Utilizing Exosomes As Innovative Microrna Biomarkers and Targeted Drug-Delivery Vehicles in Acute Myeloid Leukemia

Background: Exosomes are microvesicles that play important roles in intercellular communications in both normal and tumor cells via their cargoes, which include microRNA (miRNA) and proteins. miRNAs play critical regulatory roles in hematopoiesis, and their abnormal expression is correlated with several hematological malignancies, including acute myeloid leukemia (AML). Exosome-derived miRNAs and proteins are increasingly recognized for their prognostic biomarker potential. Exosomes are being evaluated for their potential as novel drug-delivery vehicles due to their endogenous nature and ability to carry small molecule drugs.

Aims: We aim to characterize HSC-derived exosomes, investigate the biomarker potential of exosomes derived from AML patient samples (by examining their proteomic and miRNA profiles) and utilize exosomes as innovative drug delivery vehicles with the ability to eliminate leukemic stem cells in a targeted manner.

Methods: Secreted exosomes from murine bone marrow HSCs were isolated from conditioned medium and visualized using confocal microscopy. We isolated exosomes and performed miRNA profiling, using qPCR, and LC-MS/MS proteomic analysis to characterize the constituents. We also isolated exosomes from the CD34+ cells of three AML patient samples and profiled 372 of the most abundantly expressed miRs in these cells compared to normal CD34+ cells. We analyzed the proteome of these exosomes as well. In order to assess the utility of exosomes as drug carriers, exosomes from bone marrow-derived OP9 stromal cells were transfected with Daunorubicin (1ug/ul). Normal CD34+ cells and patient-derived AML samples (from n=2 patients) were treated with varying doses of the drug-loaded exosomes. Drug-loaded exosomes uptake was tracked with a Texas Red siRNA. After 24 hours, cells were screened for apoptosis. To test the feasibility of targeted exosomes, OP9 cells were exposed to AML patient samples for 48 hours. The patient cells were then removed and, 24 hours later, "trained" stromal cell-derived exosomes were isolated from the media and transfected with Daunorubicin. These patient-specific, exosomes were plated with both the corresponding patient's CD34+ cells as well as normal CD34+ primary cells. After 24 hours, apoptosis was measured.

Results: miRNA profiling of murine bone marrow showed miR-21a, miR-92a and miR-25 were most abundant in exosomes. Proteomic LC-MS/MS analysis revealed presence of exosome-associated novel proteins such as Syntenin-1. Syntenin-1, which is known to bind IL-5R and promote myelopoiesis, was present in significantly higher levels in HSCs compared to myeloid progenitors, implying a functional role for exosome derived Syntenin-1. miRNA profiling in AML samples revealed distinct signature profiles. Importantly, exosome derived miRs such as -1290, -375, -205 and -21-that are known prognostic markers in cancers such as prostate, ovarian and hepatocellular carcinoma-were significantly upregulated in all the three exosome-derived AML samples. The drug-loaded exosomes were successful in inducing significant apoptosis in two patient samples tested. These drug-loaded exosomes also induced cell death in CD34+ normal cells when compared to control exosomes. However, the patient-trained exosomes specifically eliminated 92% of CD34+ AML patient cells, while causing significantly less cell death (44%) of normal CD34+ primary cells exposed to drug-loaded, patient-trained exosomes.

Summary/Conclusion:

Taken together, our data predict important functional roles for exosome-derived Syntenin-1 in regulating lineage specific hematopoietic differentiations. Furthermore, for the first time, we have identified highly upregulated select exosome-derived miRs from AML patient samples whose prognostic value has been recently reported for other cancers, making these miRs promising candidates for AML biomarkers as well. Finally, stromal cell-derived, drug-loaded exosomes are not only able to induce apoptosis in AML patient samples, but they can effectively be trained by leukemic cells to favor uptake resulting in targeted elimination of leukemic over normal CD34+ cells.