AML Exosomes Regulate Stromal Bystander Cells and Provide a Novel RNA Biomarker Platform

Abstract 1378

Cell membrane-derived vesicles carrying protein and RNA cargo have recently emerged as potent mediators of cell-cell communication. These vesicles traffic from their tissue of origin into the blood stream, providing a serum-based and minimally invasive biomarker platform for cancer-specific RNA and protein signatures. We were interested in evaluating vesicle production by acute myeloid leukemia (AML) cells, with an emphasis on RNA content and cell-cell trafficking. Here we demonstrate for the first time that cell lines and primary cells from AML patients release exosome-sized vesicles (30–100nm) capable of transfer to bystander cells. Analysis of their non-protein content revealed the enrichment of many coding- and non-coding RNAs with relevance to AML biology, including prognostically important transcripts encoding FLT3, NPM-1, IGF-IR, CXCR4 and MMP9. In addition, we demonstrated the presence of a diverse group of transcription factor mRNAs (MYC, MEF2C, GATA1, SHIP1, ID1, E2F1, CEPBA CEPBB) in exosomes. PCR array studies also confirmed the broad representation of cellular miRNAs. Among the 234 miRNAs represented, the levels of several (miR −9, −155 and −223 and LET-7A) were enriched over the levels seen in donor cells (U6 RNA control). When examining cell-cell trafficking of AML exosomes, we noted rapid entry into co-cultured stromal cells, with maximum uptake occurring between 2 and 3 hours. AML-derived mRNA transcripts were transferred to bystander cells without direct cell-cell contact after Transwell co-culture or direct exposure to purified exosome preparations, or via vesicle-rich media. Results show that exosome trafficking induced changes in gene expression (OPN, ANGPT, SCF, SDF-1A) and altered the secretion of cytokines and angiopoietic growth factors by bone marrow stromal cells. Additional proof-of-concept studies provided evidence for the canonical function of transferred RNA. Specifically, we found that transfer of IGF-IR mRNA led to proliferative responses in co-cultured OP9 stromal cells that were abrogated by the IGF-IR specific inhibitor picropodophyllin. Further, exosomes derived from AML cell lines (HL60 and Molm14) altered c-FOS and VEGF expression in OP9 cells as well as improving viability and promoting migration toward SDF-1A by a hematopoietic progenitor cell line (Baf3), respectively. Our observations support the concept that AML exosomes exhibit regulatory potential in the leukemic bone marrow niche via direct cytoplasmic cell-cell transfer of transcripts encoding tyrosine kinase receptors and transcription factor as well as micro- RNAs. We propose that AML exosomes contribute to the extrinsic signaling cues in the microenvironment that promote disease resistance in AML and that they are rich in biomarkers to guide diagnosis and treatment.