Plasma-Derived Exosomes in Acute Myeloid Leukemia Carry Dipeptidylpeptidase-4 and Inhibit Normal Human Hematopoiesis

Introduction: Bone marrow failure and peripheral cytopenias are common manifestations of acute myeloid leukemia (AML), that contribute significantly to disease morbidity and mortality. The mechanisms by which AML interferes with normal hematopoiesis are under intense investigation. Exosomes, the smallest (30-150nm) extracellular vesicles subsets produced by all cells, including leukemia blasts, can re-program the function of recipient cells. Newly diagnosed AML patients have higher levels of plasma exosomes that carry numerous suppressive molecules compared to those in healthy donors. We hypothesize that AML exosomes contribute to patients' cytopenias by inhibiting normal hematopoiesis.

Methods: Venous blood (20-50 mL) was obtained from 10 patients newly diagnosed with AML prior to any therapy and from age-matched healthy volunteers (NC). Exosomes were isolated from the plasma of patients, NC, or cell culture supernatants of AML cell lines (THP1, Kasumi, and ML2) by size exclusion chromatography. Protein level, number and size (qNano), and exosome morphology by transmission electron microscopy were determined. Exosome cargos were studied by Western blots for evidence of proteins involved in hematopoiesis, including dipeptidylpeptidase-4 (DPP-4/CD26), a serine protease that enzymatically cleaves the amino acids of proteins that regulate hematopoiesis. The effect of exosomes on hematopoietic progenitor cell (HPC) proliferation and differentiation were evaluated by colony forming cell (CFC) assays. CFC assays were plated in triplicate with 10-30µg of exosomes used for every 1mL of media, and the total number of colonies on the plate were counted. The functional activity of DDP-4 was measured using a DPP4-Glo Protease Assay.

Results: Exosomes isolated from the plasma of AML patients and from the supernatants of AML cell lines carry DPP-4. In addition, leukemia AML exosomes contain interleukin-3 receptor subunit alpha (CD123), erythropoietin receptor (EPO-R), tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β). AML exosomes exhibited a markedly higher DPP-4 functional activity. AML exosomes enriched with DPP-4 suppressed the differentiation and proliferation of normal HPC. The degree of inhibition was dependent on the exosome dose, with marked inhibition (up to 80%) of colony formation detected when 30µg of AML exosomes was used. Exosomes isolated from NC plasma did not inhibit HPC in CFC assays.

Conclusion: We report, for the first time, that AML exosomes enriched with DPP-4 having a high level of enzymatic activity inhibit normal human hematopoiesis in vitro . In the future, protection of normal hematopoiesis from suppressive AML exosomes with therapeutic antibodies and/or small molecule pharmacologic inhibitors will aim at decreasing the risk for infections and the need for transfusions in AML.