Translational suppression of c-Myb by exosome micro-RNA constrains hematopoietic reserve in the AML NICHE

Abstract

Hematopoietic stem and progenitor cell (HSPC) activity is regulated through the coordinate interaction of cell-autonomous programs and microenvironmental cues. The invasion of the bone marrow by myeloid leukemia cells upends this balance and actively suppresses HSPC function. The mechanism by which this occurs remains unknown. We recently demonstrated that small membrane derived-vesicles (exosomes) traffic protein and RNA from leukemia- to bystander cells, allowing for their potential contribution to this process. Herein we use NSG xenografting and in vitro approaches to show that exosome trafficking from AML blasts to stromal cells results in the loss of cell adhesion via suppression of Scf and CXCL-12. We also demonstrate that AML exosomes regulate HSPC directly, causing downregulation of CXCR4 with aggregate HSPC mobilization. We find that exosome exposure results in the suppression of critical hematopoietic transcription factors Hoxa9 and c-Myb and loss of murine progenitor activity (CFU-C) in vitro, after intrafemoral injection of purified exosomes or in an extramedullary AML xenograft model. To account for these effects we systematically explored the exosome micro (mi-) RNA content, revealing the enrichment of a select panel, including miR-146a, -150, -210 and -155, that uniquely distinguish AML- from normal CD34-derived exosomes. Direct miRNA targeting of key mRNA targets was confirmed after miRNA-mimic and exosome exposure using candidate 3’-UTR luciferase vector constructs. Our work establishes a new model for the coordinate regulation of HSPC in the leukemic microenvironment whereby leukemia derived exosomes mediate the mobilization of progenitors from specialized marrow niches concurrent with the miRNA mediated dysregulation of clonogenic HSPC. We propose that AML exosome trafficking contributes to the coordinate erosion of HSPC function in the AML niche. Hematopoietic stem and progenitor cell (HSPC) activity is regulated through the coordinate interaction of cell-autonomous programs and microenvironmental cues. The invasion of the bone marrow by myeloid leukemia cells upends this balance and actively suppresses HSPC function. The mechanism by which this occurs remains unknown. We recently demonstrated that small membrane derived-vesicles (exosomes) traffic protein and RNA from leukemia- to bystander cells, allowing for their potential contribution to this process. Herein we use NSG xenografting and in vitro approaches to show that exosome trafficking from AML blasts to stromal cells results in the loss of cell adhesion via suppression of Scf and CXCL-12. We also demonstrate that AML exosomes regulate HSPC directly, causing downregulation of CXCR4 with aggregate HSPC mobilization. We find that exosome exposure results in the suppression of critical hematopoietic transcription factors Hoxa9 and c-Myb and loss of murine progenitor activity (CFU-C) in vitro, after intrafemoral injection of purified exosomes or in an extramedullary AML xenograft model. To account for these effects we systematically explored the exosome micro (mi-) RNA content, revealing the enrichment of a select panel, including miR-146a, -150, -210 and -155, that uniquely distinguish AML- from normal CD34-derived exosomes. Direct miRNA targeting of key mRNA targets was confirmed after miRNA-mimic and exosome exposure using candidate 3’-UTR luciferase vector constructs. Our work establishes a new model for the coordinate regulation of HSPC in the leukemic microenvironment whereby leukemia derived exosomes mediate the mobilization of progenitors from specialized marrow niches concurrent with the miRNA mediated dysregulation of clonogenic HSPC. We propose that AML exosome trafficking contributes to the coordinate erosion of HSPC function in the AML niche.