AML-Derived Microvesicles as Mediators of Cell-Cell Signaling In the Leukemic Microenvironment

Abstract 1700

Directed signaling between acute myelogenous leukemia (AML) cells and the bone marrow microenvironment plays an important role in leukemia cell survival. How leukemic blasts transform the homeostatic bone marrow microenvironment is not fully explained by known ligand-receptor interactions. Microvesicles (MV) are membrane-enclosed compartments that, depending on cell specific pathways, range in diameter from 50–100 nm (exosomes) to 100–1000 nm (microparticles) and are constitutively released into circulation in many tissues. Biogenesis has been found to be upregulated in a range of cancers. We hypothesized that MV-mediated transfer of signaling components by AML blasts may contribute to intercellular signaling in the leukemic niche. In an initial screen, we characterized MV that we recovered from the culture supernatant of multiple AML-derived cell lines, as well as AML cells from patients. Live-cell imaging of primary AML blasts using the exosomal pathway-specific dye N-Rh-PE revealed that at least a portion of these vesicles could be categorized as exosomes. Consistent with the non-random incorporation of molecular cargo observed in recent studies with solid tumors, we found the protein and RNA content of MV to be distinct from cellular lysates. In a focused, candidate target approach, our experiments showed that MV contain mRNA transcripts encoding the broadly relevant AML prognostic markers FLT3-ITD, NPM1, and IGF-1R. In both cell lines and cultured primary AML cells, insulin receptor (IR) and IGF-1R mRNAs were enriched up to ∼400-fold over GAPDH in MV versus cellular fractions. The IGF-1R receptor tyrosine kinase (RTK) transduces growth and survival signals, and the IGF-I signaling pathway has recently been identified as a potentially important candidate for molecularly targeted drug therapy for AML. Corroborative evidence for a role of MV and IGF-I signaling in shaping the leukemic microenvironment comes from the detection of human IGF-1R mRNA in cDNA prepared from mouse stromal cells (OP9) co-cultured for 48 hours with primary AML cells across a cell impermeable transwell barrier. To determine if the mRNA is transferred in a biologically significant quantity, we developed a plasmid-based quantitative PCR method to measure and compare transferred transcripts to steady state IGF-1R mRNA levels in the AML cell line HL-60. Results indicated that 0.3 HL-60 cellular equivalents of the human IGF-1R transcript were detected per OP9 cell following co-culture with primary AML cells. These data imply that transfer occurs at a level that may be functionally relevant. To further explore the potential role of MV in AML-stroma signaling, experiments are underway with HL-60 cells stably transfected with a pcDNA vector encoding the human IGF1R with a C-terminal FLAG tag. These HL-60:IGF1R-FLAG cells release MV that contain the IGF1R-FLAG transcript and will serve as a model to explore the downstream intracellular RTK signaling. In conclusion, we report for the first time the production of MV containing protein and disease-specific transcripts by AML blast cells, the selective enrichment in MV of disease-relevant transcripts, and direct cell-cell transfer of leukemia cell mRNAs to bystander cells. Our findings underscore the potential role of MV in shaping the AML microenvironment through lateral transfer of RTK mRNA transcripts. We propose a role for MV during disease progression and highlight their potential as highly sensitive, cell-free, and minimally invasive AML biomarkers.