Research ArticleCANCER EXOSOMES

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

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Science Signaling  06 Sep 2016:
Vol. 9, Issue 444, pp. ra88
DOI: 10.1126/scisignal.aaf2797
  • Fig. 1 Leukemia systemically impairs HSPC function.

    (A) Engraftment of luc+ Molm-14 cells was tracked and quantified using in vivo imaging. Two representative mice (1M and 2M, left and right) were imaged at multiple time points after engraftment. Days 0, 6, 13, 17, 22, and 24 are shown. Engraftment was quantitated by radiance as photons s−1 cm−2 sr−1;. 1M and 2M are individual animal designations. (B) Schematic of mGFP incorporation into the cell membrane. Exosomes harvested from Molm-14 cells transduced with a lentiviral vector containing the mGFP expression cassette were mGFP+ (representative image, right). LTR, long terminal repeat; pA, poly-adenylate; CMV, cytomegalovirus; EV, extracellular vesicle. (C) Confocal microscopy for GFP+ vesicles in the cytosol of HSPC from mice engrafted with mGFP+ Molm-14. Bone marrow aspirates from control and xenografted mice were sorted for c-KIT+, SCA-1+, and LIN markers. Scale bars, 2 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) c-KIT+ HSPCs from C57BL/6 mice were exposed to serum or serum exosomes collected from healthy (Control) and xenografted (Xeno) mice and then plated in methylcellulose and assessed for clonogenicity. CFU-C, CFU in culture. (E) Clonogenicity of c-KIT+ cells harvested from NSG mice 48 hours after mice were injected as indicated with serum exosomes from mice bearing xenografts. Data are expressed relative to mice injected with control serum. Data are means ± SEM of a representative of cells derived from at least three mice and plated in triplicate (*P > 0.05; ***P < 0.001; n.s., not significant, by Student’s t test).

  • Fig. 2 Leukemia exosomes suppress HSPC function and are enriched with specific miRNA transcripts.

    (A) Colony-forming capacity in murine c-KIT+ cells cultured with three doses of HL-60–derived exosomes for 48 hours and plated in methylcellulose. Data are means ± SEM of a representative of cells derived from at least three mice, plated in triplicate (*P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant, by Student’s t test). (B) Expression of hematopoietic and epigenetic regulatory genes measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in c-KIT+ cells exposed to three doses of HL-60–derived exosomes (Exo). Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented relative to expression in unexposed c-KIT+ cells. (C) Microarray comparison of miRNA isolated from Molm-14, HL-60, and nonmalignant human CD34+ cells (“C”) and respective exosomes (“E”). Targets with more than twofold mean difference between producing cell and exosome are shown, robust multiarray average (RMA)–corrected, and standardized to a mean of 0 and an SD of 1. Dendrogram values are 1 − Pearson's R. (D) Principal components (PC) analysis, performed after gene selection for all (63) targets detected in all samples and demonstrating significant [false discovery rate (FDR) < 0.05] enrichment in leukemic exosomes relative to those from cells. (E) Comparison and categorization (as measured by microarray) of mature and pre-miRNA (Pre) abundance in Molm-14 cells and exosomes as detected by microarray.

  • Fig. 3 Exosome-delivered miR-155 down-regulates recipient cell abundance of c-Myb.

    (A) Fold change in miR-155 abundance by qRT-PCR in c-KIT+ cells cultured overnight with HL-60– or Molm-14–derived exosomes relative to controls. U6 small nuclear RNA (snRNA) was the reference gene. (B) Fold change in abundance of miR-155 in exosomes from the serum of xenografted mice relative to that from two control mice as assessed by qRT-PCR. 1F, 2F, etc., designate individual animal. (C) 3′UTRs of candidate targets were cloned into the psiCheck-2 vector, and miRNA targeting was determined by loss of luminescence when coexpressed with miRNA of interest, compared to coexpression with control miRNA or vehicle. Data are % RLU (relative luciferase units) of the no-miRNA control. (D) c-Myb 3′UTR luciferase assay was performed as in (C) with the exception that luminescence was read 24 hours after transfection, using HL-60–derived exosomes alongside miR-155 mimic. Data are means ± SEM from at least three independent experiments, performed in technical triplicates (*P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant, by Student’s t test).

  • Fig. 4 c-Myb suppression by exosomal miR-155 compromises HSPC clonogenicity.

    (A) Clonogenicity of murine c-KIT+ cells transfected with siRNA targeting c-Myb or control. (B) qRT-PCR quantification of c-Myb RNA in c-KIT+ cell lysates 48 hours after siRNA transfection. (C) Clonogenicity in c-KIT+ cells transfected with anti–miR-155, anti–miR-155 and anti–miR-150, or anti–miR-scramble (SCR) and then exposed to either HL-60–derived exosomes or vehicle. (D) qRT-PCR analysis of RNA extracted from cells described in (C). (E) qRT-PCR for the abundance of miR-155 wild-type and miR-155 knockdown HL-60 cells and from their purified exosomes. U6 snRNA was the reference gene. Data are fold change relative to wild-type. (F) Clonogenicity of c-KIT+ cells exposed to exosomes derived from wild-type HL-60 or miR-155 knockdown HL-60. Data are means ± SEM of a representative of cells derived from at least three mice and plated in triplicate (*P < 0.05; **P < 0.01; n.s., not significant, by Student’s t test).

  • Fig. 5 Exosome miRNA target networks overlap at key hematopoietic regulators.

    (A) Predicted targets of miR-155 identified by RISC-trap in HEK293T cells, compared with those of miR-132 and miR-137 as controls. Targets in red were not predicted by miRWalk, using the DIANA-mT, miRanda, miRDB, miRWalk, RNA22, and TargetScan in silico prediction algorithms. (B) Targets from the miR-155 RISC-trap data set were validated with the dual-luciferase assay described in Fig. 3C. Data are means ± SEM from at least three independent experiments, performed in technical triplicates (*P < 0.05; **P < 0.01; n.s., not significant, by Student’s t test). (C) Interacting partners of miR-155 targets identified by STRING database, using the targets in (A) as input queries. Results were filtered by species and interaction detection method, leaf nodes were removed, and the remaining connected nodes were color-coded. (D) Western blots against phosphorylated Ser20 of p53, p53, and GAPDH in lysates from HEK293T cells 48 hours after transfection with miR-155, cel-miR-67, or vehicle. Blots are representative of three experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/444/ra88/DC1

    Fig. S1. In vivo imaging correlates with chimerism.

    Fig. S2. Intrafemoral injections of exosomes decrease expression of hematopoietic genes.

    Fig. S3. Intravenous injections of exosomes decrease expression of hematopoietic genes.

    Fig. S4. miR-155 knockdown in HL-60 cells shows no decrease in viability or proliferation.

    Fig. S5. Exosome size profile is unaltered in miR-155 knockdown HL-60 cells.

    Table S1. The top transcripts identified by the miR-155 RISC-trap assay.

    Table S2. Primer sequences.

  • Supplementary Materials for:

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

    Noah I. Hornick, Ben Doron, Sherif Abdelhamed, Jianya Huan, Christina A. Harrington, Rongkun Shen, Xiaolu A. Cambronne, Santhosh Chakkaramakkil Verghese, Peter Kurre*

    *Corresponding author. Email: kurrepe{at}ohsu.edu

    This PDF file includes:

    • Fig. S1. In vivo imaging correlates with chimerism.
    • Fig. S2. Intrafemoral injections of exosomes decrease expression of hematopoietic genes.
    • Fig. S3. Intravenous injections of exosomes decrease expression of hematopoietic genes.
    • Fig. S4. miR-155 knockdown in HL-60 cells shows no decrease in viability or proliferation.
    • Fig. S5. Exosome size profile is unaltered in miR-155 knockdown HL-60 cells.
    • Table S1. The top transcripts identified by the miR-155 RISC-trap assay.
    • Table S2. Primer sequences.

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    Citation: N. I. Hornick, B. Doron, S. Abdelhamed, J. Huan, C. A. Harrington, R. Shen, X. A. Cambronne, S. Chakkaramakkil Verghese, P. Kurre, AML suppresses hematopoiesis by releasing exosomes that contain microRNAs targeting c-MYB. Sci. Signal. 9, ra88 (2016).

    © 2016 American Association for the Advancement of Science

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