhibitor, is the gold standard treatment for CML; nevertheless, resistance to this drug often arises, mostly caused by additional point mutations on BCR-ABL and representing a major clinical drawback. It was recently suggested that drug resistance might be horizontally transferred by EVs, from resistant to sensitive cells.
Methods: A pair of drug-sensitive BCR-ABL+ cell line (KBM5), and its drug-resistant counterpart (KBM5-STI, harboring mutated BCR-ABL) were used in this study. EVs were isolated by ultracentrifugation and characterized by Dynamic Light Scattering, Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Western Blot. The resazurin assay was used to assess drug response of drug resistant cells, drug sensitive cells and of drug sensitive cells following co-culture with EVs released by drug resistant cells. BCR-ABL levels were analysed by Western Blot.
Results: A dose-response curve to imatinib was performed in both cell lines, to confirm their different responses to the drug. Regarding EVs characterization, they had between 10 and 1000nm and presented several markers of EVs with no evidence of cellular contaminants. Interestingly, BCR-ABL protein was detected in the EVs.1–9
Conclusion: These results suggest that there is selective packaging of BCR-ABL into EVs, promoting oncogenic protein shedding. Ongoing work will clarify if the EVs released by the resistant cells have mutant BCR-ABL and if they confer drug resistance to recipient sensitive cells.