Flt3 Kinase Regulates Microvesicle Transfer of miRNA Between AML and Stromal Cells

Abstract 1492

The presence of an internal tandem duplication in the receptor tyrosine kinase Flt3 (Flt3-ITD) is found in 25–30% of cytogenetically normal AML and confers a worsened prognosis, including an increased likelihood for relapse after hematopoietic stem cell transplantation (HSCT). This tendency toward relapse, combined with the improved capacity of Flt3-ITD+ disease to resist chemotherapy, may imply mechanisms of resistance beyond those present in leukemias lacking this mutation. Microvesicles and exosomes, membrane-bound extracellular vesicles that capture cell-specific protein and RNA, have previously been isolated from the serum of CLL patients (Ghosh et al., Blood 2010 Mar 4;115(9): 1755–64). We recently observed that vesicles are constitutively produced by both AML cell lines and by AML blasts isolated from patients. Using light scatter analysis and transmission electron microscopy, we found that vesicles produced by the HL60 (Flt3-ITD-) and Molm14 (Flt3-ITD+) cell lines, as well as by AML patient cells, predominantly fall within the 30–100nm range, generally considered to specify exosomes. To demonstrate vesicle transfer to neighboring cells, we labeled vesicles produced by the HL-60 cell line with the fluorescent membrane dye PKH-26 and imaged their uptake by stromal cells. Internalization of labeled exosomes was detectable within 15 minutes following exposure, and occurred at approximately 27 particles per cell by 2 hours (n=20). This result indicates rapid uptake of exosomes by non-phagocytic bystander cells, and supports a potential role for the vesicle content in altering the cell phenotype. We next tested for the presence of certain candidate mRNAs in the AML cell lines HEL, HL-60, MOLM-14, and U937, and in vesicles produced by those cell lines. We detected several relevant mRNAs, including nucleophosmin-1 and Flt3-ITD, in the vesicle preparations. During a more global comparison of vesicle and cell-of-origin RNA spectra using bioanalyzer analysis, we found that the RNA in microvesicles from (Flt3-ITD+) MOLM14 cells contained no detectable ribosomal RNA, but a markedly increased proportion of small RNA transcripts, suggesting that miRNA content might be increased. An initial screen of cultured MOLM-14 cells in the presence and absence of a small-molecule Flt3 inhibitor (AC220) and microvesicles isolated from the culture supernatant revealed several candidate miRNAs, including let-7a, miR-99b, and miR-155, whose expression varied with the presence of inhibitor. Our results not only support a role for Flt3 kinase activation in determining the miRNA expression profile, but provide evidence for the kinase-regulated incorporation of miRNA into cell membrane vesicles. The recent description of several AML subtypes, including Flt3-ITD+ disease with unique miRNA profiles, and the role of miRNA as potent regulators of both microenvironmental function and immune responses provides strong motivation to evaluate the role of vesicles in AML therapy evasion.