Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-ofconcept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. Cancer Res; 73(2); 918-29. Ó2012 AACR.

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.

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.

Stem-like" TCF1 + CD8 + T cells (T SL ) are necessary for long-term maintenance of T cell responses and the efficacy of immunotherapy but, as tumors contain signals that should drive T-cell terminal-differentiation, how these cells are maintained in tumors remains unclear. In this study, we found that a small number of TCF1 + tumor-specific CD8 + T cells were present in lung tumors throughout their development. Yet, most intratumoral T cells differentiated as tumors progressed, corresponding with an immunologic shift in the tumor microenvironment (TME) from "hot" (T cell-inflamed) to "cold" (non-T cell-inflamed). By contrast, most tumor-specific CD8 + T cells in tumor-draining lymph nodes (dLNs) had functions and gene expression signatures similar to T SL from chronic LCMV infection, and this population was stable over time, despite the changes in the TME. dLN T cells were the developmental precursors of, and were clonally related to, their more differentiated intratumoral counterparts. Our data support the hypothesis that dLN T cells are the developmental precursors of the TCF1 + T cells in tumors which are maintained by continuous migration. Finally, CD8 + T cells similar to T SL were also present in LNs from lung adenocarcinoma patients, suggesting a similar model may be relevant in human disease. Thus, we propose that the dLN T SL reservoir has a critical function in sustaining antitumor T cells during tumor development and protecting them from the terminal differentiation that occurs in the TME.

No abstract

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 Th2 biased response and predisposes to features of AD in mice. Upon treatment with the TLR7 agonist imquimod (IMQ), Trim32 knockout (KO) mice displayed compromised psoriasiform phenotypes and defective Th17 response. Instead, IMQ treatment of Trim32 KO mice induced AD-like phenotypes with enhanced skin infiltration of eosinophils and mast cells, elevation of Th2 cytokines/chemokines expression, and reduced expression of filaggrin protein expression. Furthermore, while the induction of phosphorylated Stat3 and RelA were compromised following IMQ treatment in the KO mice, phosphorylated Stat6 was elevated. CCL20 induction by TNFα and IL-17A was reduced in Trim32 deficient keratinocytes whereas CCL5 induction by TNFα 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 AD patients. Based on Trim32 induction by IMQ, the lower levels of TRIM32 in AD skin compared to healthy control and psoriatic skin suggest a defective TRIM32 pathway in AD pathogenesis.

We presented naturalistic combinations of virtual self-movement stimuli while recording neuronal activity in monkey cerebral cortex. Monkeys used a joystick to drive to a straight ahead heading direction guided by either object motion or optic flow. The selected cue dominates neuronal responses, often mimicking responses evoked when that stimulus is presented alone. In some neurons, driving strategy creates selective response additivities. In others, it creates vulnerabilities to the disruptive effects of independently moving objects. Such cue interactions may be related to the disruptive effects of independently moving objects in Alzheimer's disease patients with navigational deficits.