Date

4-2015

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology & Immunology

Institution

Oregon Health & Science University

Abstract

Homeostatic hematopoiesis is defined by the division and differentiation of rare self-renewing stem cells, maintained and regulated by the diverse constituent cells of their microenvironment. Numerous studies have investigated the supportive roles of many types of marrow stroma, and it has been proposed that leukemogenesis is accompanied by a conversion of this space. The leukemic marrow microenviroment exhibits decreased capacity for hematopoietic stem and progenitor cell (HSPC) maintenance capability, while promoting and protecting leukemic blasts. In patients and model animals, these processes eventually result in hematopoietic failure. The mechanisms for such a conversion, however, remain unclear. HSPC support is a role that has been variously ascribed to osteoblasts, nonmyelinating Schwann cells, endothelium, and mesenchymal stromal cells, among others. As residual nonmalignant HSPC have themselves been shown to be altered in patients with acute myeloid leukemia (AML), this process requires either multiple independent specific mediators, or a highly pleiotropic mechanism. The trafficking of exosomes (small, membrane-enclosed extracellular vesicles that carry protein and nucleic acid cargo between cells) within the marrow space provides such a pleiotropic mechanism, as they have the potential to deliver regulatory messages directly to the cytoplasm of recipient cells without any known entry restriction. In these experiments, I begin to delineate the role of AML-derived exosomes in the regulation of the marrow microenvironment. Through electron and fluorescence microscopy, nanoparticle tracking analysis, and microRNA microarray, I demonstrate that AML blasts secrete exosomes containing concentrated microRNA, the makeup of which clearly distinguishes cells from exosomes and nonmalignant hematopoietic cells from AML blasts. These exosomes are taken up by both HSPC and marrow stroma, transferring sufficient microRNA to effect as much as a 10-fold increase in cellular concentration. In order to determine the physiologic impact of this exosome transfer, I evaluate exosome-exposed HSPC and stroma, both in vitro as well as through several in vivo models of exposure, using xenograft, intrafemoral injection of purified exosomes, and an extramedullary disease model to isolate the effects of exosomes from those of other secreted and contact-mediated factors. AML-derived exosomes suppress the expression of several HSPC retention and maintenance factors in stromal cells, and directly cause a decrease in the ability of HSPC to home along a CXCL12 gradient, as well as their clonogenicity (their ability to generate colonies of hematopoietic-lineage cells in culture). The marked differences between the microRNA content of malignant and nonmalignant exosomes raises the possibility of tracking these molecules as a potential biomarker of disease. Exosome-based biomarkers have particular advantages in AML, as monitoring patients in remission is of central importance to treatment (> 50% of patients relapse), and as direct observation of leukemic blasts is only reliably possible in those with advanced disease. Through a series of xenograft experiments using the Molm-14 and HL-60 cell lines, I demonstrate that exosomes present a serum-detectable microRNA signature of disease, to which both AML blasts and modified stroma contribute. This signature is durable through treatment with either cytarabine or targeted kinase inhibition. Additional experiments in an alternative AML cell line and primary patient samples provide promising support for broad applicability of serum exosome microRNA as a biomarker in AML. Finally, I pursue a mechanistic explanation of the observed effects of AML exosomes on recipient HSPC and stroma, through in-depth investigation of selected transferred microRNA. Employing RISCTrap technology and validating 3' untranslated region (3’UTR) luciferase assays and exosome transfer studies, I capture a broad sampling of targets of miR-155, working with this microRNA because of its appreciated relevance in hematopoiesis and leukemia as well as its prominence in AML-derived exosomes. This data set both confirms previously identified targets and identifies several novel potential targets. Further, it provides a starting point for an investigation of interacting partners, in order to better identify pathways regulated by this microRNA. Included targets are of known hematopoietic relevance in numerous cell types, supporting the hypothesis that exosomal microRNA is capable of co-regulating a diverse set of processes in a diverse set of cells. In total, these experiments represent a body of evidence that supports exosomes as a mechanism by which microRNA are transferred between AML blasts and multiple marrow-resident cell types. These microRNA contribute to the regulation of several molecular processes, impacting the conversion of the marrow microenvironment from a hematopoietic niche to a leukemic niche, a process with the potential to mediate a major cause of morbidity and mortality in AML patients. Further, AML exosomal microRNA are detectable in the peripheral blood, where they carry a detectable signature of disease, providing a promising avenue for development of a minimally invasive, clinically-relevant biomarker.

Identifier

doi:10.6083/M4QN65HZ

School

School of Medicine

Available for download on Saturday, April 14, 2018

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