Noah Hornick

Serum Exosome MicroRNA as a Minimally-Invasive Early Biomarker of AML

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.

Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-β and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell–cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

AML suppresses hematopoiesis by releasing exosomes that contain microRNAs targeting c-MYB

Exosomes shed from acute myeloid leukemia cells suppress the expression of a hematopoietic transcription factor in the bone marrow. AML dispatches micromanagers In patients with acute myeloid leukemia (AML), the production of healthy blood cells from hematopoietic stem cells in the bone marrow (a process called hematopoiesis) is suppressed, prompting the need for bone marrow transplants. AML cells shed extracellular vesicles called exosomes that contain molecules that suppress hematopoiesis by reprogramming the stem cell niche. Hornick et al. discovered another way that AML exosomes block this critical process by delivering microRNAs to hematopoietic stem cells. The AML-derived exosomes contained two mature microRNAs that target the mRNA encoding c-MYB, a transcription factor involved in hematopoiesis. Other targets of these AML-derived exosomal microRNAs reveal interconnected networks targeting transcripts that produce proteins that control the cell cycle. The findings suggest that disrupting this mode of intercellular communication might enhance hematopoiesis in AML patients. 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.

Alterations in acute myeloid leukaemia bone marrow stromal cell exosome content coincide with gains in tyrosine kinase inhibitor resistance.

Bone marrow stromal cells (BMSC) in acute myeloid leukaemia (AML) contribute to extrinsic drug resistance, generally attributed to cell–cell contact or secreted cytokines (Jacamo et al, 2014). However, a recent report indicates that stromal protection may also occur via other soluble factors (Yang et al, 2014). Extracellular vesicles, such as exosomes, traffic protein and RNA between cells (Valadi et al, 2007). Stromal exosomes were recently shown to confer a proliferative advantage to multiple myeloma cells via transfer of microRNA (miRNA) (Roccaro et al, 2013). The functional impact of stromal exosomes in AML has not been studied. We therefore hypothesized that AML stroma release exosomes that protect leukaemia cells and traffic a distinct subset of miRNA and cytokines. We isolated stromal cells from the marrow aspirates of 20 patients (AML-BMSC; see Table SI for patient characteristics) and five healthy controls (N-BMSC) according to a previously published protocol (Tyner et al, 2013). The cells were fibroblastic, adherent and expressed CD90, but not CD45 epitopes (Fig 1A). Reverse transcription polymerase chain reaction (RT-PCR) confirmed the expression of canonical stromal transcripts in both populations (Fig 1B) (Boxall & Jones, 2012). However, quantitative analysis revealed altered expression in AML-BMSC for CXCL12, KITLG and CXCL1, as well as for genes previously reported in modified stroma in myelodysplastic syndrome (IGFBP4, ANGPTL4) (Fig 1C) (Medyouf et al, 2014). The contribution of stromal-derived exosomes has been established in other malignancies (Roccaro et al, 2013). We isolated vesicles using ultracentrifugation and then used vesicle tracking analysis, electron microscopy and Western blotting to demonstrate a vesicle population conforming in size, morphology and tetraspanin membrane proteins to exosomes (Fig 1D) (Valadi et al, 2007). Fig 1 AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity ... Exosome biogenesis allows for selective incorporation of different RNA species (Valadi et al, 2007). We determined the spectrum of BMSC exosome RNA using a Bioanalyser (Agilent, Santa Clara, CA, USA) and observed a relatively greater abundance of small RNAs compared with the parent cell (Fig 1D). Increased levels of MIR155 and MIR375 can independently identify AML patients at high risk for recurrence, with MIR155 contributing to the pathogenesis of several other haematological malignancies (Marcucci et al, 2013; Wang et al, 2013). Reasoning that stromal cell reprogramming in AML leads to unique exosomal miRNA incorporation, we used quantitative RT-PCR to compare the MIR155 and MIR375 levels in stromal cells from 12 patients (4 normal, 8 AML) and the exosomes released during the culture period. Strikingly, all eight AML-BMSC samples showed a statistically significant fold increase in incorporation of MIR155 and MIR375 in the exosomes relative to their parent cells, while no such increase was observed for N-BMSCs (Fig 1E). The comparative enrichment in AML-BMSC exosomes persisted even after 10 passages in tissue culture devoid of leukaemia cells, suggesting a durable change in stromal cell biology (Fig 1E). We then directly compared miRNA content between exosome isolates, independent of the parent cell content, and found MIR155, but not MIR375, was consistently elevated in AML-BMSC exosomes (Fig 1F). Taken together, the data indicate that exosomes released from AML-BMSCs are selectively enriched for miRNA that signify disease risk status in AML. Cytokines and growth factors contribute to leukaemia niche function (Yang et al, 2014). We reasoned that exosome- associated and directly secreted cytokine alterations from AML-BMSCs exist. In a multiplex screening approach using bead-based technology (Luminex, Austin, TX, USA) and enzyme-linked immuno-sorbent-assay (ELISA) kits (Life Technologies, Carlsbad, CA, USA), we studied the concentration of cytokines in the exosomes and in the vesicle-depleted soluble protein fraction of conditioned media from AML-BMSCs (n = 10) and N-BMSCs (n = 3). Exosomal cytokine concentrations ranged from 1·6 pg/ml (B-FGF) to >10 000 pg/ml [interleukin (IL) 8], while other cytokines were undetected in exosomes or in the soluble protein fraction from BMSCs (IL2, IL5, IL17A, TNF, IFNG). We found statistically significant enrichment of epidermal growth factor (P < 0·05), as well as a relative depletion of hepatocyte growth factor (P = 0·08) in exosomes from AML-BMSC, changes that were not observed in the soluble protein fraction when compared to N-BMSCs (Fig 2A). Concentrations of other cytokines, whether exosome-associated or freely secreted, did not differ between the two populations. We separately analysed TGFB1, given its correlation with treatment response in AML (Hong et al, 2014). We found TGFB1 at concentrations ranging from 200 to 2000 pg/ml in exosomes from 10/10 AML-BMSC samples, but below the level of assay detection in exosomes from N-BMSCs (Fig 2B). Taken together, our results suggest that stromal cells in AML modify their exosome-associated cytokine concentrations in a manner independent from the directly secreted fraction. Fig 2 Exosomes from AML-BMSCs have altered cytokine levels and confer chemo-protection to AML cells: (A) Box-and-Whisker plot of a multiplex protein assay displaying the relative concentration of 19 cytokines in exosomes and in the vesicle-depleted soluble ... Our findings, along with existing reports on general stromal protection in AML, led us to hypothesize that stromal exosomes alter chemo-resistance in AML cells. Representing a standard component of AML therapy, we treated MOLM-14 FLT3 internal tandem duplication (FLT3-ITD+) AML cells with the nucleoside analogue cytarabine after exposure to exosomes from AML-BMSC, N-BMSC or control media (Fig 2C). The data provide the first evidence, to our knowledge, that exosomes from both AML patients and controls (Total n = 10, 6 AML-BMSC, 4 N-BMSC) protect AML cells from cytarabine. Further, when AML cells were treated with the FLT3 inhibitor AC220 after exposure, only AML-BMSC exosomes significantly protected AML cells (n = 6), while N-BMSC exosomes provided no such protective effect (n = 3) (Fig 2C). Our data in aggregate suggest that stromal cells in AML patients undergo modification that includes alterations in function as well as the protein and RNA present in the exosomes they release. We report a first demonstration that stromal cells in AML release exosomes enriched for known clinical risk factors, including TGFB1, MIR155 and MIR375. Finally, our data add stromal exosome trafficking as a candidate mechanism for extrinsic chemo-resistance within the niche in AML, with differential protection against kinase pathway inhibition observed only by AML-BMSC exosomes. Such unique protection could occur, for example, by exosomal miRNA-directed down-regulation of promoters of apoptosis or cell differentiation, thereby releasing the leukaemia cell from kinase pathway dependence. Future studies in this area may uncover new mediators of such resistance.

Trim32 Deficiency Enhances Th2 Immunity and Predisposes to Features of Atopic Dermatitis

Altered innate immunity is a feature of certain skin inflammatory diseases such as psoriasis and atopic dermatitis (AD). In this study, we provide evidence that deficiency in Trim32 (a tripartite motif [TRIM] protein with innate antiviral activity) contributes to a T helper type 2 biased response and predisposes to features of AD in mice. On treatment with the toll-like receptor 7 agonist imquimod (IMQ), Trim32 knockout mice displayed compromised psoriasiform phenotypes and defective T helper type 17 response. Instead, IMQ treatment of Trim32 knockout mice induced AD-like phenotypes with enhanced skin infiltration of eosinophils and mast cells, elevation of T helper type 2 cytokines/chemokines expression, and reduced expression of filaggrin protein expression. Furthermore, although the induction of phosphorylated Stat3 and RelA was compromised after IMQ treatment in the knockout mice, phosphorylated Stat6 was elevated. CC chemokine ligand 20 induction by tumor necrosis factor-α and IL-17A was reduced in Trim32-deficient keratinocytes, whereas CC chemokine ligand 5 induction by tumor necrosis factor-α and IL-4 was enhanced. In addition, Trim32 protein levels were elevated in mice treated with IMQ. Unlike Trim32 overexpression in psoriasis, TRIM32 levels were low in patients with AD. Based on Trim32 induction by IMQ, the lower levels of TRIM32 in AD skin compared with healthy control and psoriatic skin suggest a defective TRIM32 pathway in AD pathogenesis.