Seiichiro Yoshizawa

Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1.

Exosomes are small endosome-derived vesicles containing a wide range of functional proteins, mRNA, and miRNA. Exosomal miRNA from cancer cells helps modulate the microenvironment. In multiple myeloma (MM), the massive proliferation of malignant plasma cells causes hypoxia. To date, the majority of in vitro hypoxia studies of cancer cells have used acute hypoxic exposure (3-24 hours). Thus, we attempted to clarify the role of MM-derived exosomes in hypoxic bone marrow by using MM cells grown continuously in vitro under chronic hypoxia (hypoxia-resistant MM [HR-MM] cells). The HR-MM cells produced more exosomes than the parental cells under normoxia or acute hypoxia conditions, and miR-135b was significantly upregulated in exosomes from HR-MM cells. Exosomal miR-135b directly suppressed its target factor-inhibiting hypoxia-inducible factor 1 (FIH-1) in endothelial cells. Finally, exosomal miR-135b from HR-MM cells enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway. This in vitro HR myeloma cell model will be useful for investigating MM cell-endothelial cell interactions under hypoxic conditions, which may mimic the in vivo bone marrow microenvironment. Although tumor angiogenesis is regulated by various factors, exosomal miR-135b may be a target for controlling MM angiogenesis.

Clinical impact of down-regulated plasma miR-92a levels in non-Hodgkin's lymphoma.

Background We undertook a study to evaluate the clinical relevance of miR-92a in plasma obtained from non-Hodgkins lymphoma (NHL) patients, because the miR-17-92 polycistronic miRNA cluster plays a crucial role in lymphomagenesis and affects neo-angiogenesis. Methodology/Principal Findings Plasma miR-92a values in NHL were extremely low (<5%), compared with healthy subjects (P<.0001), irrespective of lymphoma sub-type. The very low plasma level of miR-92a increased in the complete response (CR) phase but did not reach the normal range, and the plasma level was lower again in the relapse phase. Patients in CR or CR unconfirmed with a plasma miR-92a level of less than the cut-off level showed a significantly high relapse rate compared with patients with normalized plasma miR-92a level. Conclusions/Significance The current results therefore indicate that the plasma miR-92a value could be a novel biomarker not only for diagnosis but also for monitoring lymphoma patients after chemotherapy.

Speciation of arsenic trioxide penetrates into cerebrospinal fluid in patients with acute promyelocytic leukemia

We assessed concentrations of arsenic trioxide (As(2)O(3)) and its metabolites in the plasma and cerebrospinal fluid in acute promyelocytic leukemia patients who achieved complete remission with intravenous As(2)O(3). Arsenic trioxide exists as high molecular mass proteins and low molecular mass proteins in the plasma, and metabolites seem to be able to penetrate blood-brain barrier. Methylarsonic acid (MA) in the cerebrospinal fluid is stably detected and its level was higher than that in plasma after As(2)O(3) treatment. Trivalent arsenic (AS(III)) and dimethylarsinic acid (DMA) became detectable after As(2)O(3) infusion, though the levels of arsenic metabolites in the cerebrospinal fluid was lower than plasma levels. Results suggest that a combinatory treatment of As(2)O(3) with other chemotherapeutics could be effective for APL patients with CNS involvement.

Human herpesvirus 6 reactivation on the 30th day after allogeneic hematopoietic stem cell transplantation can predict grade 2–4 acute graft‐versus‐host disease

Viral infections and their occult reactivation occasionally cause not only organ damage, but also exacerbation of acute graft‐versus‐host disease (aGVHD), which may increase transplantation‐related mortality synergistically. To determine correlations between viral reactivation and transplantation‐related complications, we performed various viral screening tests on the 30th day after allogeneic hematopoietic stem cell transplantation (HSCT), and assessed the clinical implications.

Replenishing exosomes from older bone marrow stromal cells with miR-340 inhibits myeloma-related angiogenesis

The study of bone marrow stromal cells (BMSCs) and the exosomes they secrete is considered promising for cancer therapy. However, little is known about the effect of donor age on BMSCs. In the present study, we investigated the therapeutic potential of BMSC exosomes derived from donors of different ages using an in vivo model of hypoxic bone marrow in multiple myeloma (MM). We found that donor age was strongly related to senescent changes in BMSCs. Exosomes derived from young BMSCs significantly inhibited MM-induced angiogenesis in Matrigel plugs. The exosomal microRNA (miRNA) expression profile was different between young and older BMSCs, despite similarities in the size and quantity of exosomes. Of note was the observation that the antiangiogenic effect of older BMSCs was enhanced by direct transfection of miR-340 that was preferentially expressed in exosomes derived from young BMSCs. We found that miR-340 inhibited angiogenesis via the hepatocyte growth factor/c-MET (HGF/c-MET) signaling pathway in endothelial cells. Our data provide new insights into exosome-based cancer therapy by modification of BMSC-derived exosomes.

Exosomal miRNA Signatures for Late-Onset Acute Graft-Versus-Host Disease in Allogenic Hematopoietic Stem Cell Transplantation

Recent studies have demonstrated that exosomal microRNAs (miRNAs) have the potential of facilitating molecular diagnosis. Currently, little is known about the underlying mechanism behind late-onset acute graft-versus-host disease (LA GVHD). Identifying differentially expressed miRNAs in exosomes should be useful for understanding the role of miRNAs in this disease. This study was established to investigate the relevance of miRNAs in exosomes derived from patients developing LA GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Plasma samples were collected from patients with LA GVHD (n = 5), non-GVHD (n = 5), and controls (n = 8) for exosomal miRNA expression profiling using a TaqMan low-density array; the results were validated by quantitative reverse transcription polymerase chain reaction (RT-PCR). We analyzed exosomal miRNAs differentially expressed among these three groups. MirTarBase was employed to predict potential target genes of the miRNAs specific for LA GVHD. We detected 55 miRNAs that were differentially expressed with a significant change >2.0-fold between LA GVHD and non-GVHD. Of these, we selected the 10 miRNAs (miR-423-5p, miR-19a, miR-142-3p, miR-128, miR-193b, miR-30c, miR-193a, miR-191, miR-125b, and miR-574-3p) with the most significant differential expression. Using quantitative RT-PCR, we further identified that miR-128 was significantly upregulated at the onset of LA GVHD compared with that in normal controls and is a promising diagnostic marker of LA GVHD, with an area under the curve (AUC) value of 0.975. MirTarBase analysis revealed that the predicted target genes of miR-128 are involved in the immune system and inflammation. Increased expression of miR-128 may serve as a novel, noninvasive biomarker for early LA GVHD diagnosis.

Genetic variations of bone marrow mesenchymal stromal cells derived from acute leukemia and myelodysplastic syndrome by targeted deep sequencing

Bone marrow mesenchymal stromal cells (MSCs), which support proliferation and differentiation of hematopoietic stem cells, may play a crucial role in the pathogenesis of myeloid neoplasms. To determine whether MSCs in myeloid neoplasms harbor distinct somatic mutations that may affect their function, we used a targeted gene sequencing panel containing 50 myeloid neoplasm-associated genes with coverage of ≥500. We compared the genetic alterations between MSCs and bone marrow hematopoietic (BM) cells from patients with acute leukemia (n=5) or myelodysplastic syndrome (MDS, n=5). Non-synonymous somatic mutations, such as DNMT3A-R882H and FLT3-D835Y, were only detected in BM cells with high allelic frequency. We found several non-synonymous genetic variants overlapping BM cells and MSCs, including TP53 and ASXL1, partially owing to the heterogenous cell fraction of MSC samples and lineage fidelity. We also found MSC-specific genetic variants with very low allelic frequency (7% to 8%), such as NF1-G2114D and NF1-G140. Further studies in large cohorts are needed to clarify the molecular properties of MSCs including age-related genetic alterations by targeted deep sequencing.

Determination of donor-derived killer immunoglobulin-like receptor (KIR) by sequential genotyping in hematopoietic stem cell-transplanted patients

Abstract Donor killer immunoglobulin-like receptor (KIR) and KIR–ligand mismatch is considered vital in clarifying the mechanism of natural killer (NK) cell alloreactivity in hematopoietic stem cell transplantation (HSCT). In practical terms, however, it may be difficult to analyze the KIR genotype of donor cells directly as all donor cells are used for the transplant rather than for research purposes. To accurately estimate donor KIR genotype, we determined recipient KIR genotyping sequentially, at a minimum of two time points, using 19 KIR-specific primers in 10 patients who underwent HSCT. Among 10 patients, four had a KIR–ligand mismatch in the graft versus host direction. Sequential KIR genotyping showed the genotype changes at the time of engraftment (donor-derived) as well as relapse (recipient-derived). Our results highlight the utility of sequential KIR genotyping to better understand ligand–ligand, KIR–KIR, or ligand–KIR mismatches. Further studies, including a functional assay of NK cells may clarify the underlying mechanism of KIR ligand–donor KIR mismatch in HSCT.