Persistence of Exosomes in Patients with Acute Myeloid Leukemia in Complete Remission Suppresses Hematopoiesis

Introduction: Treatment of patients with acute myeloid leukemia (AML) aims at achieving complete remission (CR) and restoration of normal bone marrow function. However, patients who achieve CR following induction chemotherapy often have incomplete count recovery (CRi< 100,000/μL platelets or <1,000/μL neutrophils in the peripheral blood). Patients with CR have better relapse-free survival and overall survival than patients with CRi. Mechanisms by which AML interferes with normal hematopoiesis are under intense investigation. Emerging evidence suggests that blast-derived exosomes suppress hematopoiesis. Exosomes, small (30-150nm) extracellular vesicles, serve as an intercellular communication system. In AML, they shuttle messages between blasts and hematopoietic progenitor cells (HPCs) and reprogram their functions, suppressing hematopoiesis. We hypothesize that exosomes contribute to cytopenias in CRi patients by inhibiting normal hematopoiesis.

Methods: Venous blood (20-50 mL) was obtained from 10 patients newly diagnosed with AML prior to therapy and at CR or CRi. Exosomes were isolated from plasma of AML patients and normal donors (n=5) by size exclusion chromatography. Protein levels, numbers and size (qNano), and exosome morphology (transmission electron microscopy) were determined. Exosome cargos were studied by Western blots for Leukemia Associated Antigens and for evidence of proteins involved in hematopoiesis, including dipeptidyllpeptidase-4 (DPP-4/CD26), a serine protease that enzymatically cleaves the proteins regulating hematopoiesis. Effects of exosomes on HPC proliferation and differentiation were evaluated in vitro by colony forming cell (CFC) assays. CFC assays were plated in triplicate. Total CFCs numbers ± exosomes (1-50 µg protein) were determined in the presence/absence of DPP-4 inhibitors.

Results: Exosomes isolated from the plasma of AML patients at diagnosis carried Leukemia Associated Antigens, DPP-4, interleukin-3 receptor subunit alpha (CD123), erythropoietin receptor (EPO-R), tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β). Blast-derived exosomes at AML diagnosis suppressed the differentiation and proliferation of normal HPCs. The degree of inhibition was exosome dose-dependent. Pharmacologic inhibition of DPP-4 activity reversed suppression of CFC formation (p<0.01). In paired analysis, inhibition of CFC formation by exosomes of the CRi patients remained elevated at levels comparable to those induced by exosomes obtained at AML diagnosis. In contrast, exosomes of CR patients induced lower CFC suppression (p<0.04) than exosomes obtained at AML diagnosis.

Conclusion: We report, for the first time, that exosomes in CRi patients inhibit normal human hematopoiesis in vitro. The data implicate AML blast-derived exosomes in suppression of normal hematopoiesis and suggest that in the future reversing the negative effects of exosomes and thus improving platelet and neutrophil counts, might emerge as a new therapeutic approach to AML.