PML-RARa Regulated Vesiculation Of Acute Promyelocytic Leukemia Cells

In acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), severe bleeding tendency coincides with upregulation of tissue factor (TF), fibrinogenopenia and disseminated intravascular coagulation (DIC). In APL, the reciprocal translocation between chromosomes 15 and 17 results in the oncogenic fusion between promelocytic leukemia gene (PML) and retinoic acid receptor alpha (RARa). Targeting the activity of PML-RARa using targeted therapy combined with chemotherapy produces a high rate of complete remission, and reversal of tissue factor (TF)-related coagulopathy. Oncogenic transformation, therapeutic responses, coagulopathy and interaction of malignant cells with their vascular microenvironment has been linked, at least in part, to the emission and uptake of extracellular vesicles (EVs, including ectosomes/microparticles and exosomes). Little is known about these processes in APL.

In this study, we explored the role of oncogenic PML-RARa in vesiculation of APL cells and the resulting changes in their procoagulant/angiogenic properties. We hypothesized that severe coagulopathy in APL may propagate to stromal (endothelial) cells via the exchange of TF-containing vesicles.

EV emission by NB4 cells derived from an APL patient with t(15;17) was measured by Nanoparticle Tracking Analysis (NTA). Ectosomes and exosomes were separated by ultracentrifucation, and the content of PML-RARa and TF in these EVs were studied at both RNA (RT-PCR) and protein and activity levels (Western, ELISA, TF-PCA). Transfer of EVs between NB4 cells and endothelium (HUVEC) was tested using membrane labeling with the PKH26 dye and flow cytometry. PML-RARa and TF in transfer to HUVECs were studied at both in RNA level and protein level, and bioassays (invasion, angiogenesis) were used to assess the consequences.

PML-RARa controls cellular vesiculation as all-trans retinoic acid (ATRA) alters emission of EVs by NB4 cells leading to preponderance of exosomes. These EVs contain both PML-RARa and TF transcripts, but only TF protein is detected in the EV cargo and transferred to endothelial cells. NB4-derived EVs render endothelial cells TF-positive and procoagulant and changes their angiogenic properties. Surprisingly, the migratory phenotype of endothelial cells is inhibited by co-culture with leukemic cells and by exposure to leukemic EVs. The nature of these effects is being studied in vitro and in vivo.

NB4 cells shed EVs as a function of their differentiation and oncogenic status. Unlike oncogenic receptors in cancer cells, PML-RARa protein is not detected in leukemic EVs, but exerts an indirect effect on their production and cargo. APL-derived EVs may render endothelial cells procoagulant. In spite of the pro-angiogenic phenotype of APL cells, their EVs inhibit migration of endothelial cells. The significance of EV interactions between leukemic and non-leukemic cells is being investigated.

No relevant conflicts of interest to declare.