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: chingchengchen@coh.org (C.-C.C.)

1

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

2

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.

3

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.

5

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

6

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.

7

8

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.

9

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

10

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.

11

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

12

following 5-FU treatments (n=8-10 mice per group in two independent experiments. *=p<0.05.

**=p<0.01, ***=p<0.001.

13

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

14

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.

15

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

18

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

19

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