Relapse remains the major cause of mortality for patients with Acute Myeloid Leukemia (AML). Improved tracking of minimal residual disease (MRD) holds the promise of timely treatment adjustments to preempt relapse. Current surveillance techniques detect circulating blasts that coincide with advanced disease and poorly reflect MRD during early relapse. Here, we investigate exosomes as a minimally invasive platform for a microRNA (miRNA) biomarker. We identify a set of miRNA enriched in AML exosomes and track levels of circulating exosome miRNA that distinguish leukemic xenografts from both non-engrafted and human CD34+ controls. We develop biostatistical models that reveal circulating exosomal miRNA at low marrow tumor burden and before circulating blasts can be detected. Remarkably, both leukemic blasts and marrow stroma contribute to serum exosome miRNA. We propose development of serum exosome miRNA as a platform for a novel, sensitive compartment biomarker for prospective tracking and early detection of AML recurrence.

Exosomes are paracrine regulators of the tumor microenvironment and contain complex cargo. We previously reported that exosomes released from acute myeloid leukemia (AML) cells can suppress residual hematopoietic stem and progenitor cell (HSPC) function indirectly through stromal reprogramming of niche retention factors. We found that the systemic loss of hematopoietic function is also in part a consequence of AML exosome-directed microRNA (miRNA) trafficking to HSPCs. Exosomes isolated from cultured AML or the plasma from mice bearing AML xenografts exhibited enrichment of miR-150 and miR-155. HSPCs cocultured with either of these exosomes exhibited impaired clonogenicity, through the miR-150- and miR-155-mediated suppression of the translation of transcripts encoding c-MYB, a transcription factor involved in HSPC differentiation and proliferation. To discover additional miRNA targets, we captured miR-155 and its target transcripts by coimmunoprecipitation with an attenuated RNA-induced silencing complex (RISC)-trap, followed by high-throughput sequencing. This approach identified known and previously unknown miR-155 target transcripts. Integration of the miR-155 targets with information from the protein interaction database STRING revealed proteins indirectly affected by AML exosome-derived miRNA. Our findings indicate a direct effect of AML exosomes on HSPCs that, through a stroma-independent mechanism, compromises hematopoiesis. Furthermore, combining miRNA target data with protein-protein interaction data may be a broadly applicable strategy to define the effects of exosome-mediated trafficking of regulatory molecules within the tumor microenvironment.

Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long‐term hematopoietic stem cells (LT‐HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT‐HSC in the BM of murine xenografts with emphasis on the role of AML‐derived extracellular vesicles (EV). AML‐EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT‐HSC quiescence. Mechanistically, AML‐EV transfer a panel of miRNA, including miR‐1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo‐phosphorylation, which in turn impairs protein synthesis in LT‐HSC. While HSC functionally recover from quiescence upon transplantation to an AML‐naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double‐strand breaks and evidence of a sustained DNA‐damage response. In sum, AML‐EV contribute to niche‐dependent, reversible quiescence and elicit persisting DNA damage in LT‐HSC.

Successive adaptation of the bone marrow (BM) from homeostatic hematopoietic microenvironment to a self-reinforcing niche is an integral aspect of leukemogenesis. Yet, the cellular mechanisms underlying these functional alterations remain to be defined. Here, we found that AML incursion precipitates compartmental endoplasmic reticulum (ER) stress and an unfolded protein response (UPR) in both leukemia and stromal cells. We observed that extracellular vesicles (EV) transmit ER stress in vivo from the AML xenograft to BM stroma, whereby the upregulation of core UPR components drives subsequent osteolineage differentiation of mesenchymal stem cells (MSC). Finally, we show that the underlying mechanism involves quantitative incorporation and cell-cell transfer of Bone Morphogenic Protein 2 (BMP2), a potent osteogenic signal, by AML-EVs. Corroborative studies in AML patient samples support the translational relevance of AML-EVs as a platform for BMP trafficking and source of compartmental crosstalk. Transmissible ER stress was previously identified as a source of chemoresistance in solid tumor models, and this work reveals a role in remodeling the BM niche in AML.

The bone marrow stroma maintains hematopoiesis and coordinately regulates regenerative responses through dynamic interactions with hematopoietic stem and progenitor cells. Recent studies indicate that stromal components in the bone marrow of leukemia patients undergo a process of successive adaptation that in turn exerts dramatic effects on the hematopoietic stem cell compartment and promotes leukemic drug resistance. Therefore, functional changes in discrete marrow stromal populations can be considered an aspect of leukemia biogenesis in that they create an aberrant, self-reinforcing microenvironment. In this review, we will describe the current understanding of the remodeling of the hematopoietic stem cell niche following invasion by leukemia cells. We place emphasis on existing evidence of how mesenchymal stem cells and their progeny facilitate neoplastic growth and describe available models and analytical techniques to understand the conversion of the niche toward disease persistence. STEM CELLS 2018;36:304-312 SIGNIFICANCE STATEMENTThere is increasing awareness that the bone marrow microenvironment is a critical contributor to therapeutic resistance and relapse progression in hematological malignancies. Experimental evidence discussed herein highlights key aspects of the stromal conversion by leukemia and the resulting aberrant signaling within the niche. This article discusses the advantages and limitations of available experimental model system and emphasizes open scientific questions and opportunities.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

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