Enrichment and Trafficking of BCR-ABL Oncoprotein in Exosomes Promotes the IL-3 Independent Proliferation of Bystander Cells,

Abstract 3751

Chronic Myeloid Leukemia (CML) accounts for 15% of all adult leukemias worldwide and is characterized by the BCR-ABL t(9;22)(q34;q11) translocation. Experimental overexpression of BCR-ABL also provides evidence for oncoprotein-mediated induction of instability phenotypes and rescue from apoptosis in non-leukemic cells. Recent work with Chronic Lymphoid Leukemia (CLL) reveals the circulation of cell membrane-derived vesicles in the serum of patients and suggests in vitro signaling effects in the CLL microenvironment. Such lipid-enclosed vesicles, termed exosomes (30–100nm), are released by normal and malignant cell types and have been shown to traffic RNA and proteins between cells, presumably contributing to disease progression. These studies prompted us to evaluate a potential role for vesicles in transferring BCR-ABL oncoprotein to bystander cells and test the hypothesis that oncoprotein transfer may contribute to signaling in the leukemic microenvironment. K562 cells, a BCR-ABL-positive human CML cell line, have been shown to constitutively release membrane-derived vesicles and serve as a model for understanding their biogenesis. Using electron microscopy, we have confirmed exosome release from K562 cells, with >90% of vesicles ranging from 30–100nm in size (n=291). Comparison of cell of origin and exosome content confirmed the previously reported differential global protein profiles and showed colocalization of the exosomal marker CD63 with BCR-ABL by immunofluorescent imaging. Further, quantitative reverse transcription PCR indicated a 35-fold enrichment of BCR-ABL transcript in exosomes compared to the K562 cytosol. Because BCR-ABL is known to rescue certain cytokine-starved cell types from apoptosis, we next tested whether K562 cells could transfer BCR-ABL-enriched exosomes to bystander cells and subsequently foster their proliferation. To demonstrate trafficking of the BCR-ABL-enriched exosomes to bystander cells, a 0.4uM Boyden transwell chamber was used to coculture K562 and recipient cells while preventing direct cell-to-cell contact. Through semi-quantitative reverse transcription PCR we have successfully shown transfer of BCR-ABL transcript to two murine-derived cell lines, BaF3 (pro-B) and OP9 (stromal) cells, resembling cell types present in the leukemic microenvironment. Furthermore, transwell coculture with exosome-producing K562 cells promoted the canonical survival of BaF3 cells following IL-3-deprivation. This is consistent with previous studies where BCR-ABL overexpression rescues IL-3-deprived BaF3 cells from cell cycle arrest and apoptosis. Our work demonstrates the potential of exosome trafficking in affecting the phenotype of bystander cells in the CML microenvironment. Additionally, exosome enrichment encourages further research toward utilizing BCR-ABL-enriched vesicles as a minimally invasive serum biomarker for disease progression.