Acute myeloid leukemia transforms the bone marrow niche into a leukemia-permissive
microenvironment through exosome secretion
Bijender Kumar1,2,3, Mayra Garcia1,2,3, Lihong Weng1,2,3, Xiaoman Jung1,2,3, Jodi L. Murakami1,2,3,4,
Xingbin Hu1,6, Tinisha McDonald1,2, Allen Lin1,2,3, Ashish R. Kumar7, David L. DiGiusto8, Anthony
S. Stein1,2,3, Vinod A. Pullarkat1,2,3, Susanta K. Hui5, Nadia Carlesso1,2,3, Ya-Huei Kuo1,2,3, Ravi
Bhatia9, Guido Marcucci1,2,3 and Ching-Cheng Chen1,2,3,4*
1Divison of Hematopoietic Stem Cell and Leukemia Research of Beckman Research Institute,
2Department of Hematology and Hematopoietic Cell Transplantation,
3Gehr Family Center for Leukemia Research,
4Irell & Manella Graduate School of Biological Sciences,
5Department of Radiation Oncology,
City of Hope, Duarte, CA 91010, USA
6Department of Transfusion Medicine, Xijing Hospital, Fourth Military Medical University, Xi’an
7100032, PR China
7Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH 45229, USA
8Department of Pediatric Transplantation and Regenerative Medicine, Stanford School of
Medicine. Stanford, CA 94305, USA
9Division of Hematology and Oncology, University of Alabama at Birmingham, Birmingham, AL
35294, USA
CONTACT
*Corresponding author: [email protected] (C.-C.C.)
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Supplementary Figure 1. Characterization of mice that received primary MLL-AF9
leukemia cells or normal human CD34+ cells. (a, b) Mouse spleen from DKO mice engrafted
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with human AML (MV411, KG1A) cell lines (a) or B6 mice engrafted with mouse AML (MLL-
AF9) cells (b). (c) 8- to 12-week-old B6 mice were irradiated and injected with 10,000 murine
AML (MLL-AF9) cells. The litter was sacrificed when transplanted mice were moribund or 4
weeks after transplantation. Spleen weight of control and MLL-AF9-transplanted B6 mice (n=5–
6 mice per group in at least two independent experiments). (d) Gating strategy for LT-HSC
analysis. (e) Frequency of the LT-HSC population in marrow of control and (MLL-AF9) B6 mice
(n=5–8 mice per group in at least three independent experiments). (f) Frequency of the LT-HSC
population in marrow of neonatal DKO mice injected with vehicle (control) or 500,000 normal
human CD34+ cells intrahepatically 8 weeks after transplantation (n=5 mice per group in two
independent experiments). (g) Gating strategy for BM stromal cell analysis. (h) Frequency of
Sca1+, CD146+ and CD166+ cells in the stromal compartment of control and MLL-AF9-
transplanted B6 mice (n=7–13 mice per group in at least three independent experiments). (i)
Frequency of Sca1+, CD146+ and CD166+ cells in the stromal compartment of neonatal DKO
mice injected with vehicle (control) or normal human CD34+ cells (n=5 mice per group in two
independent experiments). (j) Immunofluorescence staining of bone sections from control or
MLL-AF9-tranplanted B6 mice. Staining of CD146 and MECA32 (left) are shown. Number of
CD146+MECA32- (right) cells in the view field determined by immunofluorescent staining.
ns=not significant.
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4
Supplementary Figure 2. Plasma exosome and OCN levels in AML patients and
characterization of AML-derived exosomes. (a) Healthy control (n=12) or AML patients
separated based on FLT3-ITD mutation status (FLT3-ITD neg, n=18; FLT3-ITD, n=13). Plasma
exosome numbers (left) and plasma OCN levels (right). (b) Healthy controls and AML patients
separated based on disease risk status (control, n=12; Intermediate risk, n=12; poor risk, n=22).
Plasma exosome numbers (left) and plasma OCN levels (right). (c) Protocol for isolation of
exosomes from the supernatant fractions of normal human PBMC or AML cells using
ultracentrifugation. (d) Representative nanoparticle tracking analysis of exosomes isolated from
normal human PBMC or primary AML cells cultures. (e) Mean size of purified exosome
measured by NanoSight in healthy control (n=5) and AML patients (n=18). (f) Western blot
analysis of AML (MV411, KG1A, COH193, COH195)-derived exosomes for common exosome-
specific markers TSG101 and CD63, and β-actin loading control. (g-h) Images (g) and size
distribution (h) of AML-derived exosomes, generated by transmission electron microscopy. (i)
200 g CFSE-labeled exosomes were injected into mice intravenously, and BM cells were
harvested 10 h later. Representative FACS analysis showing internalization of CFSE-labeled
exosomes (left) and percent CFSE+ cells (right, n=3 mice in three independent experiments)
within in indicated BM subpopulations. ns=not significant, *=p<0.05, **=p<0.01, ***=p<0.001,
****=p<0.0001.
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Supplementary Figure 3. Characterization of mice that received PBMC or AML-derived
exosomes. (a) Mouse spleen of 6- to 8-week-old B6 mice injected with either normal human
PBMC-derived or AML-derived exosomes. Mice were euthanized 30 days after initial injection.
(b) Representative FACS profiles of LT-HSC compartments. (c) Frequency of LT-HSC in spleen
(n=6–8 mice per group in at least three independent experiments). (d) Femurs of mice receiving
normal PBMC-derived or AML-derived exosomes (n=6–8 mice in at least two independent
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experiments). Quantitative -CT analysis of cortical wall thickness in compact bone region,
relative bone volume (BV/TV), thickness of trabecular bone, number of trabeculae, and space
between trabeculae in trabecular bone region. (e) Adult B6 mice were injected with four weekly
doses of normal PBMC-derived exosomes or PBS (vehicle control) intravenously. Mice were
euthanized 30 days after initial injection. Frequency of Sca1+, CD146+, and CD166+ cells in the
stromal compartment. ns=not significant. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.
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Supplementary Figure 4. AML and AML-derived exosomes modulate gene expression in
BM stromal cells. (a) qRT-PCR showing expression levels of indicated genes (n=4–12 in at
least three independent experiments). BM Sca1+, CD146+, or CD166+ stromal cells from DKO
mice treated with vehicle control or engrafted with AML cell lines (MV411, KG1A, NB4, MLL-
AF9) or patient samples (COH101, COH105). (b)Sorted BM Sca1+, CD146+ and CD166+ cells
from normal B6 mice were cultured directly with PBMC-derived or AML (MV411, KG1A)-derived
exosomes. Adherent cells were harvested and assayed for gene expression by qRT-PCR. n=4–
12 in at least three independent experiments. (c, d) Human BM stromal cells were cultured with
either vehicle control or AML cell lines (MV411, KG1A) or patient samples (COH101, COH103,
COH105) (c) or normal PBMC-derived or corresponding AML-derived exosomes (d). ns=not
significant, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.
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Supplementary Figure 5. AML and AML exosomes alter lineage fate of the stromal cells
and DKK1 blocks osteoblast differentiation. (a, b) Neonatal DKO mice were injected with
vehicle control or AML cells (NB4, COH101) intrahepatically. Sorted Sca1+ stromal cells from
BM of transplanted mice were then cultured under osteogenic conditions for 21 days. Alizarin
Red staining was performed to assess Ca2++ deposition after osteogenic induction (a). qRT-PCR
of osteoblast-specific gene OCN (b). (c, d) qRT-PCR of adipogenic (a; PPAR) and
chondrogenic (b; ACAN) genes in Sca1+ stromal cells isolated from BM of normal B6 mice and
treated with PBMC-derived or AML (MV411, KG1A)-derived exosomes, after 2 weeks culture in
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adipogenic or chondrogenic conditions. (e) Sorted Sca1+, CD146+, CD166+ stromal cells from
BM of normal B6 mice were cultured with PBMC-derived or AML (MV411, KG1A)-derived
exosomes. The expression of DKK1, a suppressor of normal hematopoiesis and osteogenesis,
was assayed by qRT-PCR. (f, g) Sca1+ stromal cells isolated from BM of normal B6 mice and
cultured under osteogenic conditions with vehicle (control) or 10 ng/mL of recombinant DKK1.
Alizarin Red staining after osteogenic induction (h) and qRT-PCR of osteogenic (OCN) genes
(e). (f) Sorted Sca1+ cells from BM of normal B6 mice were cultured with PBMC-derived or AML
(MV411)-derived exosomes, with or without DKK1 inhibitor (Way262611). Alizarin Red staining
after osteogenic induction. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.
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Supplementary Figure 6. LT-HSCs from AML-derived exosome-treated mice show
increased cell cycle entry and decreased stem cell activities. (a) B6 mice received three
weekly doses of normal PBMC-derived or KG1A-derived exosomes before sacrifice. (b)
Representative cell cycle analysis assessing intracellular Ki67 and DNA content (PI) staining in
hematopoietic progenitors (LSKs) and LT-HSCs. (c, d) Percentage of cells in G0 (c) and S/G2/M
(d) phases in LSK cells and LT-HSCs (n=5 mice per group in two independent experiments). (e)
Mice received three weekly doses of either normal PBMC-derived or AML (KG1A)-derived
exosomes prior to weekly 5-FU treatments. (f) Kaplan-Meier survival curve analysis of mice
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following 5-FU treatments (n=8-10 mice per group in two independent experiments. *=p<0.05.
**=p<0.01, ***=p<0.001.
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Supplementary Figure 7. AML-derived exosome treatment accelerate stromal
compartment changes; disruption of exosome production does not alter AML cell cycle
and apoptosis status. (a) Three weekly doses of normal PBMC-derived or AML (KG1A)-
derived exosomes were injected intravenously into 6- to 8-week-old DKO mice. Mice were
irradiated and injected with 20 million KG1A cells. Frequency of Sca1+, CD146+, and CD166+
cells in the stromal compartment of treated mice 20 days after KG1A transplantation (n=5 mice
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per group in at least two independent experiments. (b-c) MV411 AML cells were transduced
with lentiviral shRNA against Rab27a, a protein involved in exosome release. Cell cycle (b) and
apoptosis analysis (c) of lentiviral-transduced MV411 cells. ns=not significant, *=p<0.05.
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Supplementary Table 1. Clinical characteristics of AML patients
Sample ID Age Sex Diagnosis Disease
Status Risk Status Cytogenetic Gene MutationWBC
counts (x 103)
Blasts % in PB
COH101 49 F de novo Untreated intermediate t(15:17)FLT-3 ITD Pos., FLT-3
D835 Neg., NPM1 Neg., PML-RARa Pos
92.6 91
COH103 18 M de novo Untreated Poor NormalFLT-3 ITD Pos., FLT-3
D835 Pos., NPM1 Neg., CEBPA Pos.
76.8 84
COH105 19 F de novo Refractory Poor t(7;21) FLT-3 ITD Pos., FLT-3 D835 Neg., 77.9 86
COH107 66 F de novo Untreated Poor Complex FLT-3 ITD Pos., FLT-3 TKD Neg., IDH 1/2 Neg. 26.2 65
COH109 54 M de novo Relapsed intermediate Normal C-KIT Neg, FLT-3 Neg, NPM1 Neg, 3.2 26
COH111 62 F de novo Relapsed Poor der(17)t(12;17), +19
FLT-3 ITD Pos., FLT-3 TKD Neg., IDH 1/2 Neg. 13.9 54
COH113 56 M de novo Refractory Poor t(2;17), der(8)t(8;8)
FLT-3 ITD Pos., FLT-3 TKD Neg., 112.1 85
COH115 60 M secondary Untreated Poor Trisomy 8
FLT-3 ITD Pos., FLT-3 TKD Pos., NPM1 Neg., CEBPA Neg., IDH1/2
Neg.
32.9 43
COH117 58 F de novo Untreated Poor NormalFLT-3 ITD Pos., FLT-3 TKD Neg., NPM1 Neg., CEBPA Pos., IDH2 Neg.
97 53
COH119 54 M de novo Relapsed intermediate Normal C-KIT Neg, FLT-3 Neg, NPM1 Neg, 1.8 47
COH121 65 F de novo Untreated Poor Complex FLT-3 ITD Pos., FLT-3 TKD Neg., NPM1 Neg.,
CEBPA Neg.15.8 43
COH123 70 M de novo Refractory Poor del(5q), del(7q), del(20q)
CEBPA Neg, FLT-3 Neg, NPM1 Neg, 0.5 96
COH125 42 M de novo Untreated Poor NormalFLT-3 ITD Pos., FLT-3 TKD Neg., NPM1 Pos., Kit Neg., CEBPA Neg.
29.9 56
COH127 62 M secondary Untreated Poor Monosomy 7
FLT-3 ITD Neg., FLT-3 TKD Neg., NPM1 Neg., BCR/ABL Neg., IDH2
Pos.,
21 9.6
COH129 37 M de novo Refractory Poor t(12;22) FLT-3 ITD Pos., FLT-3 D835 Neg., 1.2 NA
COH131 44 F de novo Refractory Poor Normal FLT-3 ITD Pos., FLT-3 D835 Neg., CEBPA Neg. 16.3 48
COH133 50 M de novo Relapsed Poor
t(1;11) (q21;q23), +19 [19]/[20], KMT2A (MLL) translocation,
Trisomy 8
FLT3 ITD Neg., FLT3-TKD Pos., NPM1 Neg., IDH1 Neg., IDH2 Neg.,
2.5 16
COH135 65 M de novo Relapsed intermediate Normal FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Pos. 2.8 14
COH137 76 F secondary Relapsed/ Refractory intermediate Normal FLT-3 Neg, NPM1 Neg.,
IDH1 Neg., IDH2 Neg. 0.9 0
16
COH139 32 M de novo Untreated intermediate Normal
CEBPA Pos, FLT-3 ITD Neg, FLT3 TKD Neg.,
NPM1 Neg, ,IDH1 Neg., IDH2 Neg.
6.4 14
COH141 71 M secondary Untreated intermediate Normal
FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Neg.,
CEBPA Neg., IDH1 Neg., IDH2 Neg.
27.5 10
COH143 23 F de novo Relapsed/ Refractory Poor Complex Not done 0.9 46
COH145 64 M de novo Untreated intermediate Normal FLT-3 Neg, NPM1 Neg, 1 7
COH147 27 F de novo Untreated intermediate Normal
FLT3 ITD Neg., FLT3 TKD Neg., FLT3 TKD
Neg., C-Kit Neg., CEBPA Pos, NPM1 Neg., IDH1
Neg., IDH2 Neg.,
98.5 94.0%
COH149 52 F de novo Relapsed Poor Normal
FLT3 ITD Pos., FLT3 TKD Neg., NPM1 Pos., IDH1 Neg., IDH2 Neg.,
MPL Neg.
24.3 18
COH151 58 F de novo Relapsed Poor t(2;18), del(3) Trisomy 8
FLT-3 ITD Pos., FLT-3 TKD Neg 46.4 66
COH153 66 M de novo Relapsed intermediate Trisomy 8 Not done 42.9 93
COH155 52 M de novo Untreated Poor Del(5)
FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Neg.,
C-Kit Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.,
41.4 62.0%
COH157 21 M de novo Untreated intermediate Del(9)
FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Neg.,
C-Kit Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.
62 90.0%
COH159 45 M secondary Untreated Poor t(3;3), del(11) FLT3 ITD Neg., FLT3 TKD Neg. IDH1 Neg.,
IDH2 Neg.11.8 60
COH161 61 F de novo Untreated intermediate Normal
FLT3-ITD Neg, FLT3-TKD Neg., IDH1 Neg., IDH2 Neg., NPM1 Neg.
CEBPA Neg.,
5.1 10
COH163 29 F de novo Untreated Poor Trisomy 8
FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Neg.,
C-Kit Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.
65.2 99
COH165 66 M de novo Relapsed/ Refractory intermediate Trisomy 8 Not Done 5.4 83
COH167 60 M de novo Untreated Poor Complex
FLT3-ITD Neg., FLT3-TKD Neg., NPM1 Neg.,
C-Kit Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.
6.4 25
17
COH169 59 F de novo Untreated Poor Normal
FLT3 ITD Pos., FLT3 TKD Neg., CEBPA Neg., NPM1 Pos., IDH1 Neg.,
IDH2 Neg.,
50.4 87.0%
COH171 75 F de novo Untreated intermediate Normal
FLT3-ITD Neg., FLT3 TKD Pos., NPM1 Pos., C-KIT Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.
47.5 55
COH173 45 M de novo Untreated Poor Complex,Trisomy 8
FLT3 ITD Neg., FLT3 TKD Neg., C-Kit Neg.,
CEBPA Neg., IDH1 Neg., IDH2 Pos.
21.1 18
COH175 54 M de novo Refractory Poor Normal
FLT3 ITD Pos., FLT3 TKD Neg., NPM1 Neg., C-Kit Neg., IDH1 Neg.,
IDH2 Neg.
1.5 6
COH177 77 M de novo Untreated Poor Complex
FLT3 ITD Pos., FLT3 TKD Neg., IDH1 Neg.,
IDH2 Neg., C-KIT Neg., NPM1 Neg.,
5.1 32
COH179 51 M de novo Relapsed/ Refractory intermediate Normal
FLT3 ITD Neg., FLT3 TKD Neg., IDH1 Neg.,
IDH2 Neg.18 96
COH181 45 M secondary Refractory intermediate Trisomy 8
FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Neg.,
CEBPA Neg., IDH1 Neg., IDH2 Neg., BCR-ABL
Neg.,Positive for ASXL1, ETV6,EZH2, PTPN11
and RUNX1,Negative for JAK2, MPL, and CALR
8.7 15
COH183 60 F de novo Relapsed Poor Normal
FLT3 ITD Pos., FLT3 TKD Neg., NPM1 Pos.,
CEBPA Neg., IDH1 Neg., IDH2 Neg.
38.3 57
COH185 58 M de novo Untreated Poor t(9;22)
LT3-ITD Neg., FLT3 TKD Neg., NPM1 Neg.,
CEBPA Neg., IDH1 Neg., IDH2 Neg., BCR-ABL
Pos.
7.5 11
COH187 43 F secondary Relapsed Poor RUNX1/RUNX1T1 translocation
FLT3 ITD Pos., FLT3 TKD Neg., NPM1 Pos.,
C-Kit Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.
8 81
COH189 71 F de novo Untreated Poor Aneusomy for Chomosome 4
FLT3 ITD Pos., FLT3 TKD Neg., NPM1 Pos.,
C-Kit Neg., CEBPA Neg., IDH1 Neg., IDH2 Neg.,
PML-Rara Neg.
49.2 73
COH191 38 F de novo Relapsed Poor der(7)t(?1;7), inv(16), del(13)
FLT3 ITD Neg., FLT3 TKD Neg., NPM1 Neg., IDH1 Neg., IDH2 Neg.
32.1 71
COH193 60 F de novo Untreated Poor BCR/ABL1 translocation
FLT3-ITD Neg., FLT3-TKD Neg., IDH1 Neg., IDH2 Neg., NPM1 Neg.
CEBPA Neg.,
191.2 83
COH195 71 M ce novo Relapsed PoorTrisomy
del(5q) [1] ;SL 45, idem, -Y, del(9) [4]
Not Done 2.4 0
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Supplementary Table 2. TaqMan probes used in current study
Gene TaqMan Assay IDCXCL12 Mm00445553_m1DKK1 Mm00438422_m1GAPDH (mouse) Mm99999915_g1IGF1 Mm00439560_m1IL-6 Mm00446190_m1IL-7 Mm01295803_m1KITL Mm00442972_m1Osteocalcin (OCN) Mm03413826_mHCCL3 Mm00441259_g1Col1A1 Mm00801666_g1PPAR Mm00440940_m1ACAN Mm00545794_m1Rab27a (human) Hs00608302_m1GAPDH (human) Hs02758991_g1IL-6 (human) Hs00985639_m1CXCL12 (human) Hs03676656_mHOCN (human) Hs01587814_g1Col1A1 (human) Hs00164004_m1
Supplementary Table 3. Antibodies used in Current study
Antigen Vendor Catalogue # DilutionCD16/32 BioLegend 101302 1:200
CD45 BioLegend 103128 1:100CD31 BioLegend 102418 1:100
Ter119 BioLegend 116210 1:500Sca1 BioLegend 108134 1:20
CD146 BioLegend 134705 1:25CD166 R&D
SystemsFAB1172P 1:50
c-Kit BioLegend 105826 1:100Sca1 BioLegend 108114 1:100
CD150 BioLegend 115912 1:100CD48 BioLegend 103426 1:100CD48 BioLegend 103414 1:100
CD135 BioLegend 135306 1:50CD3 BioLegend 100334 1:100CD4 BioLegend 100428 1:200
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CD8 BioLegend 100725 1:100CD11b BioLegend 101224 1:200B200 BioLegend 103227 1:200GR-1 BioLegend 108430 1:200
Ter119 BioLegend 116232 1:100TSG101 Sigma SAB2702167 1:100
CD63 Santa Cruz sc-15363 1:100-Actin Cell Signaling 4967S 1:100Sca1 BioLegend 108102 1:50
CD146 BioLegend 134702 1:50MECA32 BioLegend 120504 1:100
Osteocalcin Clontech M173 1:100Goat-anti-rat-Alexa 555 ThermoFishe
rA-21434 1:200
Goat-anti-rat-Alexa 488 ThermoFisher
A-11006 1:200
Goat-anti-rabbit-Alexa 555 ThermoFisher
A-21429 1:200
Ki67 BioLegend 652410 1:100
20