View
2
Download
0
Category
Preview:
Citation preview
1
Chronic myelogenous leukemia cells remodel the bone marrow niche 1
via exosome-mediated transfer of miR-320 2
Xiaotong Gao1#, Zhuo Wan1#, Mengying Wei2,3#, Yan Dong1, Yingxin Zhao1, Xutao Chen4, 3
Zhelong Li5, Weiwei Qin1*, Guodong Yang2,3*, Li Liu1,* 4
1Department of Hematology, Tangdu Hospital, Fourth Military Medical University, Xi’an, 5
710038, People’s Republic of China. 6
2State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi’an, 710032, 7
People’s Republic of China. 8
3Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 9
Xi’an, 710032, People’s Republic of China. 10
4Department of Implantation, School of Stomatology, Fourth Military Medical University, 11
Xi’an, 710032, People’s Republic of China. 12
5Department of Ultrasound Diagnostics, Tangdu Hospital, Fourth Military Medical 13
University, Xi’an, 710038, People’s Republic of China. 14
# These authors contributed equally to this work. 15
*Correspondence and requests for materials should be addressed to Li Liu (email: 16
heamatol@fmmu.edu.cn). Correspondence may also be addressed to Guodong Yang (email: 17
yanggd@fmmu.edu.cn) and Weiwei Qin (email: vivianq1126@126.com) 18
All the authors declare no potential conflicts of interest. 19
2
Abstract 20
Rationale: Reciprocal interactions between leukemic cells and bone marrow mesenchymal 21
stromal cells (BMMSC) remodel the normal niche into a malignant niche, leading to leukemia 22
progression. Exosomes have emerged as an essential mediator of cell-cell communication. 23
Whether leukemic exosomes involved in bone marrow niche remodeling remains unknown. 24
Methods: We investigated the role of leukemic exosomes in molecular and functional changes 25
of BMMSC in vitro and in vivo. RNA sequencing and bioinformatics were employed to screen 26
for miRNAs that are selectively sorted into leukemic exosomes and the corresponding RNA 27
binding proteins. 28
Results: We demonstrated that leukemia cells significantly inhibited osteogenesis by BMMSC 29
both in vivo and in vitro. Some tumor suppressive miRNAs, especially miR-320, were enriched 30
in exosomes and thus secreted by leukemic cells, resulting in increased proliferation of the 31
donor cells. In turn, the secreted exosomes were significantly endocytosed by adjacent 32
BMMSC and thus inhibited osteogenesis at least partially via β-catenin inhibition. 33
Mechanistically, miR-320 and some other miRNAs were sorted out into the exosomes by RNA 34
binding protein heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), as these miRNAs 35
harbor the recognition site for HNRNPA1. 36
Conclusion: HNRNPA1-mediated exosomal transfer of miR-320 from leukemia cells to 37
BMMSC is an important mediator of leukemia progression and is a potential therapeutic target 38
for CML. 39
Key words: chronic myelogenous leukemia, exosomes, bone marrow mesenchymal stromal 40
cells, miR-320, HNRNPA1 41
3
Graphical Abstract 42
HNRNPA1-mediated exosomal transfer of miR-320 from leukemia cells to BMMSC is an 43
important mediator of leukemia progression and niche remodeling. 44
45
4
Introduction 46
Chronic myelogenous leukemia (CML) results from the transformation of normal 47
hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Disruption of normal 48
hematopoiesis occurs even before outgrowth of leukemic cells, causing anemia, 49
thrombocytopenia, neutropenia and osteoporosis, which are the major causes of morbidity and 50
mortality of CML. The bone marrow hematopoietic microenvironment (“niche”) is a unique 51
composition of mesenchymal and non-mesenchymal cells, which supports the self-renewal of 52
normal and malignant hematopoietic cells [1]. Reciprocal interactions between leukemic cells 53
and the niche are essential for the leukemic progression and resistance to drug treatment [1]. In 54
addition, emerging evidence has shown that myeloid malignancies induce molecular and 55
functional changes in the niche [2-5], switching the normal hematopoietic cell favouring niche 56
to leukemic cell favouring one [6-8]. However, the mechanisms by which leukemic cells 57
remodel the bone marrow niche have not been adequately defined. 58
Exosomes are recognized as pivotal mediators of cell-cell communication, through 59
transmission of intracellular cargo such as miRNAs [9, 10]. Exosomes loaded with miRNAs 60
are secreted by cancer cells and internalized by neighbouring cells in the malignant niche, 61
affecting the phenotype of niche cells [11-15]. Specific miRNAs are selectively sorted into 62
exosomes, through mechanisms that are still poorly understood [16]. It has been reported that 63
RNA-binding proteins such as heterogeneous nuclear ribonucleoproteins A2B1 (HNRNPA2B1) 64
and Q (HNRNPQ) are involved in exosomal RNA export by recognizing and binding specific 65
motifs in miRNA sequences [17, 18]. Notably, HNRNPA1 is induced by BCR/ABL tyrosine 66
kinase in a dose- and kinase-dependent manner [19]. In addition, HNRNPA1 controls the 67
nuclear export of mRNAs that are indispensable for the leukemia phenotype of CML [20]. All 68
of these suggest that leukemic cells might selectively sort certain miRNAs into the exosomes 69
during the progression of CML. 70
Here, by using leukemic cell lines, a murine leukemic model, and patient samples, we 71
found that leukemic cells selectively transport growth-suppressing miRNAs into exosomes via 72
heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), resulted in promoted CML cell 73
5
growth. In addition, the leukemia cell-derived exosomes could be endocytosed by the 74
surrounding BMMSC, leading to inhibited osteogenesis of the recipient cells and thus 75
remodelled bone marrow niche favourable for CML progression. 76
Material and methods 77
Animals 78
Double-transgenic SCL-tTa X TER-BCR/ABL mice [21] were backcrossed for at least eight 79
generations into the C57/BL6 background and were maintained in the presence of 0.5 g/L 80
doxycycline in the drinking water. BCR/ABL expression was induced in 5-week-old double-81
transgenic mice (BA) by doxycycline withdrawal for at least 4 weeks. All mice were 82
maintained under specific pathogen free conditions and the experimental procedures were 83
performed in accordance with guidelines and protocols approved by the Institutional Animal 84
Experiment Administration Committee. 85
Cell line and human samples 86
Human CML cell lines K562 and LAMA84 were purchased from ATCC and cultured in RPMI 87
1640 medium (Hyclone, Loga, UT, USA) with 10% FBS and penicillin–streptomycin 88
(Hyclone). 89
Human bone marrow and peripheral blood specimens were obtained from 41 patients with 90
newly diagnosed CML; 24 patients were in chronic phase (CML-CP) and 17 patients in blast 91
crisis (CML-BC). Specimens from 11 age-matched healthy donors were used as control. All 92
subjects signed an informed consent form. All specimen acquisition was approved by the Ethics 93
Committee of Fourth Military Medical University. 94
Isolation and expansion of bone marrow mesenchymal stromal cells (BMMSC) 95
BMMSC were isolated from the bone marrow of CML-CP patients and age-matched healthy 96
donors after informed written consent as previously described [22]. Briefly, the mononuclear 97
cell fraction was isolated by density gradient centrifugation using Human Lymphocyte 98
Separation Medium (Dakewe Biotech, Shenzhen, China), re-suspended in alpha-MEM (Gibco, 99
Carlsbad, CA, USA) containing 5% human platelet lysate (HELIOS, Atlanta, GA, USA), L-100
6
glutamine, and penicillin–streptomycin (Hyclone), and plated at an initial density of 1×106 101
cells/cm2. Three days later, non-adherent cells were removed and the remaining monolayers of 102
adherent cells were cultured in fresh medium until they reached confluence. The cells were 103
harvested by trypsinization and sub-cultured at densities of 5000–6000 cells/cm2. BMMSC 104
were identified as CD34-/CD45-/HLA-DR-/CD29+/CD90+/CD105+ using flow cytometry. 105
Multipotent differentiation capacity was demonstrated by osteogenic, adipogenic and 106
chondrogenic inductions as described below. For osteogenesis analysis, BMMSC were used 107
during third to fifth passages for experiments. For miR-320a expression analysis in the 108
BMMSCs, the primary culture was used. 109
Exosome purification and characterization 110
For exosome isolation from K562 and LAMA84 cells, RPMI 1640 medium with 10% 111
exosome-depleted FBS (ultracentrifugation at 120,000g for 16 h) was used. 112
For exosome isolation from mice or patients’ hematopoietic cells, bone marrow 113
mononuclear cells were collected from either mice femurs or patients’ bone marrow samples 114
and then cultured using StemSpan SFEM II medium (Stemcell Technologies, Vancouver, 115
Canada) without FBS. Exosomes were isolated by differential centrifugation. In detail, 116
supernatant fractions collected from 48-72 hr cell cultures were centrifuged at 800g for 5 min 117
and 2000g for 10 min. The supernatant was then filtered using 0.22 μm filters, followed by a 118
final centrifugation at 10,0000g for 2 hr. The supernatant was aspirated and the pellet was re-119
suspended in PBS. The concentration of exosomes was determined by total exosomal proteins 120
using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), according 121
to the manufacturer’s instruction. For exosome isolation from serum, serum was collected from 122
all blood samples using a 2-step centrifugation protocol (2000g for 10 min, 10000g for 10 min) 123
to remove cellular debris. Serum was then incubated with ExoQuickTC exosome precipitation 124
solution (System Biosciences, Johnstown, PA, USA) according to manufacturer’s instructions. 125
Exosomes were stored at 4°C and analyzed or used for experimental procedures within one 126
week after harvest. 127
NanoSight and transmission electron microscopy were used to determine exosome size 128
7
distribution, concentration and morphology. For biomarker analysis of exosomes, the exosome 129
pellet was dissolved in lysis buffer for western blot (see below). 130
Fluorescently labeling of exosomes for in vitro and in vivo tracing 131
For in vitro analysis of the uptake of leukemic exosomes, exosomes in suspension were labeled 132
with 10 μM DiI (Invitrogen, Carlsbad, CA, USA) in a 37 oC water bath for 20 minutes and 133
repelleted by ultracentrifugation at 100,000g for 2 hr. DiI-labeled exosomes were added into 134
culture medium of normal BMMSC, which were plated on round cover slips (Electron 135
Microscopy Sciences, Hatfield, PA, USA) at 2×103/cm2, and incubated for another 24 hr. 136
Images of exosome uptake were acquired with a confocal laser scanning microscope. 137
For in vivo tracking of exosomes, exosomes prepared from cell lines K562, HeLa (human 138
cervical carcinoma), and A549 (human non-small cell lung cancer) were stained with 10 μM 139
DiR (Invitrogen, Carlsbad, CA, USA) similar as the DiI method mentioned above. About 200 140
μg of DiR-labeled K562, HeLa or A549-derived exosomes were intravenously injected in 141
C57BL/6 mice for 24 hours. The localization of the exosomes in organs was detected by 142
imaging using the IVIS® Lumina II in vivo imaging system (PerkinElmer, Waltham, MA, USA). 143
Flow cytometry was used to analyze the exosome distribution in different cell populations 144
in the bone marrow. Briefly, 200 μg of DiO-labeled exosomes were intravenously injected in 145
C57BL/6 mice, and bone marrow cells were harvested 24 hours later. DiO positive cells in 146
bone marrow mesenchymal stromal cells (CD45- Ter119- CD31-), endothelial cells (CD45- 147
Ter119- CD31+), granulocyte (Mac1+ Gr1+), B cells (B220+), and T cells (CD3+) were 148
analyzed by flow cytometry. 149
Colony forming unit-fibroblast assay 150
The colony forming unit-fibroblast (CFU-F) assay was performed as previous described [23]. 151
Briefly, normal or BA mice were sacrificed by CO2 and their femurs were cleaned from muscle 152
and crushed into pieces to release bone marrow cells. The marrow-free bone pieces were further 153
digested with 3% collagenase (Type I, Sigma-Aldrich, Saint Louis, MO USA) at 37 oC for 40 154
min. 2 x 104 cells were plated into a 60 mm dish in MesenCultTM Expansion Medium (Stemcell 155
Technologies). The medium was removed after 14 days culture and cells were fixed with 4% 156
8
PFA for 5 min at room temperature and then stained with crystal violet for 15 min. After 157
washing, colonies with more than 50 cells per colony were counted. 158
Flow cytometric analysis 159
Single cell suspensions from bone marrow, spleen, and peripheral blood were analyzed by flow 160
cytometry using monoclonal antibodies listed in the supplemental materials (Table S2). All 161
samples were first stained for 20 min at 4 oC with CD16/32 Fc blocking antibody (Biolegend, 162
San Diego, CA, USA) unless indicated otherwise. Stained cells were collected using Beckman 163
FC500 or BD FACSCalibur and analyzed using FlowJo software (FlowJo, Ashland, OR, USA). 164
Quantitative real-time PCR 165
Total RNA from cells or exosomes was extracted with Tripure Isolation Reagent (Roche, Basel 166
Switzerland) following the manufacturer’s instructions. RNA purity and concentration were 167
assessed with a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, 168
USA). miRNA expression was measured by quantitative real-time PCR (qPCR) using miRcute 169
miRNA First-Strand cDNA Synthesis Kit and miRcute miRNA SYBR Green qPCR Detection 170
Kit (Tiangen, Beijing, China). U6 was used as an internal control. For analysis of mRNA 171
expression, 1 μg total RNA was reverse transcribed using Transcriptor Reverse Transcriptase 172
Kit (Roche) and qPCR was performed using FastStart Essential DNA Green Master Kit 173
(Roche). GAPDH was used for normalization. All kits were used per manufacturers’ 174
instructions. The primer sequences are listed in Table S1. qPCR was run using the Roche 175
LightCycler 96 qPCR system (Roche) with each reaction run in triplicate. The change in 176
miRNA or mRNA expression was calculated by normalizing to the control sample as described 177
previously [24]. 178
Western blot 179
Cells or exosomes were homogenized in lysis buffer (20 mM Tris [pH7.5], 150 mM NaCl, 1% 180
Triton X-100 2 mM sodium pyrophosphate,25 mM β-glycerophosphate) supplemented with 181
Protease Inhibitor Cocktail (Roche). Protein extracts were separated in 10% or 12% SDS-182
PAGE and transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked 183
with 5% nonfat milk for 1 hr and then incubated overnight with primary antibodies at 4 oC. 184
9
The membrane was then incubated 1 hr with peroxidase-conjugated secondary antibodies at 185
room temperature and visualized using the ECL Prime Western Blotting Detection Reagent 186
(GE Healthcare,Buckinghamshire UK). All the details of antibodies are listed in Table S2. 187
Co-culture assay 188
K562 cells pre-transfected with 50 nM C. elegans specific miRNA (cel-miR-39) were placed 189
on the insert of a trans-well plate (0.4 μm polycarbonate filter, Corning, Beijing China), while 190
the normal BMMSC was placed on the lower chamber. The medium was additionally treated 191
with or without GW4869 (10 μM, Sigma-Aldrich) for 24 hr during co-culture with BMMSC. 192
At the end of the experiment, BMMSC were washed with PBS and cel-miR-39 expression in 193
BMMSC were detected by qPCR. 194
Small RNA (sRNA) sequencing and analysis 195
Total RNA was extracted from K562 cells and exosomes. The preparation of sRNA libraries 196
and deep sequencing were performed as follows. Briefly, about 500 ng purified RNA from each 197
group was used to prepare the sRNA libraries with Small RNA Sample Pre Kit (Illumina, San 198
Diego, CA, USA). Then miRNA expression was analyzed by small RNA sequence using HiSeq 199
2500 (Illumina). The differential miRNAs expression was shown by heatmap performed using 200
Cluster3.0 and Java Treeview. Multiple alignment of the mature miRNA sequence of cells and 201
exosomes was performed with ClustalX2 and FigTree software. 202
An unbiased search to identify over-represented motifs on exosome-enriched miRNAs 203
was performed with Improbizer as previous described [25]. Zero-or-one occurrence model was 204
used to find over-represented 4-8 nucleotide-long motifs. The remaining miRNAs annotated in 205
miRBase V22 were used as background. A Markov model of order 0 was assumed for the 206
background sequences. The cladogram was generated with the Weblogo software 207
(http://weblogo.threeplusone.com/). 208
Plasmid construction and lentivirus production 209
For knockdown of HNRNPA1 in K562 cell line, lentivirus expression vector pLKO.1 was used 210
to express shRNAs for human HNRNPA1 (TRCN0000006583, TRCN0000006586, 211
10
TRCN0000368907) or scrambled shRNA as control. For overexpression of HNRNPA1, human 212
HNRNPA1 coding sequence was amplified from cDNA of K562 cell line and inserted into the 213
lentivirus expression vector pCDH-CMV-MCS-EF1a-PURO with BamHI and EcoRI sites. The 214
same vector expressing eGFP was used as control. 215
For overexpression and knockdown of miR-320a in K562 cell line, we used the lentivirus 216
expression vector pLKO.1 to express the mature sequence of miR-320a or a sponge sequence 217
that contain 6 repeats of miR-320a binding sites. Detailed sequences used to generate 218
constructs were listed in Table S3. 219
Lentivirus was produced by transfecting HEK293T cells with lentivirus expression vector 220
along with psPAX2 and pMD2.G packaging plasmids. Supernatants containing lentivirus 221
particles were collected 48 hr after transfection, filtered and stored. For virus infection, virus 222
containing medium were added to cells, and after 6 hr incubation, medium was changed to 223
RPMI 1640 complete medium for another 48 h. The lentivirus infected cells were selected 224
using 3μg/ml puromycin for 2-4 weeks. 225
Cell cycle analysis 226
For evaluation of K562 cells’ cell cycle distribution, cells were fixed with 70% ice-cold ethanol 227
for 1 h, treated with RNase A (100 μg/ml) for 30 min at 37oC, and then stained with 20 μg/ml 228
propidium iodide for 30 min at 37oC. Cells were analyzed with BD FACSCalibur flow 229
cytometer. 230
EdU incorporation assay 231
EdU was added to the culture medium for a final concentration of 20 μM and incubated at 37 232
oC for 30 min. Cells were fixed and stained with EdU In Vitro Imaging Kit (Sangon, Shanghai, 233
China), according to manufacturer’s instruction. The percentage of EdU positive cells was 234
determined using ImageJ software. 235
Cell Counting Kit-8 assay 236
Cell proliferation were also analyzed using the Cell Counting Kit-8 (CCK-8) (Dojindo, Mashiki, 237
Kumamoto Japan). Briefly, K562 cells with different treatments were plated in 96-well plates 238
11
at 400/well. The cells were normally cultured for 1 to 7 days before 10 μl CCK-8 reagent was 239
added and incubated for another 2 hours. Absorbance was measured at 450 nm using a 240
SpectraMax 96-well plate reader (Molecular Devices, Sunnyvale, CA, USA). 241
In vitro osteogenic differentiation assay 242
Osteogenic differentiation was assayed as previously described [26]. Briefly, pre-confluent 243
BMMSC were supplemented with osteogenic medium, α-MEM containing 100 nM 244
dexamethasone (Sigma-Aldrich), 50 µg/mL L-ascorbic acid 2-phosphate, and 10 mM β-245
glycerophosphate (Sigma-Aldrich), plus either 5% human platelet lysate (HELIOS) for human 246
BMMSC or 10% FBS for mouse BMMSC, for 28 days. After differentiation, cells were fixed 247
with 4% PFA and stained with Alizarin red S (Sigma-Aldrich). For mineralization 248
quantification, destaining was conducted by adding a 10% cetylpyridinium chloride (Sigma-249
Aldrich) solution. Absorbance was measured in a 96-well plate reader at 570 nm. For alkaline 250
phosphatase (ALP) staining, cells were induced with osteogenic medium for 14 days and ALP 251
activity was measured using BCIP/NBT Alkaline Phosphatase Color Development Kit (Sangon, 252
Shanghai, China) according to manufacturer’s instructions. 253
Transmission electron microscopy 254
Exosomes were fixed with 2% paraformaldehyde, loaded on 200-mesh Formvar-coated grids, 255
and then counterstained and embedded as previously described [27]. Grids were dried and 256
visualized on a transmission electron microscope at 80 kV. 257
Micro-computed tomography (microCT) analysis of bone density 258
Distal femoral metaphases were scanned by microCT (eXplore Locus SP, GE Healthcare, 259
Milwaukee, WI, USA) under the following specifications: voltage, 80kV; current, 80 mA; 260
exposure time, 3000 ms; total rotation, 360o; rotation angle increment, 0.4o; resolution rate, 21 261
μm. Bone morphometric indices were assessed using 3D image reconstructions, included 262
cortical wall thickness (mm), bone volume relative to tissue volume (BV/TV, %), trabecular 263
thickness (mm), trabecular number (1/mm) and trabecular separation (mm). 264
RNA immunoprecipitation 265
12
RNA immunoprecipitation was performed with cytoplasmic extracts. Briefly, K562 cells or 266
freshly isolated bone marrow cells derived from BCR/ABL+ mice were lysed in ice cold lysis 267
buffer (20 mM Tris [pH7.5], 150 mM NaCl, 1% Triton X-100 2 mM sodium pyrophosphate, 268
25 mM β-glycerophosphate) supplemented with Protease Inhibitor Cocktail (Roche) and 40 269
U/ml Protector RNase Inhibitors (Roche) for 20 min. The lysates were then centrifuged for 15 270
min at 12,000g and supernatant was collected. About 1 mg protein extract was incubated with 271
10 μg mouse anti-HNRNPA1 monoclonal antibody (9H10, Abcam) or 10 μg mouse 272
immunoglobulin G [IgG] (Abcam) for 12 hr at 4 oC at a vertical shaking table. After that, 30 273
μl protein A sepharose (Abcam) was added for another 2 hr, followed by three washes with ice 274
cold lysis buffer. Co-immunoprecipitated RNA was extracted using Tripure Isolation Reagent 275
(Roche). miRNA qPCR analysis was performed with SYBR green mix (Roche) after reverse 276
transcription using miRcute kit (Tiangen). The miRNA fold enrichment in immunoprecipitated 277
samples was expressed as percentage against input and compared to IgG control. 278
Biotinylated miRNA Pull-Down 279
Biotinylated miRNA pull-down was performed with cytoplasmic extracts. Briefly, 20 nmol 280
biotinylated single stand miRNA oligonucleotides (GenePharma, Shanghai, China) were re-281
suspended in 100 μL lysis buffer supplemented with Protease Inhibitor Cocktail (Roche) and 282
40 U/ml Protector RNase Inhibitors (Roche) and incubated with 400 μL protein extract ( about 283
1mg) for 4 hr at 4 oC on a vertical shaking table. Streptavidin sepharose beads (Cell Signaling 284
Technology), pre-washed three times with lysis buffer, were added to the mixture and incubated 285
for an additional 2 hr at 4 oC. The beads were then washed three times with 1 mL lysis buffer 286
each. Beads were mixed with protein loading buffer and heated at 95 oC for 10 min to allow 287
collection of bound proteins for western blot or mass spectrometry (MS) analysis. 288
Statistical analysis 289
Data were analyzed with GraphPad Prism7 software. Unpaired two-tailed t test was used to 290
compare data for two groups. One-way ANOVA test was used to compare data for more than 291
two groups. Data are presented as the mean±SEM. Differences were considered significant 292
when the p value was < 0.05 (* p<0.05, ** p<0.01, *** p<0.001). 293
294
13
Results 295
Chronic myelogenous leukemia inhibits osteogenesis of bone marrow mesenchymal 296
stromal cells (BMMSC) 297
The SCL-tTA x TRE-BCR/ABL double transgenic (BA) mice have been widely used as a 298
mouse model of chronic phase CML [8, 21, 28]. As expected, BCR/ABL expression following 299
doxycycline withdrawal results in accumulation of myeloid progenitors and mature 300
granulocytes in the bone marrow, spleen and peripheral blood (Figure S1A and 1B) and 301
splenomegaly (Figure S1C), within in one month. With the occurrence of the CML, micro-302
computed tomography (micro-CT) revealed abnormal bone formation in BA mice after one 303
month induction of BCR/ABL expression. Cortical wall thickness, bone volume relative to 304
tissue volume (BV/TV) and trabecular thickness were significantly decreased, together with an 305
increase of trabecular separation (Figure 1A). Consistent with the observed osteoporosis, 306
BMMSC from induced BA mice exhibited a significant decline of total CFU-F activity per 307
femur as compared to controls (Figure 1B). When cultured under osteoinductive condition, 308
BMMSC from BA mice showed reduced capacity for mineralized nodule formation (Figure 309
1C). Similarly, BMMSC derived from CML patients also showed reduced capacity for 310
osteogenesis (Figure 1D). These data indicate impairment of osteogenic differentiation and 311
proliferation capacity of BMMSC in both CML mouse model and CML patients. 312
Exosomes secreted by leukemia cells are efficiently taken up by BMMSC 313
Exosomes derived from cancer cells have been reported to mediate the remodeling of cancer 314
microenvironments, suggesting possible involvement of leukemic exosomes in tuning 315
BMMSC. Exosomes isolated from K562 culture medium by sequential ultracentrifugation 316
exhibited typical cup-shaped morphology by transmission electron microscopy (Figure 2A), 317
with a size distribution of 50 to 150 nm in diameter analyzed by nanoparticle tracking analysis 318
(Figure 2B). Leukemic exosomes were positive for specific exosomal marker TSG101 but 319
negative for GM103, a marker for Golgi complex (Figure 2C). Coomassie blue staining of total 320
protein showed different protein electrophoresis patterns between leukemia cells and leukemic 321
exosomes (Figure 2D). As revealed by confocal fluorescence microscopy, the labeled 322
14
exosomes were efficiently taken up by BMMSC (Figure 2E). Consistently, ex vivo imaging 323
also revealed that these CML-derived exosomes preferentially migrated into the bone marrow, 324
compared with exosomes from other sources, such as cell lines (HeLa and A549) of human 325
solid tumors (Figure 2F). Consistently, intravenous injection of exosomes loaded with a C. 326
elegans specific miRNA (cel-miR-39) also revealed abundant expression of cel-miR-39 in 327
femurs (Figure 2G). To further determine whether leukemic exosomes indeed endocytosed into 328
BMMSC, we transfected K562 cells with cel-miR-39, followed by co-culture with BMMSC in 329
a 0.4 µm transwell plate. The appearance of cel-miR-39 in BMMSC demonstrates that the cel-330
miR-39 was delivered from the K562 cells in the upper well to the BMMSC in the lower well 331
(Figure 2H). In addition, the exosome secretion inhibitor GW4869 significantly lowered the 332
delivery of cel-miR-39 to BMMSC (Figure 2H), further indicating that K562-derived 333
exosomes are efficiently endocytosed into BMMSC. Next, we examined whether BMMSC are 334
the main recipient cells of the leukemic exosomes in the bone marrow (BM) cells. DiO labeled 335
K562 exosomes were intravenously injected into normal C57BL/6 mice. Internalization of the 336
labeled exosomes into mouse BM cells was demonstrated after 24 hr by FACS. Bone marrow 337
mesenchymal stromal cells (CD45- Ter119- CD31-) exhibited the highest fluorescence 338
intensity than other BM cells, such as endothelial cells (CD45- Ter119- CD31+), granulocyte 339
(Mac1+ Gr1+), B cells (B220+), and T cells (CD3+) (Figure S2A), confirming that leukemic 340
exosomes were more efficiently taken up by BMMSC. 341
Exosomes from leukemia cells inhibit BMMSC osteogenesis 342
We next explored whether leukemic exosomes can modulate osteogenesis of human BMMSC 343
in vitro. BMMSC treated with leukemic exosomes exhibited decreased capacity of osteogenic 344
differentiation as shown by alkaline phosphatase (ALP) and Alizarin Red staining, compared 345
to healthy human serum-derived exosomes or PBS-treated BMMSC (Figure 3A). In addition, 346
the expression of RUNX2, a major osteogenic marker, was downregulated in a dose-dependent 347
manner by treatment with leukemic exosomes (Figure 3B). Furthermore, genes associated with 348
osteogenesis (OPN and COL1A1) or hematopoiesis (CXCL12 and KITL) were also 349
downregulated in BMMSC treated with leukemic exosomes in a dose-dependent manner 350
(Figure 3C and Figure S3A). 351
15
Next, we assumed that leukemic exosomes might also inhibit BMMSC functions in vivo. 352
C57BL/6 mice (6 to 8 weeks old) were intravenously injected with 50 µg of either leukemic 353
exosomes or control exosomes, derived from BA mice or control mice, twice per week for four 354
consecutive weeks. Mice that received leukemic exosomes exhibited significant loss of 355
trabecular bone and whole bone volume in the femurs (Figure 3D and Figure S3B), similar to 356
the phenotype of transgenic BA mice. Moreover, there was a significant decrease of CFU-F 357
frequencies of BMMSC from leukemic exosome-treated mice, compared to normal controls 358
(Figure 3E). Taken together, these results demonstrate that exosomes secreted by leukemia cells 359
can significantly inhibit the function of BMMSC in vitro and in vivo. 360
Specific repertoires of miRNAs are sorted into leukemia exosomes 361
MiRNAs have been reported to be key exosome cargo that can be actively secreted into 362
exosomes and delivered to target cells, resulting in direct modulation of their mRNA targets. 363
In order to clarify whether miRNAs are the key functional mediators in the observed function 364
of leukemic exosomes, we thus performed small RNA sequencing and analyzed the 365
intracellular and exosomal profile of the miRNome in K562 cells (Figure 4A and Figure S4A). 366
In particular, 148 miRNAs were enriched and 165 miRNAs were under represented in leukemic 367
exosomes compared to the cellular fraction. These miRNAs were further analyzed based on 368
fold changes and overall TPM (transcripts per million reads) (at least two data >1000). Forty 369
seven of the miRNAs met these criteria and were selected for further analysis (Figure S4B, 370
Table S5). A heat map of the selected miRNAs demonstrated the profound difference between 371
leukemic cells and exosomes (Figure 4B). The expression profile wase further confirmed by 372
qPCR analysis of selected miRNAs in both K562 cells (Figure 4C) and LAMA84 cells (Figure 373
S4C). These results showed that distinct subsets of miRNAs were either enriched or diminished 374
in leukemic exosomes. 375
ClustalW was used to find the sequence characteristics of the enriched/deminished 376
miRNAs. As shown in Figure 4D, a group of miRNAs that were enriched in exosomes harbored 377
a similar mature sequence, including miR-320a, miR-320b, miR-320c, miR-320d, miR-3180-378
3p, miR-128-3p and miR-423-5p. An unbiased search for over-represented sequence motifs 379
using Improbizer [25] detected a conserved minimal AGAGGG motif (Figure 4E). About 50% 380
16
of exosome-enriched miRNAs contain at least 4 nucleotide of the motif but only 9.7% of cell-381
enriched miRNAs (Table S5). These data indicated that specific repertoires of miRNAs with 382
certain motifs were sorted into leukemia exosomes 383
Exosomal miR-320 plays an essential role in the observed effects on both leukemia cells 384
and BMMSC 385
Among these miRNAs that were enriched in leukemic exosomes, we focused on the miR-320 386
family for three reasons: (1) family members such as miR-320a, miR-320b, miR-320c and 387
miR-320d were specifically loaded into leukemic exosomes (Figure 4B, 4D, S4B); (2) miR-388
320 family contains the AGAGGG motif which may control their exosomal sorting; and (3) 389
previous research has shown that miR-320 can directly target the BCR/ABL oncogene [29], 390
and β-catenin [30, 31] which is a key regulator of osteogenesis. We then investigated the 391
functional effect of miR-320 on both leukemic cells and BMMSC. The 3’-UTR of BCR/ABL 392
contains a binding site that can be recognized by all four miR-320 as predicted from a miRNA 393
target database (Figure 5A). As expected, miR-320a mimics transfection significantly 394
decreased the endogenous expression of BCR/ABL in K562 cells (Figure 5B). Forced 395
expression of miR-320a has significantly inhibited K562 cell proliferation (Figure 5C). Cell 396
cycle assessment suggested that miR-320a can cause G1 phase arrest in the cell cycle (Figure 397
5D), which was further confirmed by EdU incorporation assay (Figure 5E). 398
Notably, the 3’-UTR of β-catenin also contains a binding site that can be recognized by 399
all four miR-320 (Figure 6A). β-catenin is the transcriptional regulator of canonical Wnt 400
signaling, which can promote the maturation of osteoblastic precursor cells into mature 401
osteoblasts. Knocking down of β-catenin in BMMSC by siRNAs inhibited the osteogensis as 402
measured by ALP staining (Figure S5A), and reduced the expression of osteogenesis (OPN, 403
COLIA1) or hematopoiesis (CXCL12, KitL) related genes (Figure S5B). K562 derived 404
exosomes significantly decreased the expression of β-catenin in BMMSC, which was rescued 405
by prior miR-320 knockdown in K562 cells (Figure 6B and 6C). Accordingly, the inhibited 406
osteogenic differentiation capacity of BMMSC by exosomes derived from K562 cells was 407
compromised when miR-320a was knocked down in the parental cells (Figure 6D). To further 408
confirm the role of aberrant differentiation of BMMSC induced by miR-320, we have knocked 409
17
down miR-320 (miR-320 KD) in K562 cells, and then injected 50 µg of the derived exosomes 410
into the C57BL/6 mice, twice per week for four consecutive weeks. Compared with the control 411
groups, exosomes from miR-320 KD K562 cells had minimal effects on the bone abnormalities 412
(Figure 6E). These data indicate that miR-320 is essential for the observed effects of K562 413
exosomes on both K562 cells and BMMSC. 414
HNRNPA1 mediates exosomal sorting of miR-320 415
To unravel the molecular mechanisms that control the active sorting into exosomes of specific 416
miRNAs, especially the miR-320 family, K562 cell lysates were incubated with streptavidin 417
beads coated with biotinylated miR-320a. The bound proteins were subjected to high-418
throughput identification by mass spectrometry (MS). Non-coated beads and beads with 419
biotinylated miR-374b-5p (which was a cell-enriched miRNA) served as negative control. 420
Among the enriched proteins precipitated with biotinylated miRNA-320a (Table S4), we 421
focused on HNRNPA1, a ubiquitously expressed RNA-binding protein recognizing the motif 422
UAGGG(A/U) (Figure S6A). 423
Consistently, HNRNPA1 was abundantly expressed in both leukemic cells and their 424
exosomes, as confirmed by both immunofluorescence confocal microscopy (Figure 7A) and 425
western blot (Figure 7B). Specific binding of HNRNPA1 to miR-320a was verified with 426
miRNA pull-down followed by western blot analysis of HNRNPA1 (Figure 7C). HNRNPA1 427
can be efficiently pulled down by biotinylated miR-320a, in comparison to biotinylated miR-428
374b-5p, demonstrating specific binding of HNRNPA1 and miR-320a in cells. The result was 429
further confirmed with immunoprecipitation of HNRNPA1 from cytoplasmic lysates of both 430
K562 cells (Figure 7D) and bone marrow mononuclear cells from BA mice (Figure S6B). The 431
effects of HNRNPA1 knockdown and overexpression (Figure S6C) on miR-320 sorting into 432
exosomes were further assessed. After HNRNPA1 knockdown, miR-320 was significantly 433
increased in the cellular components but decreased in the exosomes (Figure 7E). In contrast, 434
overexpression of HNRNPA1 increased the exosomal enrichment but decreased the 435
intracellular abundance of miR-320 (Figure 7F). No significant change of cellular and 436
exosomal expression of cell-enriched miR-374b-5p was found upon manipulating HNRNPA1 437
expression. Consistent with the role of HNRNPA1 in exosomal sorting of miR-320, knockdown 438
18
of HNRNPA1 significantly decreased the growth rate of K562 cells, which can be rescued by 439
miR-320 inhibitor (Figure 7G). Notably, inhibition of BCR/ABL by Imatinib decreased both 440
the cellular and exosomal HNRNPA1 protein level (Figure S6D), together with increased miR-441
320 in the cells while decreased level in the exosomes (Figure S6E). Collectively, these data 442
demonstrate that HNRNPA1 functions importantly in the sorting of miR-320 into leukemic 443
exosomes, and Imatinib might inhibit leukemia cell growth partially via interfering the pathway. 444
Abnormal expression of HNRNPA1 and exosomal miR-320 in clinical CML samples 445
Finally, we asked whether our findings in the leukemic cell line and murine model had clinical 446
relevance. HNRNPA1 mRNA and protein levels were markedly increased in CP (chronic 447
phase)-CML cells and became even higher in BC (blast crisis) -CML cells (Figure 8A and 8B), 448
compared to normal BM cells. Moreover, BC-CML patients had a much lower miR-320 449
expression in intracellular levels but higher in plasma exosomal levels, compared to CP-CML 450
patients (Figure 8C), which might be explained by the higher expression of HNRNPA1 in BC-451
CML cells. In addition, some other differentially miRNAs identified in the RNA-seq data were 452
also found expressed in a similar pattern in the CML cells and plasma exosomes (Figure S7A). 453
Consistent with the assumption that exosomal miR-320 might be transported from leukemic 454
cells to BMMSC, there were higher intracellular level of miR-320 in primary BMMSC derived 455
from CML patients than from healthy donors (Figure 8E). 456
Discussion 457
In this study, we have found that the exosomes secreted from leukemia cells reduced the tumor 458
suppressive miR-320 in the donor cells, resulting promoted cell growth. Moreover, we found 459
that leukemic exosomes can decrease the osteogenic differentiation potential of BMMSC both 460
in vitro and in mouse model, resulting in reduced trabecular bone volume. The study 461
demonstrate that the exosome derived from leukemia cells is an important contributor of niche 462
remodeling 463
The functional importance of bone marrow niche remodeling for failure of normal 464
hematopoiesis in myeloproliferative neoplasms has become increasingly evident [5, 7, 8, 32]. 465
During the progress of myeloproliferative neoplasms, the functions of bone marrow 466
19
mesenchymal stromal cells (BMMSC) are profoundly impaired. AML-derived BMMSCs are 467
molecularly and functionally altered with decreased proliferation and osteogenic differentiation, 468
which contribute to hematopoietic insufficiency [2, 3, 33-35]. The mesenchymal niche of CML 469
is also found aggressively modified into a self-reinforcing leukemic niche that impairs normal 470
hematopoiesis [5, 7, 32]. Previous studies have suggested an essential role for tumor cell-471
derived exosomes in reprogramming the tumor microenvironment. Kumar et al [36] found that 472
acute myeloid leukemia cell-derived exosomes can transform the bone marrow niche into a 473
leukemia-permissive microenvironment by impairing the normal functions of BMMSC. Here, 474
we further revealed that CML secreted exosomes could simultaneously promote the CML cell 475
growth and remodel the bone marrow niche toward leukemia permissive microenvironment via 476
inhibiting osteogenesis. 477
Exosomes derived from CML cells can shuttle miRNAs, amphiregulin and even bcr/abl 478
mRNAs to nearby stromal cells [37-39], resulting in reprogramming of niche cell functions. In 479
our study, deep sequencing of small RNAs in leukemic cells and their exosomes shows a 480
profound difference between intracellular and exosomal miRNAs. Among the exosome-481
enriched miRNAs, a higher proportion has previously been identified as tumor suppressive 482
miRNAs [29, 30, 40, 41], including miR-320 family, miR-3180-3p, miR-128-3p and miR-423-483
5p, compared to cell-enriched miRNAs. Based on our observation, leukemia cells are more 484
likely to selectively export these suppressive miRNAs to promote tumor cell growth. Although 485
we here focused on the role of tumor suppressor miR-320, the function of other miRNAs should 486
not be excluded. In fact, all of the encapsulated miRNAs might work together to remodel the 487
niche, while secretion of these miRNAs via exosomes also promotes the donor cell 488
proliferation. 489
To date, different pathways and molecules have been described to involved in the sorting 490
of specific miRNAs into exosomes. For example, heterogeneous nuclear ribonucleoprotein 491
A2B1 (HNRNPA2B1) and heterogeneous nuclear ribonucleoprotein Q (HNRNPQ) can bind to 492
miRNAs containing a certain sequence (called EXO motif) and guide their inclusion into 493
exosomes [17, 18]. In addition, major vault protein (MVP) is also reported to mediate the 494
loading of miRNAs into exosomes [42]. We here found that HNRNPA1 at least selectively 495
20
sorted out miR-320 into exosomes via the AGAGGG motif in the miRNAs. HNRNPA1 is a 496
highly abundant RNA-binding protein that has been implicated in diverse cellular functions 497
related to RNA metabolism including nucleo-cytoplasmic shuttling [43], alternative splicing 498
regulation [44], mRNA stability and miRNA processing [45]. Indeed, some leukemia-related 499
factors such as BCL-XL and SET are processed by HNRNPA1 [20]. HNRNPA1 expression is 500
also induced by BCR/ABL protein in a dose and kinase-dependent manner [20]. Consistent 501
with previous research, HNRNPA1 levels were higher in CP-CML cells, and even higher in 502
BC-CML cells, compared to normal bone marrow cells. Together, our findings suggest a novel 503
mechanism of how HNRNPA1 promotes leukemia progression: by selectively sorting tumor 504
suppressor miRNAs into exosomes. As leukemia stem cells (LSC) are the origin of CML, it is 505
thus worthwhile to confirm whether the HNRNPA1 mediated miR-320 sorting into exosomes 506
occurs in LSCs. It is also important to compare the differences between HSC and LSC derived 507
exosomes, while the study is limited by the technical difficulty of in vitro culture of HSCs and 508
LSCs without feeder cells. Single cell sequencing might be the solution. 509
In summary, this study has revealed that HNRNPA1 promotes cell growth via exosomal 510
sorting of tumor suppressive miRNAs (mainly miR-320) in CML, while the secreted exosomes 511
in turn function to remodel the niche to a leukemic favorable microenvironment via inhibiting 512
osteogenesis of BMMSC (Figure 8E). Thus, HNRNPA1 and the exosomal miRNAs provide 513
novel therapeutic targets to restore the normal function of the bone marrow niche. 514
Acknowledgements 515
We thank Dr F. Lu for her assistance with the Nikon microscope system, J. Kang for her help 516
with the electron microscopy. We are grateful to the technical help of Q. Chen and J. Zhang 517
from the Department of Ultrasound Medicine in Tangdu Hospital, Fourth Military Medical 518
University. This work was supported by the Nature Science Foundation of China 519
(NSFC31573244 to L Liu, NSFC31771507 to G Yang), Key Projects of Shaanxi Province 520
(2018ZDXM-SF-063 to L Liu) and State Key Laboratory of Cancer Biology Projects 521
(CBSKL2017Z10 to G. Yang). 522
Author contributions 523
21
X.G, Z.W, and M.W conducted the experiments. Y. D, Y. Z, X. C, Z. L, and W. Q performed 524
the gene expression analysis, exosome purification, intravenous injection and tissue dissection. 525
G. Y and L. L designed the experiments, analyzed the data, and wrote the manuscript. 526
References 527
1. Schepers K, Campbell TB, Passegue E. Normal and leukemic stem cell niches: insights and 528
therapeutic opportunities. Cell Stem Cell. 2015; 16: 254-67. 529
2. Geyh S, Rodriguez-Paredes M, Jager P, Khandanpour C, Cadeddu RP, Gutekunst J, et al. 530
Functional inhibition of mesenchymal stromal cells in acute myeloid leukemia. Leukemia. 531
2016; 30: 683-91. 532
3. von der Heide EK, Neumann M, Vosberg S, James AR, Schroeder MP, Ortiz-Tanchez J, et 533
al. Molecular alterations in bone marrow mesenchymal stromal cells derived from acute 534
myeloid leukemia patients. Leukemia. 2017; 31: 1069-78. 535
4. Kim JA, Shim JS, Lee GY, Yim HW, Kim TM, Kim M, et al. Microenvironmental 536
remodeling as a parameter and prognostic factor of heterogeneous leukemogenesis in acute 537
myelogenous leukemia. Cancer Res. 2015; 75: 2222-31. 538
5. Schepers K, Pietras EM, Reynaud D, Flach J, Binnewies M, Garg T, et al. Myeloproliferative 539
neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. 540
Cell Stem Cell. 2013; 13: 285-99. 541
6. Colmone A, Amorim M, Pontier AL, Wang S, Jablonski E, Sipkins DA. Leukemic cells 542
create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells. 543
Science. 2008; 322: 1861-5. 544
7. Zhang B, Ho YW, Huang Q, Maeda T, Lin A, Lee SU, et al. Altered microenvironmental 545
regulation of leukemic and normal stem cells in chronic myelogenous leukemia. Cancer Cell. 546
2012; 21: 577-92. 547
8. Welner RS, Amabile G, Bararia D, Czibere A, Yang H, Zhang H, et al. Treatment of chronic 548
myelogenous leukemia by blocking cytokine alterations found in normal stem and 549
progenitor cells. Cancer Cell. 2015; 27: 671-81. 550
9. Tkach M, Thery C. Communication by Extracellular Vesicles: Where we are and where we 551
need to go. Cell. 2016; 164: 1226-32. 552
22
10. van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular 553
vesicles. Nat Rev Mol Cell Biol. 2018; 19: 213-28. 554
11. Zhou W, Fong MY, Min Y, Somlo G, Liu L, Palomares MR, et al. Cancer-secreted miR-555
105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell. 2014; 25: 501-556
15. 557
12. Bruns H, Bottcher M, Qorraj M, Fabri M, Jitschin S, Dindorf J, et al. CLL-cell-mediated 558
MDSC induction by exosomal miR-155 transfer is disrupted by vitamin D. Leukemia. 2017; 559
31: 985-8. 560
13. Zhang L, Zhang S, Yao J, Lowery FJ, Zhang Q, Huang WC, et al. Microenvironment-561
induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature. 562
2015; 527: 100-4. 563
14. Hornick NI, Doron B, Abdelhamed S, Huan J, Harrington CA, Shen R, et al. AML 564
suppresses hematopoiesis by releasing exosomes that contain microRNAs targeting c-MYB. 565
Sci Signal. 2016; 9: ra88. 566
15. Boyiadzis M, Whiteside TL. The emerging roles of tumor-derived exosomes in 567
hematological malignancies. Leukemia. 2017; 31: 1259-68. 568
16. Zietzer A, Werner N, Jansen F. Regulatory mechanisms of microRNA sorting into 569
extracellular vesicles. Acta Physiol (Oxf). 2018; 222: e13018. 570
17. Villarroya-Beltri C, Gutierrez-Vazquez C, Sanchez-Cabo F, Perez-Hernandez D, Vazquez 571
J, Martin-Cofreces N, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into 572
exosomes through binding to specific motifs. Nat Commun. 2013; 4: 2980. 573
18. Santangelo L, Giurato G, Cicchini C, Montaldo C, Mancone C, Tarallo R, et al. The RNA-574
binding protein SYNCRIP is a component of the hepatocyte exosomal machinery controlling 575
microRNA sorting. Cell Rep. 2016; 17: 799-808. 576
19. Perrotti D, Neviani P. From mRNA metabolism to cancer therapy: chronic myelogenous 577
leukemia shows the way. Clin Cancer Res. 2007; 13: 1638-42. 578
20. Iervolino A, Santilli G, Trotta R, Guerzoni C, Cesi V, Bergamaschi A, et al. hnRNP A1 579
nucleocytoplasmic shuttling activity is required for normal myelopoiesis and BCR/ABL 580
leukemogenesis. Mol Cell Biol. 2002; 22: 2255-66. 581
21. Koschmieder S, Gottgens B, Zhang P, Iwasaki-Arai J, Akashi K, Kutok JL, et al. Inducible 582
23
chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a 583
transgenic model of BCR-ABL leukemogenesis. Blood. 2005; 105: 324-34. 584
22. Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, et al. 585
Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery 586
vehicles for anticancer agents. J Natl Cancer Inst. 2004; 96: 1593-603. 587
23. Abbuehl JP, Tatarova Z, Held W, Huelsken J. Long-term engraftment of primary bone 588
marrow stromal cells repairs niche damage and improves hematopoietic stem cell 589
transplantation. Cell Stem Cell. 2017; 21: 241-55.e6. 590
24. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. 591
Nat Protoc. 2008; 3: 1101-8. 592
25. Ao W, Gaudet J, Kent WJ, Muttumu S, Mango SE. Environmentally induced foregut 593
remodeling by PHA-4/FoxA and DAF-12/NHR. Science. 2004; 305: 1743-6. 594
26. Ambrosi TH, Scialdone A, Graja A, Gohlke S, Jank AM, Bocian C, et al. Adipocyte 595
accumulation in the bone marrow during obesity and aging impairs stem cell-based 596
hematopoietic and bone regeneration. Cell Stem Cell. 2017; 20: 771-84.e6. 597
27. Wang J, Hendrix A, Hernot S, Lemaire M, De Bruyne E, Van Valckenborgh E, et al. Bone 598
marrow stromal cell-derived exosomes as communicators in drug resistance in multiple 599
myeloma cells. Blood. 2014; 124: 555-66. 600
28. Reynaud D, Pietras E, Barry-Holson K, Mir A, Binnewies M, Jeanne M, et al. IL-6 controls 601
leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia 602
development. Cancer Cell. 2011; 20: 661-73. 603
29. Xishan Z, Ziying L, Jing D, Gang L. MicroRNA-320a acts as a tumor suppressor by 604
targeting BCR/ABL oncogene in chronic myeloid leukemia. Sci Rep. 2015; 5: 12460. 605
30. Yuan SX, Wang J, Yang F, Tao QF, Zhang J, Wang LL, et al. Long noncoding RNA 606
DANCR increases stemness features of hepatocellular carcinoma by derepression of 607
CTNNB1. Hepatology. 2016; 63: 499-511. 608
31. Kang DW, Noh YN, Hwang WC, Choi KY, Min do S. Rebamipide attenuates Helicobacter 609
pylori CagA-induced self-renewal capacity via modulation of beta-catenin signaling axis in 610
gastric cancer-initiating cells. Biochem Pharmacol. 2016; 113: 36-44. 611
32. Arranz L, Sanchez-Aguilera A, Martin-Perez D, Isern J, Langa X, Tzankov A, et al. 612
24
Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms. 613
Nature. 2014; 512: 78-81. 614
33. Huang JC, Basu SK, Zhao X, Chien S, Fang M, Oehler VG, et al. Mesenchymal stromal 615
cells derived from acute myeloid leukemia bone marrow exhibit aberrant cytogenetics and 616
cytokine elaboration. Blood Cancer J. 2015; 5: e302. 617
34. Jacamo R, Davis RE, Ling X, Sonnylal S, Wang Z, Ma W, et al. Tumor Trp53 status and 618
genotype affect the bone marrow microenvironment in acute myeloid leukemia. Oncotarget. 619
2017; 8: 83354-69. 620
35. Kornblau SM, Ruvolo PP, Wang RY, Battula VL, Shpall EJ, Ruvolo VR, et al. Distinct 621
protein signatures of acute myeloid leukemia bone marrow-derived stromal cells are 622
prognostic for patient survival. Haematologica. 2018; 103: 810-21. 623
36. Kumar B, Garcia M, Weng L, Jung X, Murakami JL, Hu X, et al. Acute myeloid leukemia 624
transforms the bone marrow niche into a leukemia-permissive microenvironment through 625
exosome secretion. Leukemia. 2018; 32: 575-87. 626
37. Ramos TL, Sanchez-Abarca LI, Lopez-Ruano G, Muntion S, Preciado S, Hernandez-627
Ruano M, et al. Do endothelial cells belong to the primitive stem leukemic clone in CML? 628
Role of extracellular vesicles. Leuk Res. 2015; 39: 921-4. 629
38. Corrado C, Saieva L, Raimondo S, Santoro A, De Leo G, Alessandro R. Chronic 630
myelogenous leukaemia exosomes modulate bone marrow microenvironment through 631
activation of epidermal growth factor receptor. J Cell Mol Med. 2016; 20: 1829-39. 632
39. Taverna S, Amodeo V, Saieva L, Russo A, Giallombardo M, De Leo G, et al. Exosomal 633
shuttling of miR-126 in endothelial cells modulates adhesive and migratory abilities of 634
chronic myelogenous leukemia cells. Mol Cancer. 2014; 13: 169. 635
40. Aslan D, Garde C, Nygaard MK, Helbo AS, Dimopoulos K, Hansen JW, et al. Tumor 636
suppressor microRNAs are downregulated in myelodysplastic syndrome with spliceosome 637
mutations. Oncotarget. 2016; 7: 9951-63. 638
41. Yu WW, Jiang H, Zhang CT, Peng Y. The SNAIL/miR-128 axis regulated growth, invasion, 639
metastasis, and epithelial-to-mesenchymal transition of gastric cancer. Oncotarget. 2017; 8: 640
39280-95. 641
42. Teng Y, Ren Y, Hu X, Mu J, Samykutty A, Zhuang X, et al. MVP-mediated exosomal 642
25
sorting of miR-193a promotes colon cancer progression. Nat Commun. 2017; 8: 14448. 643
43. Pinol-Roma S, Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and 644
cytoplasm. Nature. 1992; 355: 730-2. 645
44. Tavanez JP, Madl T, Kooshapur H, Sattler M, Valcarcel J. hnRNP A1 proofreads 3' splice 646
site recognition by U2AF. Mol Cell. 2012; 45: 314-29. 647
45. Kooshapur H, Choudhury NR, Simon B, Muhlbauer M, Jussupow A, Fernandez N, et al. 648
Structural basis for terminal loop recognition and stimulation of pri-miRNA-18a processing 649
by hnRNP A1. Nat Commun. 2018; 9: 2479. 650
26
Figure legends 651
652
Figure 1. Functional inhibition of bone marrow mesenchymal stromal cells in chronic 653
myelogenous leukemia. (A) Representative micro-CT cross-section images of femurs from 654
BCR/ABL+ (BA) mice and control (Ctrl) mice. Bar graphs demonstrate average cortical wall 655
thickness in compact bone regions, relative bone volume (BV/TV), number of trabeculae and 656
space between trabeculae in trabecular bone regions of control and BCR/ABL+ mice. Data 657
are expressed as mean ± SEM (n=6 mice per group). (B) Representative images show 658
decreased frequency of CFU-Fs in BMMSC derived from BA mice vs. control mice. Data are 659
expressed as mean ± SEM (n=9 mice per group). (C) Decreased osteogenic differentiation 660
potential of BMMSC from BA mice vs. control mice. Osteogenesis was indicated by alizarin 661
red staining on day 28. Data are expressed as mean ± SEM of three independent experiments. 662
(D) Alizarin red staining photomicrographs of decreased osteogenic differentiation potential 663
of BMMSC from CML patient (CML-BMMSC) vs. age-matched healthy control (Normal-664
BMMSC). Data presented are representative of 3 normal control and CML patients 665
respectively. ** p<0.01 by t test. 666
27
667
Figure 2. Leukemia cell-secreted exosomes are efficiently taken up by BMMSC. (A) 668
Transmission electron microscopy images of exosomes derived from K562 cells. (B) 669
Nanoparticle tracking analysis shows size distribution of K562-derived exosomes. (C) Western 670
blot analysis of exosome-specific marker TSG101 and Golgi-specific marker GM130 in lysates 671
from whole cells (Cell) or exosomes (EXO). GAPDH serves as internal control. (D) Coomassie 672
blue-stained sulfate polyacrylamide gel after electrophoretic separation of total lysates from 673
K562 cells (Cell) and derived exosomes (EXO). (E) Confocal fluorescence image of the 674
endocytosis of DiI-labeled exosomes by primary human BMMSC. (F) Ex vivo fluorescence 675
imaging of excised femurs from C57BL/6 mice injected with DiR-labeled exosomes from 676
different cell lines: K562, CML cell line; Hela, human cervical cancer cell line; and A549, 677
human adenocarcinoma cell line. Data are expressed as mean ± SEM (n=3 mice per group). (G) 678
28
qPCR analysis of cel-miR-39 level in femurs of C57BL/6 mice. Femurs were harvested 24 679
hours after tail vein injection of exosomes derived from indicated cell lines loaded with cel-680
miR-39. NC: negative control. Data are expressed as mean ± SEM of three independent 681
experiments. (H) Schematic of co-culture system in Transwell (membrane pore diameter, 0.4 682
μm) to measure exosome transfer (Left panel). BMMSC were co-cultured with K562 cells 683
transfected with cel-miR-39. The culture medium was added with or without exosome secretion 684
inhibitor GW4869. Cel-miR-39 in BMMSC was quantified by qPCR (right panel). Data are 685
expressed as mean ± SEM of three independent experiments. * p<0.05, ** p<0.01 by one way 686
ANOVA. 687
29
688
Figure 3. Leukemia cell-secreted exosomes inhibit BMMSC function in vitro and in vivo. 689
(A) ALP and Alizarin red staining analysis of osteogenesis. BMMSC cultured in osteogenic 690
condition were additionally treated with PBS, or exosomes derived from either healthy human 691
serum (Serum-EXO) or cultured K562 cells (K562-EXO) every 3 days, followed by ALP 692
staining after 14 days or by alizarin red staining after 28 days. Images are representative of 693
three independent experiments. (B) Western blot analysis of RUNX2 expression in human 694
BMMSC treated as indicated. Cells were cultured in osteogenic condition for 3 days. Images 695
are representative of three independent experiments. (C) qPCR analysis of mRNA expression 696
of indicated genes in BMMSC treated same as above. Data are presented as the mean ± SEM 697
30
of three independent experiments. (D) microCT analysis of the femurs from C57BL/6 mice 698
treated as indicated. Mice were intravenously injected with 50 μg of indicated exosomes twice 699
per week for four consecutive weeks (Nor-EXO: exosomes derived from bone marrow 700
mononuclear cells (BM-MNC) of control normal mice; CML-EXO: exosomes derived from 701
the BM-MNC of BA mice). Mice treated with PBS served as control. Micro-CT was used to 702
illustrate the effect on cross-sections of femurs in trabecular bone regions and to quantify 703
relative bone volume (BV/TV) and number of trabeculae. Data are expressed as the mean ± 704
SEM (n=6 mice per group). (E) Representative images show the CFU-Fs of the BMMSC 705
derived from mice with indicated treatments (left panel). Data are presented as the mean ± 706
SEM of three different experiments from 3 mice per group. * p<0.05, ** p<0.01 by one way 707
ANOVA. 708
31
709
Figure 4. Specific repertoires of miRNAs are sorted into leukemia exosomes. (A) Deep 710
sequence data visualization by scatter plot comparing miRNAs in K562 cells and derived 711
exosomes. (B) Heat map depicting differentially expressed miRNAs between cells (Cell) and 712
exosomes (EXO) from human K562 cells. These miRNAs had overall TPM (transcripts per 713
million reads) >1000 in at least one group and fold change difference >2. (C) Verification of 714
32
selected miRNAs using qPCR. Data are presented as the mean ± SEM of three independent 715
experiments. ** p<0.01 by t test. (D) Cladogram showing the multiple alignment of mature 716
sequences of 14 leukemic exosome-enriched miRNAs (red) and 31 cell-enriched miRNAs 717
(black). (E) Over-represented motifs on exosome-enriched miRNAs. The core AGAGGG 718
shows high information content. 719
33
720
Figure 5. Exosomal miR-320 plays an essential role in leukemia cell growth. (A) Schematic 721
diagram of the putative binding sites of the miR-320 family in the 3’ untranslated region (UTR) 722
of bcr/abl. (B) BCR/ABL (P210) protein level, measured by western blot, in K562 cells 723
transfected with different dose of miR-320a mimics or scramble (NC) for 48 hr. Images are 724
representative of three independent experiments. (C) Proliferation of K562 cells with miR-725
320a (miR-320 OE) or scrambled miRNA (miR-scramble) overexpression, as analyzed by 726
CCK-8 assay. (D) Cell cycle phase analysis of K562 cells transfected with either miR-320a 727
(miR-320 OE) or scrambled control (miR-scramble). The percentage of cells in the G1, S, and 728
G2 phases is shown in the bar graph. Data are presented as the mean ± SEM of three 729
independent experiments. (E) EdU incorporation assay of K562 transfected with either miR-730
320a (miR-320 OE) or scrambled miRNA (miR-scramble). Images are representative of three 731
independent experiments. * p<0.05, ** p<0.01 by t test or two way ANOVA. 732
34
733
Figure 6. Exosomal miR-320 plays an essential role in osteogenesis of BMMSC. (A) 734
Schematic diagram of the putative binding sites of the miR-320 family in the 3’UTR of β-735
catenin. (B) Expression of β-catenin in BMMSC with indicated treatments. Images are 736
representative of three independent experiments. (C) Expression of β-catenin in BMMSC with 737
indicated exosome treatments. Serum-EXO: healthy human serum exosomes; miR-scramble-738
EXO and miR-320 KD-EXO: exosomes from control or miR-320 knocked down K562 cells 739
(infected with control or miR-320 sponge); EXO and EXO+miR-320: exosomes from 740
HEK293T cells loaded without or with miR-320. Images are representative of three 741
independent experiments. (D) Alizarin red staining showing the osteogenic differentiation of 742
BMMSC treated with indicated exosomes. BMMSC treated with healthy human serum 743
exosomes (Serum-EXO) served as control. Images are representative of 3 independent 744
experiments. (E) Micro-CT analysis of the femurs of mice treated as indicated. C57BL/6 mice 745
35
were tail vein injected with 50 μg of indicated exosomes twice per week for four consecutive 746
weeks. Mice treated with PBS served as control. Relative bone volume (BV/TV), trabecular 747
thickness, trabecular separation, and trabecular numbers were further analyzed. Data are 748
presented as the mean ± SEM (n=6 mice per group). * p<0.05, ** p<0.01, *** p<0.001 by one 749
way ANOVA. 750
36
751
Figure 7. HNRNPA1 mediates exosomal sorting of miR-320. (A) Confocal microscopy 752
image shows the co-localization of HNRNPA1 (green) and specific exosome marker TSG101 753
(red) in K562 cells. (B) Western blot showing HNRNPA1 and CD9 expression in both cellular 754
(Cell) and exosomal (EXO) lysates. GAPDH serves as internal control. (C) Western blot 755
showing HNRNPA1 pulled down by biotinylated miR-320 in whole cellular extracts (WCE) 756
of K562 cells, with biotinylated miR-374b-5p serving as control. Images are representative of 757
three independent experiments. (D) RNA-IP analysis of miR-320 bound by HNRNPA1. K562 758
cytoplasmic extracts were incubated with IgG or antiHNRNPA1 and the immunoprecipates 759
were isolated for qRT-PCR analysis. miR-374b-5p serves as a negative control. Data are means 760
± SEM of three independent experiments. (E) qRT-PCR analysis of intracellular (left) and 761
exosomal (right) levels of selected miRNAs in HNRNPA1 knock down (shHNRNPA1) or 762
control K562 cells (shControl). The values were normalized to U6 levels in cells or exosomes 763
and shown as mean ± SEM of three independent experiments. (F) qRT-PCR analysis of 764
37
intracellular (left) and exosomal (right) levels of selected miRNAs in control (GFP) or 765
HNRNPA1 overexpression K562 cells. Data are means ± SEM of three independent 766
experiments. (G) Proliferation of K562 cells treated as indicated were further analyzed by 767
CCK-8 assay. Data are expressed as mean ± SEM of 3 different experiments. * p<0.05, ** 768
p<0.01, *** p<0.001 by t test. 769
770
38
771
Figure 8. Abnormal expression of HNRNPA1 and miR-320 in chronic myelogenous 772
leukemia patients. mRNA (A) and protein (B) expression of HNRNPA1 in primary human 773
bone marrow mononuclear cells (BM-MNC) of normal control, chronic phase (CML-CP) or 774
blast crisis CML (CML-BC). (C) BM-MNC cellular and plasma exosomal expression of miR-775
320 in CML-CP or CML-BC patients. (D) Intracellular expression level of miR-320 in 776
BMMSC derived from healthy control and CML patients. (E) Graphic illustration of the study: 777
39
HNRNPA1 mediated exosomal transfer of miR-320 from leukemia cell to mesenchymal 778
stromal cell simultaneously promotes leukemia cell growth while inhibits mesenchymal 779
stromal cell osteogenesis. * p<0.05, ** p<0.01 by t test or one way ANOVA. 780
Recommended