Leukemia (2014) 28, 1666–1675 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

ORIGINAL ARTICLE BCR-ABL1–positive microvesicles transform normal hematopoietic transplants through genomic instability: implications for donor leukemia

X Zhu1,4,YYou1,4,QLi1, C Zeng1,FFu1, A Guo2, H Zhang2, P Zou1, Z Zhong1, H Wang3,YWu1,QLi1, F Kong1 and Z Chen1

Malignant transformation of normal hematopoietic transplants induced by residual leukemia cells is considered as a pivotal mechanism of donor cell leukemia (DCL). The effects of leukemia cell–derived microvesicles (MVs) in this transformation were examined. We found that MVs derived from K562 leukemia cells contained the breakpoint cluster region–Abelson leukemia gene human homolog 1 (BCR-ABL1) mRNA. Following incubation with BCR-ABL1–positive MVs, mononuclear cells derived from normal transplants exhibited a leukemia-like malignant phenotype both in vitro and in vivo. Horizontal transfer of BCR-ABL1 mRNA from MVs into the recipient cells was critical to the transformation. Relative genomic instability was observed and considered the main mechanism in the recipient cells. MVs contributed to genomic instability by two distinct pathways: via consequent overexpression of activation-induced cytidine deaminase and , which mediated DNA breakage and recombination; and via upregulation of methyltransferases and global DNA hypermethylation. We demonstrated that BCR-ABL1–positive MVs could initiate malignant transformation of normal hematopoietic transplants through genomic instability, which might serve as a convenient and operable model for investigating leukemogenesis, especially for DCL. Furthermore, MVs themselves could act as an early warning indicator and a novel tool to detect and prevent the occurrence of DCL.

Leukemia (2014) 28, 1666–1675; doi:10.1038/leu.2014.51

INTRODUCTION radiation and/or could release intact portions of Allogeneic hematopoietic stem cell transplantation remains the nucleic acids, which might be incorporated into nearby donor 11 main treatment modality for curing hematological .1 cells. In addition, the notion of cell fusion as a mechanism of DCL 12 Post-transplantation relapse of leukemia, the most common cause has been discussed for over a century. However, both notions of treatment failure, is frequently observed, with incidence varying fail to elucidate and demonstrate in vitro models of the based on the type of primary .2 Most relapses refer to extra abnormal karyotype and phenotype conversion involved primary disease progression, and in some cases, leukemia is in DCL. derived from the donor cells, thus it is termed donor cell leukemia Microvesicles (MVs) are released by eukaryotic cells, especially 13 (DCL).3 Emerging evidence suggests that DCL might represent up tumor cells, by outer budding. MVs released by to 45% of all post-transplant leukemia ‘relapses’.4 Although it is a cancer cells have implications for tumor growth, , rare complication, insight into the mechanism of DCL will provide and immune response both in vitro and in vivo. The more information on the prognosis of allogeneic hematopoietic biological functions of MVs are due to their complexity of stem cell transplantation and, more importantly, yield further bioactive cargo, including proteins, RNA, microRNA (miRNA) and clues into leukemogenesis of normal cells, as leukemia in the DNA derived from parent cells.14,15 MVs are thought to deposit general population is usually sporadic and unpredictable. It has paracrine information and create paths of least resistance to been reported that occult leukemia or a pre-leukemic state in transfer their cargo to adjacent or remote cells.16,17 Our previous donors, therapy-related stromal abnormalities and DNA repair work demonstrated that MVs derived from K562 leukemia errors in stem/progenitor cells might explain the mechanism of cells contained the dominant onco-mRNA of breakpoint cluster DCL.5–8 However, most researchers have expanded on this idea region–Abelson leukemia gene human homolog 1 (BCR-ABL1) recently speculating that some non-viral leukemogenic materials and could deliver it to other cells. On the other hand, chronic might be released from residual leukemia cells and fuse with myeloid leukemia is the primary neoplasm that most frequently incoming donor cells, resulting in transformation.9,10 Here, we are leads to DCL (26–36%).18 Consequently, the aim of this study confronted with the puzzle of how residual leukemia cells was to investigate whether BCR-ABL1–positive MVs participate in communicate and exchange materials with normal cells derived the transformation of normal hematopoietic transplants via from the donor. Generally, the genomic damage inflicted by intercellular transferring of onco-materials.

1Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 2Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, People’s Republic of China and 3Department of Hematology, the Central Hospital, Wuhan, People’s Republic of China. Correspondence: Professor P Zou or Professor Z Chen, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Road, Wuhan 430022, People’s Republic of China. E-mail: [email protected] or [email protected] 4These authors contributed equally to this work. Received 20 September 2013; revised 19 January 2014; accepted 24 January 2014; accepted article preview online 31 January 2014; advance online publication, 25 February 2014 BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1667 MATERIALS AND METHODS 95 1C for 30 s, primer annealing at 55–60 1C for 30 s and elongation at 72 1C Cell culture and MV isolation for 15 s. MSI was determined by loci multiplex PCR in an ABI 3730xl Genetic analyzer (Applied Biosystems Inc., Foster City, CA, USA). The human chronic myeloid leukemia blast crisis cell line K562, acute lymphoblastic leukemia cell line Jurkat, lymphoma cell lines Raji and JeKo and acute myeloid leukemia cell lines HL-60, THP-1 and KG-1a were Intracellular reactive oxygen species (ROS) purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured in RPMI 1640 containing 15% fetal bovine serum at Intracellular ROS were detected using a Reactive Oxygen Species Assay Kit (S0033, Biyuntian Inc., Beijing, China) and measured with a multimode 37 1Cin5%CO2. For MV isolation, cells were centrifuged at 1000 g for 10 min. The plate reader (EnSpire, Austin, TX, USA). All procedures were conducted in supernatant was centrifuged at 5000 g for 20 min to remove cellular debris, accordance with the kit instructions. and the remaining supernatant was centrifuged at 13 000 g for 60 min to obtain MVs. To confirm that we had isolated MVs, random samples were Total DNA methylation analyzed by a fluorescence-activated cell sorter. The morphology of the precipitate was visualized using transmission electron microscopy and DNA was extracted with an EpiQuik Nuclear Extraction Kit (Epigentek). confocal microscopy, stained with 5 mmol/l carboxyfluorescein diacetate Total DNA methylation was performed with a MethylFlash Methylated DNA succinimidyl ester (CFSE; Life Technologies, Carlsbad, CA, USA). Quantification Kit (P-1035-48, Epigentek). All procedures were conducted Mononuclear cells (MNCs) were extracted from the peripheral in accordance with the kit instructions. mobilization; cord blood, peripheral blood and bone marrow of healthy volunteers and cultured in StemSpan SFEM (No. 09600, STEMCELL Real-time PCR Technologies, Vancouver, BC, Canada). Total RNA was extracted with an E.Z.N.A. Blood RNA Kit (Omega Biotek Inc., Norcross, GA, USA). For reverse transcription, 2 mg total RNA was primed Transformation of MNCs from normal hematopoietic transplants with a random hexamer mixture as the primer using Moloney Murine by MVs Leukemia Virus Reverse Transcriptase (Promega, Madison, WI, USA). Real- The retained MVs were suspended in serum-free RPMI 1640 (to avoid MVs time was performed with 10 ml SYBR Green PCR Master Mix (Applied derived from the fetal bovine serum). To generate intact MVs for the cell- Biosystems Inc.), 2 ml primers (Invitrogen), 6.5 ml RNase-free H2O and 1.5 ml based assays and other experiments, the MV-containing RPMI 1640 was cDNA as a template in a final reaction volume of 20 ml. Fluorescence filtered by using a Millipore Steriflip polyvinylidene difluoride filter intensity was measured using a Stratagene Mx3000P QPCR System (Agilent (Billerica, MA, USA) with pore sizes of 1.0 or 0.45 mm (to filter cells and Technologies Inc, Santa Clara, CA, USA). cell debris) and 0.1 mm (to filter MVs as negative controls). MVs were quantified according to their total RNA content and copies of BCR-ABL1 mRNA. The MNCs were adjusted to 4 Â 106 cells per well in six-well plate, Western blotting and 400 ng MVs (containing RNA) was added to the cells three times a day Fifty microgram protein samples for each well were separated by sodium for 14–23 days. dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to To confirm the specificity, equal amounts of BCR-ABL1–negative MVs nitrocellulose membranes. The membranes were blocked with 5% non-fat (derived from Jurkat, Raji, JeKo, THP-1, KG-1a cells) were added at equal milk and incubated overnight with monoclonal antibody (1:500) at 4 1C. frequency to MNCs as control groups. In selected experiments, K562-MVs Blots were developed using a horseradish peroxidase–conjugated rabbit were treated with 10 U/ml RNase A (Ambion, Austin, TX, USA) for 3 h at anti-human secondary antibody (1:500) and a chemiluminescent detection 37 1C; the reaction was stopped by ultrafiltration and centrifuged at kit (Amersham Biosciences, Piscataway, NJ, USA). 10 000 g for 1 h at 4 1C. To decrease the BCR-ABL1 mRNA in the MVs, K562 cells were transfected with hsa-miR-203 mimics and negative control miRNA mimics (RiboBio, Guangzhou, China) at a final concentration of Statistical analysis 10 nmol/l using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For SPSS 10.0 (Chicago, IL, USA) was used for statistical analysis. Non- selected transformation process, we added 0.5 mM imatinib to the parametric and unpaired t-test comparisons were used to compare groups; MNC–K562-MV mixture each day. the rates between groups were compared by the chi-squared test. Two- sided Po0.05 was defined as being statistically significant. Characteristics of transformed cells The morphology of the transformed cells was observed using Wright’s stain. The phenotype was analyzed using fluorescein isothiocyanate– RESULTS labeled antibodies to CD34, CD14, CD15, CD38, CD45, CD11b, CD19, CD20, Description of K562 cell-derived MVs CD64, CD71, CD117 and myeloperoxidase purchased from BD Pharmingen A major issue in the MV literature is the somewhat confusing (Franklin Lakes, NJ, USA). Karyotyping was performed by G-banding. BALB/ nomenclature, especially regarding exosomes and MVs. In this c-nu mice aged 3–4 weeks were purchased from Huafukang (Beijing, 13 China). All animal experiments were considered ethically acceptable paper, the nomenclature proposed by Raposo and Stoorvogel is according to the Hubei Province animal experiment regulations. Trans- used. MVs, also called ectosomes or , form mostly by formed cells (5 Â 108/ml, 0.2 ml) were subcutaneously injected into the reverse budding: fission of the plasma membrane stalk detaches right shoulders of the mice for tumor formation. Neoplasms were analyzed the cytoplasmic protrusions (Figures 1a and 1b). Their lipid morphologically and by immunohistochemistry of human CD45 composition, irregular shape and density are major features that antibodies. distinguish MVs from exosomes. Exosomes are small (30–100 nm in diameter) membranous vesicles formed by the inward budding DNA breaks and microsatellite instability (MSI) in recipient cells of multivesicular bodies and released from the cell. The MVs DNA double-strand breaks were assessed by agarose gel electrophoresis measured 100–1000 nm, thus we could observe them under using genomic DNA isolated from recipient cells. Briefly, cells were washed, confocal microscopy when stained with a fluorescent dye trypsinized and centrifuged at 1500 g for 5 min. After 3-h incubation at (Figure 1c). Unlike exosomes, the surfaces of most MVs are 50 1C, samples were treated with RNase A for 1 h at 37 1C. The DNA was annexin V–positive (Figure 1e). extracted with phenol:chloroform and precipitated with 0.3 mol/l sodium Our previous work determined that K562-MVs contained acetate and ethanol at À 20 1C. DNA samples (10 mg) were separated on a BCR-ABL1 mRNA and protein. Moreover, we found that 2% agarose gel containing 5 mg/ml ethidium bromides and analyzed by K562-MVs contained 977 miRNAs. miRNAs with significant autoradiography. To evaluate MSI, DNA was extracted with an EpiQuik Nuclear Extraction expression in MVs were involved in the processes of immune Kit (OP-0002-01, Epigentek, Farmingdale, NY, USA) and PCR-amplified at escape, hematopoiesis and differentiation (Figure 1d). Interestingly, eight microsatellite loci (NR-21, BAT-26, NR-27, BAT-25, NR-24, MONO-27, miR-203 and miR-125b, which were reported to modify the D9S179 and D22S315). After initial denaturation at 95 1C for 5 min, DNA function of BCR-ABL1, were significantly different in K562-MVs amplification was performed for 25–30 cycles consisting of denaturation at (Supplementary Figure S1).

& 2014 Macmillan Publishers Limited Leukemia (2014) 1666 – 1675 BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1668

Figure 1. Characteristics of K562-MVs. (a, b) Scanning electron microcopy revealing MVs (arrows) as 0.1–1-mm vesicles shed from K562 cell membranes that contain cytoplasm and organelles. (c) Confocal microscopy of CFSE-stained MVs. (d) Microarray analysis of comparison of miRNAs in K562 cells and their derived MVs revealing that 111 miRNAs (72 downregulated and 39 upregulated in MVs) were significantly different in K562 cells and K562-MVs; onco-miRNA such as miR-26a and miR-17 were involved. (e) Flow cytometry showing that MVs were o1 mm particles in diameter and the surface of MVs was positive for annexin V. P1–P5 referred to 1, 3, 0.8, 0.5 and 0.3 mm beads for gate, respectively. P6–P9 was performed to describe the expression of annexin V in P1, P3, P4 and P5, respectively.

BCR-ABL1–positive MVs transformed normal hematopoietic transplants Table 1. Transformation induced by K562-MVs After consecutive incubation with MVs for at least 14 days, a new Groups Cell Time (days) BCR-ABL1 group of leukemia-like cells could be observed in the MNCs number derived from normal transplants (Table 1). The morphology of these cells was similar to that of monoblasts, possessing a roughly Mobilization 4 Â 106 13.16±1.52 7.982 Â 104 circular nucleus, high cytoplasmic ratio, delicate lacy chromatin Bone marrow 3 À 4 Â 106 18.32±1.70 2.123 Â 105 7 6 and abundant basophilic cytoplasm (Figures 2a and 2b). Cord blood 3 Â 10 23.63±3.14 1.718 Â 10 Peripheral blood 4 Â 106 Failure Failure Immunophenotypically, these leukemia-like cells expressed mye- 6 loid (CD15, CD71) markers and were negative for mature Mesenchymal stem cells 4 Â 10 Failure Failure markers such as CD14 and B-cell marker CD19 Abbreviations: BCR-ABL1, breakpoint cluster region–Abelson leukemia (Figure 2d and Supplementary Figure S2). The characteristic gene human homolog 1; MV, microvesicle. Note: Transformation time t(9;11), typically associated with monoblasts, was absent from the indicates the first time abnormal cells were observed based on transformed cells. The chromosome number of these cells ranged morphology. from 64 to 103, and the modal number was 80–83. Most of the cells were triploid or hypotetraploid and consistently or frequently contained several structural aberrations (Figure 2c). In addition, almost the same morphological characteristics with the trans- the frequency of ecotropic viral integration site 1 (EVI1) and formed cells (Figure 2g). Immunofluorescence assay of human homeobox gene 11 (HOX11) positivity was the highest in the anti-CD45 antibodies demonstrated human CD45–positive cells in molecular packing of the acute myeloid leukemia cells (Figure 2e). the tumors (Figure 2h1); the nuclei of these cells were stained Fourteen days after subcutaneous transplantation into nude with DAPI (4,6-diamidino-2-phenylindole) to avoid background mice (without cyclophosphamide), the leukemia-like cells formed fluorescence (Figure 2h2). The secondary host mice into which tumors at the injection sites (n ¼ 10; Figure 2f), and there were no the tumor cells were subcutaneously transplanted presented neoplasms in mice transplanted with K562 cells as a control obvious cachexia and spleen enlargement after 20 days (n ¼ 5; (n ¼ 10). Malignant cells in the tumors from the mice shared Figures 2i and 2j). Leukemia-like malignant cells could be

Leukemia (2014) 1666 – 1675 & 2014 Macmillan Publishers Limited BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1669

Figure 2. Morphology, immunophenotype, cytogenetics, molecular packing and tumor formation in Balb/C-nu mice of transformed cells. Transformed cells contained a roughly circular nucleus, high cytoplasmic ratio, delicate lacy chromatin and abundant basophilic cytoplasm (a, b). (c) Chromosome numbers of transformed cells ranged 64–103, with a modal number of 80–83. Most cells were triploid or hypotetraploid and consistently or frequently contained several structural aberrations. (d) Transformed cells expressed CD15, CD71 variably and were negative for CD14, CD19 and CD56. (e) The frequency of EVI1 and HOX11 positivity was highest in molecular packing of the acute myeloid leukemia cells; fms-related tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) mutations were observed in some cases. (f) Transformed cells formed tumors (arrow) at the injection site 14 days after subcutaneous transplantation (n ¼ 10). (g) Near-identical morphological features of the malignant cells in the tumors from the mice as compared with transformed cells. Immunofluorescence assay of human anti-CD45 antibodies (red) demonstrating that the tumors contained human CD45-positive cells (h1); nuclei were stained with DAPI (blue) to avoid background fluorescence (h2). Secondary host mice exhibiting obvious cachexia (i) and spleen enlargement (j) 20 days after transplantation (n ¼ 5). Leukemia-like malignant cells in the bone marrow (k) confirmed by immunofluorescence assay of human anti-CD45 antibodies (l1, l2).

& 2014 Macmillan Publishers Limited Leukemia (2014) 1666 – 1675 BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1670 observed in bone marrow of the mice (Figure 2k), which was BCR-ABL1 mRNA from MVs was translated into protein in the confirmed by immunofluorescence assay of human anti-CD45 recipient cells. This was proven by the significant P210 reduction antibodies (Figure 2l). in the recipient cells when K562-MVs were treated with RNase or miR-203 mimics (Figure 3b). Horizontal transfer of BCR-ABL1 into normal hematopoietic transplants by MVs Sequencing and clustering of mRNA involved in transformation Under confocal microscopy, we found that K562-MVs labeled with mRNAs from cells on days 0, 7, 14, and 21 of the transformation CFSE presented in the cytoplasm of the recipient cells (Figure 3a, process were sequenced to investigate the main events of the arrow). To investigate whether MV-associated proteins or RNA was responsible for the transformation capability, we digested K562- MVs with RNase A. However, it is unlikely that was due to protein, as the MVs lost their transforming abilities following RNase Table 2. Process of transformation treatment (Table 2). Among the RNAs, we considered the Groups Recipient Cell Tumor oncogene BCR-ABL1 a possible candidate, because this fusion cells number formation gene has been reported to transform normal cells and non–BCR- ABL1 MVs did not exhibit this ability (Table 2). Using non-MV MV from BCR-ABL1–negative cellsa Mobilization 4 Â 106 No 6 supernatant of K562 (exosomes included) and non–BCR-ABL1 MVs K562-MV treated with RNase Mobilization 4 Â 10 No Decreased BCR-ABL1 in K562-MVb Mobilization 4 Â 106 No as a control, we found that K562-MVs could deliver BCR-ABL1 K562-MV withdrawal at day 7 Mobilization 4 Â 106 No mRNA and P210 protein into the recipient cells (Figures 3b and K562-MV withdrawal at day 14 Mobilization 4 Â 106 Yes 3e). After incubation with 400 ng MVs (containing RNA) once, the Recipient cells incubated with imatinib Mobilization 4 Â 106 No 6 c exogenous fusion mRNA was present in recipient cells for almost Recipient cells treated with decitabine Mobilization 4 Â 10 Yes 48 h (Figure 3c). That BCR-ABL1 induced transformation was Abbreviations: BCR-ABL1, breakpoint cluster region–Abelson leukemia supported by the finding that MVs from K562 cells transfected gene human homolog 1; MV, microvesicle. aBCR-ABL1–negative cells were with miR-203 mimics, which decreased BCR-ABL1 mRNA in the KG-1a, THP-1, Jurkat, Raji, JeKo, and HL-60 cells. bK562-MVs overexpressing MVs, failed to transform the MNCs (Figure 3d and Table 2). To miR-203 were constructed by transfecting miR-203 mimics into K562 cells. identify the role of P210 protein, imatinib was added to the cDecitabine treatment could not halt the transformation but resulted in an recipient cells. Interestingly, recipient cells incubated with imatinib 18-day delay; thus, transformation spanned 32 days when decitabine was could not exhibit the transformation (Table 2), indicating that added.

Figure 3. Horizontal transfer of BCR-ABL1 into recipient cells from K562-MVs. (a) Confocal microscopy images showing that K562-MVs could infuse or be internalized by recipient cells. CFSE-labeled MVs (green) were present in the cytoplasm of recipient cells (arrow). (b) K562-MVs delivered BCR-ABL1 mRNA into recipient cells (Po0.05), control, non-MV supernatant and non–BCR-ABL1 MVs. (c) Exogenous fusion mRNA from MVs persisted in recipient cells for almost 48 h after incubation with 400 ng (containing RNA) K562-MV once (Po0.05). (d) Incubation with miR-203 mimic–transfected K562 cells and RNase-treated K562-MVs resulted in decreased BCR-ABL1 mRNA in MVs and recipient cells (Po0.05). (e) Significant reduction of P210 protein in recipient cells when K562-MVs were treated with RNase or miR-203 mimics (Po0.05).

Leukemia (2014) 1666 – 1675 & 2014 Macmillan Publishers Limited BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1671 MV-induced leukemogenesis (methods referred in the Supple- double-strand break induction, increased over time, peaking on mentary Materials). Of the genes for which there was a significant day 14 (Supplementary Figure S4B). Moreover, we observed more difference, 172 were related to DNA mismatch repair, non- DNA breakage in K562-MVs than in MVs derived from other cells homologous end-joining, DNA replication, nucleotide excision on days 3 and 10 (Figure 4c). RAD51b and RAD18 protein and and repair, base excision and homologous recombination and mRNA, important elements required for DNA repair and homo- were upregulated during transformation (Supplementary Figure logous recombination, were both elevated in the same trend S3). Some genes were downregulated during transformation. For during transformation (Figures 4g and 4h). Moreover, RAD51b example, ataxia-telangiectasia mutated, which is a cell cycle expression was significantly higher than RAD18 expression. checkpoint kinase and phosphorylates a wide variety of down- MSI stems from small bases insertions/deletions within nucleo- stream proteins such as the tumor-suppressor proteins P53 and tide repeats due to replication errors and is associated with breast cancer 1 and the DNA repair protein NBS1, was greatly tumorigenesis, especially colorectal cancer.22 On days 0 and 21, downregulated in day-21 cells. we analyzed the sizes of six common microsatellites and two leukemia-associated MSI on chromosomes 9 and 22, respectively. There was a significant difference for the sizes of MONO-27 and Genomic instability of recipient cells NR-21 but not for the other four microsatellites (Figure 4d). In the Genomic instability, the most important hallmark of cancer, is case of the two leukemia-associated MSI, the size of D22S315 19–21 associated with tumorigenesis. Indeed, there were numerical exhibited an apparent mutation at 131.05 (Figure 4e), whereas and structural aberrations in our leukemia-like cells (Figure 2f). D9S179 remained unchanged (Figure 4f). Thus, we next assessed whether genomic instability of recipient cells was induced. We found that there was increased DNA breakage during Mechanisms of genomic instability induced by K562-MVs transformation in the vast majority of recipient cells incubated K562-MVs increased global DNA methylation levels in recipient with K562-MVs (Figure 4a). There was a significant decrease in cells. Hypermethylation of tumor-suppressor and ‘stability’ genes DNA breakage when MVs derived from K562 cells transfected with also contributes to chromosomal instability (CIN).23,24 We miR-203 mimics were added (Figure 4b) and when recipient cells confirmed an increase in global DNA methylation in K562-MV– were incubated with imatinib (Supplementary Figure S4A). During treated cells that peaked on day 3 as compared with the control, transformation, g-H2AX protein, a highly sensitive marker for suggesting that MVs induced methylation before leukemic

Figure 4. Genomic instability was induced in the recipient cells. (a) There was ongoing DNA breakage during transformation in recipient cells cultured with K562-MVs (Po0.05). (b) There was a significant decrease in DNA breakage when MVs derived from miR-203 mimic–transfected K562 cells were added. There was more DNA breakage in K562-MVs on days 3 (c1) and 10 (c2). There was a significant difference between the sizes of the MONO-27 and NR-21 microsatellites in recipient cells on days 0 (d1) and 21 (d2). For the two leukemia-associated MSI sizes on chromosomes 9 and 22, the size of D22S315 exhibited a mutation at 131.05 (e), whereas D9S179 remained unchanged (f), respectively. (g, h) RAD51b and RAD18 protein and mRNA were both elevated in the same trend during transformation; RAD51b expression was significantly higher than RAD18 expression.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1666 – 1675 BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1672 transformation (Figure 5a). Promoter hypermethylation of the treated with RNase and miR-203 mimics (Figures 6c and 6d). tumor-suppressor genes P53 and RIZ1, which are frequently Among MVs derived from different cells, increased AICDA induced regulated by methylation, was observed in K562-MV–treated cells by K562-MVs was observed on day 3 (Figure 6e). Besides, the (Figures 5b and 5c). However, the level of the oncogene MYC, MNCs from the mobilization exhibited the highest AICDA level on which promotes the cell cycle, increased during transformation day 3 (Figure 6f). (Figure 5d). We next investigated changes in the DNA methyl- ROS have also been reported to induce genomic instability by transferase (DNMT) level by measuring the mRNA and protein oxidative DNA damage and were increased by the BCR-ABL1 levels of DNMT3a and DNMT3b. DNMT3a and DNMT3b mRNA and oncogene.27,28 A significant increase in ROS was observed on days protein were consistently elevated during transformation (Figures 3 and 21 during transformation, which might be associated with 5e and 5f). DNMT3b expression was consistently higher than the DNA breakage during the process (Figure 6g). Similar to DNMT3a expression. The mRNA and protein of these methyl- AICDA, there was no significant difference when K562-MVs were transferases decreased when K562-MVs were treated with RNase treated with RNase and miR-203 mimics (Supplementary Figures (Figure 5g). However, decreasing BCR-ABL1 mRNA levels in MVs S6a and b). Compared with MVs derived from other cells, K562- with miR-203 mimics did not affect the protein expression of MVs increased ROS on day 3 (Figure 6h). In the recipient cells, DNMT3a or DNMT3b (Figure 5h), indicating that exogenous BCR- higher levels of ROS were observed in MNCs isolated from ABL1 might not be the most important compound in induction of mobilization on day 3 than in bone marrow and cord blood MNCs the methyltransferases (Figures 5j and 5i). The mismatch between (Figure 6i); on day 10, however, bone marrow MNCs exhibited mRNA and protein might be explained by the fact that MVs are rapidly increased levels of ROS (Figure 6j). A possible reason is that packages of bioactive molecules that contain not only contribut- bone marrow MNCs represent a complex group that is composed ing factors but also detractors, such as miR-29 (Supplementary of cells at different stages of development; ROS was elevated not Figure S5). only in the hematopoietic cells but also in other cells, for example, the . K562-MVs increased activation-induced cytidine deaminase (AICDA) and ROS. AICDA, which is both essential and sufficient for forming antibody memory, is also linked to tumorigenesis of B DISCUSSION lymphomas and myeloid leukemias.25 AICDA caused the Tumor-derived MVs represent a remarkable system for short- and accumulation of genetic alterations in tumor-related genes.26 long-range intercellular communication. A number of studies have During transformation, AICDA mRNA and protein expression levels set the stage for further investigations into the importance of MVs were increased in cells incubated with K562-MVs (Figures 6a and in tumor progression.14,29,30,31 We have shown here that exposing 6b). The induction might be associated with the transfer of BCR- hematopoietic grafts to bioactive leukemia MVs, which are ABL1, as the level of AICDA decreased when K562-MVs were constitutively shed by BCR-ABL1–positive cells, could cause

Figure 5. Methylation was involved in transformation. (a) Compared with the control, global DNA methylation of K562-MV–treated cells increased, peaking on day 3 (Po0.05). Promoter hypermethylation with low levels of expression of the tumor-suppression genes p53 (b) and RIZ-1 (c). (d) Increased level of the oncogene myc during transformation (Po0.05). (e, f) DNMT3a and DNMT3b mRNA and protein were consistently elevated during transformation (Po0.05); DNMT3b expression was consistently higher than DNMT3a expression. (g) DNMT3a and DNMT3b mRNA and protein decreased when K562-MVs were treated with RNase or when K562 cells were transfected with miR-203 mimics (Po0.05). (h) Decreased BCR-ABL1 mRNA in MVs by miR-203 mimics did not affect protein expression of DNMT3a or DNMT3b (Po0.05). (j, i) K562-MVs were not the most influential factor in DNMT3a and DNMT3b expression (Po0.05).

Leukemia (2014) 1666 – 1675 & 2014 Macmillan Publishers Limited BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1673

Figure 6. Alteration of AICDA and ROS during transformation. Expression of AICDA mRNA (a) and protein (b) increased in K562-MV– transfected cells during transformation (Po0.05). (c, d) AICDA levels decreased when K562-MVs were treated with RNase or miR-203 mimics (Po0.05). (e) K562-MVs induced a significant increase in AICDA on day 10. (f) Mobilization led to the highest AICDA levels on day 3 (Po0.05). (g) A significant increase in ROS was observed on days 3 and 21 during transformation (Po0.05). (h) K562-MVs elevated ROS on day 3 (Po0.05). (i) Higher levels of ROS were observed in MNCs isolated from mobilization on day 3 than in bone marrow and cord blood MNCs (Po0.05), but the ROS levels rapidly increased in bone marrow MNCs on day 10 (j). recipient cells to acquire a transformed phenotype. These findings donors bearing BCR-ABL1 is rare. Here, we proved that MVs help to further our understanding of the communication between horizontally transferred mRNA and protein into donor cells; as a leukemia cells and hematopoietic cells in the same niche. The result, exogenous BCR-ABL1 was expressed in normal grafts. The process of malignant transformation might also serve as a rapid, MV-borne BCR-ABL1 transcripts have a finite lifespan after being convenient, operable and controllable model for the investigation added to normal recipient cells and need to be replenished of leukemogenesis. Although most of our data have been limited continuously. However, exogenous BCR-ABL1 was no longer thus far to cell culture experiments in vitro, MV shedding by cancer essential when the recipient cells have already acquired a cells represents a tumor-relevant process both in vivo and transformed phenotype. These results indicated that tyrosine in vitro.32,33 Consequently, the chronic MV shedding by cancer kinase inhibitors could prevent the occurrence of chronic myeloid cells into their microenvironment might provide a continuous leukemia–associated DCL via inhibition of p210 protein in supply of MVs to nearby recipient cells to induce and maintain a recipient cells, but the resulting DCL cells are likely not sensitive transformed phenotype. This effect prompts the idea that the to tyrosine kinase inhibitors. Decreasing BCR-ABL1 mRNA copies in formation of a secondary tumor (such as DCL) would not MVs and MV cessation delayed or even halted transformation, necessarily depend solely on exogenous oncogenic factors. indicating that desultory and irregular supplementation of MVs Rather, aberrant transformation of normal cells that have been in vivo owing to MV clearance and/or low tumor burden might be exposed to MVs shed by cancer cells should also be considered, one explanation for sporadic DCL in patients. Thus, monitoring which reveals new insights into the cellular basis of DCL and the MVs and/or their content would be a warning indicator for the potential to translate this knowledge into innovative approaches occurrence of secondary malignancies after hematopoietic stem for diagnostics and personalized therapy. cell transplantation. We further identified the mRNA of BCR-ABL1 as an essential One aspect that merits further consideration involves the component of K562-MVs that functionally mediated the trans- potential connection of exogenous BCR-ABL1 and transformation forming activities. However, the probability of cells from healthy of normal hematopoietic transplants. Clustering analysis of

& 2014 Macmillan Publishers Limited Leukemia (2014) 1666 – 1675 BCR-ABL1–positive MVs transform normal grafts X Zhu et al 1674 sequencing data indicated that most of the significantly transformation and the ultimately important extension of animal expressed mRNAs participated in genomic instability. Indeed, models remain to be investigated further. most reported DCL cases presented genomic instability or an abnormal karyotype; 28% of ensuing DCL phenotypes closely resembled that of the original disease.10,34 There were numerical CONFLICT OF INTEREST and structural aberrations in our transformed cells, and this The authors declare no conflict of interest. oncogenic effect is the result of consequent overexpression of MYC, AICDA and ROS induced by MVs, which mediated the upregulation of DNA breakage and recombination, thereby ACKNOWLEDGEMENTS hastening the onset of transformation. The overexpression could We thank Professor Zhijian Xiao (Institute of Haematology and Blood be halted when BCR-ABL1 was downregulated in the MVs or Hospital, Chinese Academy of Medical Sciences, Tianjin, China) for his kind supply of recipient cells, consistent with the findings that both AICDA and Imatinib. This work was supported by grants from the National Natural Science ROS were induced by the oncogene BCR-ABL1.35 To our Foundation of China (Nos. 81170497 and 81170498). knowledge, few researchers have highlighted the connection between MVs derived from cancers and CIN or MSI of recipient REFERENCES cells, which could be a novel mechanism of MV-induced biological functions. 1 Hamilton BK, Copelan EA. Concise review: the role of hematopoietic stem cell transplantation in the treatment of acute myeloid leukemia. 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