Supplementary Materials for · 2020. 4. 6. · Table S3. Patient- and disease-specific...
Transcript of Supplementary Materials for · 2020. 4. 6. · Table S3. Patient- and disease-specific...
stm.sciencemag.org/cgi/content/full/12/538/eaax5104/DC1
Supplementary Materials for
IL-6 blockade reverses bone marrow failure induced by human
acute myeloid leukemia
Tian Yi Zhang, Ritika Dutta, Brooks Benard, Feifei Zhao, Raymond Yin, Ravindra Majeti*
*Corresponding author. Email: [email protected]
Published 8 April 2020, Sci. Transl. Med. 12, eaax5104 (2020)
DOI: 10.1126/scitranslmed.aax5104
This PDF file includes:
Fig. S1. PB leukemic burden does not correlate with cytopenias or BM leukemic burden. Fig. S2. Leukemic burden estimated by variant allelic frequency does not correlate with severity of cytopenias. Fig. S3. Representative markers and gating strategy to determine human AML engraftment in NSG mice. Fig. S4. Conventional NSG AML xenografts do not develop BM failure evidenced by lack of cytopenias. Fig. S5. Representative markers and gating strategy to determine human normal CB-CD34+ cell engraftment in NSG mice. Fig. S6. CBCs in NSGsham and NSGspln- mice transplanted with primary AML samples or normal CB-CD34+ cells. Fig. S7. Splenectomized NSG mice engrafted with human primary AML exhibit shortened survival. Fig. S8. Surgical splenectomy does not increase leukemic burden in NSG mice engrafted with human AML. Fig. S9. Representative flow plots for the evaluation of mouse HSPCs in NSGspln- PDX mice. Fig. S10. Mouse HSPCs in NSGspln--PDX mice do not exhibit increased apoptosis/cell death or displacement into PB. Fig. S11. Mouse HSPCs are similarly depleted in NSGsham-PDX mice. Fig. S12. Mouse proerythroblasts in NSGspln--PDX mice exhibit similar rates of apoptosis/cell death. Fig. S13. Representative markers and gating strategy used to sort purified populations of human AML blasts. Fig. S14. Representative markers and gating strategy used to sort purified populations of human CB-CD34+ cells. Fig. S15. Viability of human AML in culture. Fig. S16. Human AML blasts do not block erythroid differentiation by inhibition of cell cycle entry or induction of cell death.
Fig. S17. RNA-seq of purified populations of human AML blasts and CB-CD34+ cells shows an inflammatory transcriptome signature in AML. Fig. S18. Effects of the addition of recombinant human IL-6 on normal CB-CD34+ progenitors undergoing erythroid differentiation. Fig. S19. Siltuximab treatment does not decrease leukemic burden in NSGspln- mice engrafted with human AML. Fig. S20. Siltuximab treatment does not decrease leukemic burden in various organs of NSGspln- mice engrafted with human AML. Fig. S21. Siltuximab treatment initiated after establishment of disease also improves anemia and overall survival in human AML xenografts. Fig. S22. IL-1β and CCL3 production in NSGspln--PDX mice and AML conditioned medium. Table S1. Summary of characteristics of patients whose clinical data were analyzed in Fig. 1 (A to D). Table S2. Summary of characteristics of patients whose clinical data were analyzed in figs. S1 and S2. Table S3. Patient- and disease-specific characteristics of human AML samples investigated here. Table S4. Human AML conditioned medium suppresses mouse progenitor colony formation. Table S5. Human AML conditioned medium suppresses human progenitor colony formation.
A
B
C
Fig. S1.
Fig. S1. PB leukemic burden does not correlate with cytopenias or BM leukemic burden.
(A) hemoglobin (R2=0.0046, p=0.016), (B) platelet count (R
2=0.015, p=9.4e-06), and (C) PB
blast percentage (R2=0.22, p=1.5e-65) with BM blast percentage at time of diagnosis.
R2=Pearson correlation of determination.
Fig. S2.
A.
B.
Fig. S2. Leukemic burden estimated by variant allelic frequency does not correlate with
severity of cytopenias. Correlation of (A) hemoglobin and (B) platelet count with VAF of
recurrently mutated genes in AML. R2=Pearson correlation of determination.
Fig. S3.
Fig. S3. Representative markers and gating strategy to determine human AML
engraftment in NSG mice. Femoral aspirates were obtained at 12 weeks after transplantation
and stained with a panel of human myeloid- and lymphocyte-specific antibodies followed by
flow cytometry analysis. The presence of mCD45-hCD45
+CD33
+CD3
-CD19
- cells is indicative
of successful engraftment by human AML. Mice with bi-lineage engraftment (with additional
presence of mCD45-hCD45
+CD33
-CD3
-CD19
+ cells) after transplantation would be indicative of
engraftment with normal human multipotent progenitors rather than leukemic cells and were
therefore excluded from analysis.
FSC-A
SS
C-
A 50K 100
K 150K
200K
250K
0
50K
100
K
150
K
200
K
250
K
SSC-A
SS
C-H
50K 100
K 150K
200K
250K
0 50K
100
K
150
K
200
K
250
K Single Cells
PI/Ter119 (PE- Texas Red)
SS
C-H
0 1x10
3 1x104 1x10
5
50K
100
K
150
K
200
K
250
K
hC
D45 (
PE
-C
y7)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
mCD45 (PB)
CD
19 (
AP
C)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
CD33 (PE)
Myeloid
B lymph
CD
19 (
AP
C)
0 1x10
3 1x104 1x10
5 1x1
03
1x1
04
1x1
05
CD33 (PE)
Myeloid
T lymph
Fig. S4.
A.
B.
Fig. S4. Conventional NSG AML xenografts do not develop BM failure evidenced by lack
of cytopenias. (A) Platelet and (B) WBC counts were measured via tail vein every two weeks,
and results at 12 weeks are shown (n.s. = not significant).
0 12 0 12 0 120
100
200
300
400
Time after transplant (weeks)
Pla
tele
t (1
e9/L
)
SU540 SU266 SU209
n.s. n.s. n.s.
0 12 0 12 0 120
1
2
3
4
5
Time after transplant (weeks)
WB
C (
1e9/L
)
SU540 SU266 SU209
n.s. n.s.n.s.
Fig. S5.
Fig. S5. Representative markers and gating strategy to determine human normal CB-
CD34+ cell engraftment in NSG mice. Femoral aspirates were obtained at 12 weeks after
transplantation and stained with a panel of human myeloid- and lymphocyte-specific antibodies
followed by flow cytometry analysis. Bilineage engraftment with mCD45-hCD45
+CD33
+CD19
-
CD3-
and mCD45-hCD45
+CD33
- CD19
+CD3
- cells demonstrates successful engraftment by
normal CB-CD34+ cells.
FSC-A
SS
C-
A 50K 100
K 150K
200K
250K
0
50K
100
K
150
K
200
K
250
K
SSC-A
SS
C-H
50K 100K
150K
200K
250K
0
50K
100
K
150
K
200
K
250
K Single Cells
PI (PE- Texas Red)
SS
C-H
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
1x1
02
1x1
02
CD
19 (
AP
C)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
CD33 (PE)
hC
D45 (
PE
-C
y7)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
mCD45 (PB)
T lymph
Myeloid
CD
19 (
AP
C)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
CD33 (PE)
Figure. S6.
A.
B.
SU54
0
SU55
5
SU20
9
SU48
0
SU26
6
SU35
3
SU35
1
SU45
6CB1
CB2
CB3
0
5
10
15
20
Hem
oglo
bin
(g/d
L)
Sha
m
SU54
0
SU57
5
SU55
5
SU20
9
SU48
0
SU26
6
SU35
3
SU35
1
SU49
6
SU45
6CB1
CB2
CB3
0
100
200
300
400
500
Pla
tele
t (1
e9/L
)
****
n.s.
C.
Fig. S6. CBCs in NSGsham
and NSGspln-
mice transplanted with primary AML samples or
normal CB-CD34+ cells. (A) NSG
sham mice engrafted with primary AML or normal CB-CD34
+
cells do not develop BM failure up to 6 months (8-9 months of age) after transplantation. (B)
Thrombocytopenia and (C) leukopenia develop in NSGspln-
mice engrafted with AML but not
CB-CD34+ HSPCs. All AML samples are color coded to reflect individual AML or CB samples
reported in Figure 2D.
Sha
m
SU54
0
SU57
5
SU55
5
SU20
9
SU48
0
SU26
6
SU35
3
SU35
1
SU49
6
SU45
6CB1
CB2
CB3
0
1
2
3
4
5
WB
C (
1e9/L
)
****
n.s.
Fig. S7
A.
B.
0 10 20 30 40
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU496
NSGsham-SU496
*p = 0.01
0 10 20 30 40
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU456
NSGsham-SU456
*p = 0.02
0 10 20 30
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU351
NSGsham-SU351
*p = 0.02
0 10 20 30 40
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU353
NSGsham-SU353
*p = 0.01
0 10 20 30
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU266
NSGsham-SU266
*p = 0.01
0 10 20 30
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU480
NSGsham-SU480
*p = 0.01
0 10 20 30 40
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU209
NSGsham-SU209
*p = 0.01
0 10 20 30 40
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU555
NSGsham-SU555
*p = 0.01
0 10 20 30
0
50
100
Weeks following engraftment
Perc
ent S
urv
ival %
NSGspln--SU575
NSGsham-SU575
**p = 0.002
Sample
ID
Median Survival (weeks) P
value Splenectomized Sham
SU540 9.5 36 0.0001
SU575 8 26 0.0002
SU555 11 34 0.01
SU209 11 34 0.009
SU480 9 25 0.01
SU266 13 25 0.01
SU353 13 26 0.01
SU351 11 27 0.02
SU496 12 31 0.01
SU456 17 35 0.02
Fig. S7. Splenectomized NSG mice engrafted with human primary AML exhibit shortened
survival.
(A) Kaplan Meier curves indicating overall survival of NSGsham
(n=3-5) and NSGspln-
(n=5) mice
engrafted with 9 independent primary AML samples as indicated in each plot. (B) Summary of
medial survival of each group of NSGsham
and NSGspln-
. P values were calculated by log-rank
test.
Fig. S8
A. B.
C.
SU540 SU266 SU2090
20
40
60
80
100
Hum
an A
ML B
M
engra
ftm
ent %
SU540 SU266 SU2090
5
10
15
Hum
an A
ML P
B e
ngra
ftm
ent %
Nonsplenectomy
Splenectomized
NSGsham-PDX NSGspln-PDX
10X
40X
10X
40X
Lung
Liv
er
Kid
ney
Heart
NSGsham-PDX NSGspln-PDX
Fig. S8. Surgical splenectomy does not increase leukemic burden in NSG mice engrafted
with human AML. Human AML disease burden in the (A) BM and (B) PB of NSGsham
(n=5)
and NSGspln-
(n=5) mice engrafted with 3 independent primary AML samples. (C)
Representative IHC stain of human CD45 (brown) in the lung, kidney, liver, and heart to
estimate organ infiltration of human AML in NSGsham
and NSGspln-
mice. Scale bars represent
100 µm (10x top panel) and 50 µm (40x bottom panel).
Fig. S9
FSC-A
SS
C-
A 50
K 100K
150K
200K
250K
0
50K
100
K
150
K
200
K
250
K
SSC-A
SS
C-H
50K 100K
150K
200K
250K
0
50K
100
K
150
K
200
K
250
K Single Cells
hC
D45
(Am
Cyan)
0 1x10
3 1x104
1x105
1x1
03
1x1
04
1x1
05
mCD45 (PE-Cy7)
mCD45
PI (PE- Texas Red)
Lin
(P
E-C
y5)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
Lin-
Sca-1 (APC)
c-K
it
(FIT
C)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
04
1x1
05
CD34 (PB)
FcR
g
(PE
)
0 1x10
3 1x104 1x10
5
1x1
03
1x1
05
1x1
04
CD48 (AlexaFluor 700)
CD
150 (
Qdot 605)
1x104 1x10
5
1x1
04
1x1
05
0
HSC MPP
CMP
GMP
MEP
Fig. S9. Representative flow plots for the evaluation of mouse HSPCs in NSGspln-
PDX mice.
Splenectomized NSG mice transplanted with human AML blasts or normal human CB-CD34+
cells were euthanized at pre-determined time points and mononuclear cell fractions prepared as
described. An aliquot of the cells from each mouse was stained with a panel of antibodies
designed to evaluate various progenitor populations.
Fig. S10
A.
B.
Fig. S10. Mouse HSPCs in NSGspln-
-PDX mice do not exhibit increased apoptosis/cell death
or displacement into PB. (A) The annexin V+/PI
+ fractions of HSPCs from mononuclear cell
fractions of each mouse in Figure 3B-F were used to determine the degree of apoptosis/cell
death. (B) The frequency of mouse HSPCs in the PB of NSGspln-
-PDX mice was determined at
12 weeks after engraftment.
CB-CD34+ AML0
5
10
15
Dead
Cells (
%)
Lin-cKit+Sca-1- Lin-cKit+Sca-1+0.00
0.02
0.04
0.06
0.08
Mo
use H
SP
Cs in
PB
(%
)
Fig. S11
Fig. S11. Mouse HSPCs are similarly depleted in NSGsham
-PDX mice. Absolute numbers of
HSC (AML 10325.6 vs CB 1112185; p<0.0001), MPP (AML 16037 vs CB 1456167;
p<0.001), CMP (AML 29335.8 vs CB 4864358; p<0.0001), MEP (AML 65735.6 vs CB
2186247; p<0.0001), and GMP (AML 38147 vs CB 2525538; p<0.0012) in NSGsham-
PDX
(n=5) or NSGsham
-CB-CD34+ (n=5) mice 8 weeks after transplantation.
0 500 1000 1500 2000 2500
CB-CD34+
AML
# of cells
****
HSC
0 500 1000 1500 2000 2500
CB-CD34+
AML
# of cells
MPP
****
0 2000 4000 6000 8000
CB-CD34+
AML
# of cells
CMP
***
0 1000 2000 3000 4000
CB-CD34+
AML
# of cells
MEP
****
0 2000 4000 6000
CB-CD34+
AML
# of cells
GMP
****
Fig. S12
Fig. S12. Mouse proerythroblasts in NSGspln-
-PDX mice exhibit similar rates of
apoptosis/cell death. The percentages of annexin V+/PI
+ proerythroblasts from each
mononuclear cell fractions of each mouse in Fig. 3M-P were used to determine the degree of
apoptosis/cell death.
CB-CD34+ AML0
2
4
6
8
Dead C
ells
(%
)
Fig. S13.
Fig. S13. Representative markers and gating strategy used to sort purified populations of
human AML blasts. Patient samples were stained to identify human AML blasts expressing
CD45mid/low
CD33mid
CD3-. Blast cells were sorted and post-sort analysis was conducted to assess
purity.
Post-Sort Blasts
FSC-A
SS
C-A
SSC-AS
SC
-HPI (PE-Texas Red)
SS
C-A
Annexin V (AlexaFluor 647-A)
SS
C-A
CD45 (Pacific Blue-A)
SS
C-A
CD33 (PE-A)
CD
3(A
PC
-Cy7
-A)
50K 100K 150K 200K 250K0
50
K1
00
K1
50
K2
00
K2
50
K
50K 100K 150K 200K 250K0
50
K1
00
K1
50
K2
00
K2
50
K
0
50
K1
00
K1
50
K2
00
K2
50
K
0
50
K1
00
K1
50
K2
00
K2
50
K
0
50
K1
00
K1
50
K2
00
K2
50
K
1x103 1x104 1x105
1x103 1x104 1x105
1x103 1x104 1x105 1x103 1x104 1x105
1x1
03
1x1
04
1x1
05
0
Non-Debris
96.8
Single Cells 96.9 PI Negative 80.7
Annexin V Negative
76.3Blast Gate 64.5 CD33 mid,
CD3 Negative 97.9
CD33 (PE-A)
CD
3(A
PC
-Cy7
-A)
1x103 1x104 1x105
1x1
03
1x1
04
1x1
05
0
CD33 mid, CD3 Negative 98.1
Fig. S14.
Fig. S14. Representative markers and gating strategy used to sort purified populations of
human CB-CD34+ cells. Cord blood was enriched for CD34
+ cells using positive magnetic
selection, and cells were stained using antibodies specific for hCD3 and hCD34 to isolate
purified populations of CB-CD34+
progenitors by flow cytometry. A post-sort analysis was
conducted to assess purity.
Post-Sort CD34+
FSC-A
SS
C-A
SSC-A
SS
C-H
DAPI (Pacific Blue-A)
SS
C-A
CD3 (APC-Cy7-A)
SS
C-A
CD34 (APC-A)
SS
C-A
50K 100K 150K 200K 250K0
50
K1
00
K1
50
K2
00K
25
0K
50K 100K 150K 200K 250K0
50
K1
00
K1
50
K2
00K
25
0K
0
50
K1
00
K1
50
K2
00
K2
50
K
1x103 1x104 1x105 0
50
K1
00
K1
50
K2
00K
25
0K
1x103 1x104 1x105 0
50
K1
00
K1
50
K2
00
K2
50
K
1x103 1x104 1x105
CD34 (APC-A)
SS
C-A
0
50
K1
00
K1
50
K2
00
K2
50
K
1x103 1x104 1x105
Non-Debris78.9
Single Cells 96.5
DAPI 87.5 CD3 Negative 98.1 CD34+ Positive 46.1
CD34+ Positive 100
Fig. S15.
A.
B.
Fig. S15. Viability of human AML in culture. (A) Percentages of live (PI-) human AML cells
in culture (14-21 days). (B) Percentages of live (PI-) human AML blasts flow purified as
described at the end of 72 h culture in SFEM supplemented with TPO, SCF, and FLT3L for
generation of conditioned medium.
SU54
0
SU57
5
SU55
5
SU20
9
SU48
0
SU26
6
SU35
3
SU35
1
SU49
6
SU45
6
SU32
0
80
85
90
95
100
% L
ive C
ells (
PI-
)
SU54
0
SU57
5
SU55
5
SU20
9
SU48
0
SU26
6
SU35
3
SU35
1
SU49
6
SU45
6
SU32
0
80
85
90
95
100
% L
ive C
ells (
PI-
)
Fig. S16.
A.
B.
SU54
0
SU55
5
SU57
5
SU36
9
SU35
3
SU29
0
SU26
6
0
20
40
60
% E
DU
Up
take
Media Control
AML coculture
C.
SU54
0
SU55
5
SU57
5
SU36
9
SU35
3
SU29
0
SU26
60
2
4
6
8
10
% C
ell d
eath
Media Control
AML coculture
*
*
Fig. S16. Human AML blasts do not block erythroid differentiation by inhibition of cell
cycle entry or induction of cell death. (A) Absolute number of CD71+/GPA
+ normoblasts in
culture on day 6 of the transwell assay in the absence or presence of purified AML blasts.
*p<0.01 by unpaired t-test. (B) Percentage of EDU+ or (C) PI
+ CB-CD34
+ cells in the transwell
assay in the absence or presence of purified AML blasts. *p<0.05 by unpaired t-test.
Fig. S17.
A.
B.
Fig. S17. RNA-seq of purified populations of human AML blasts and CB-CD34+ cells
shows an inflammatory transcriptome signature in AML. (A) Heatmap representation of
unsupervised hierarchal clustering of the differentially expressed transcripts between purified
populations of primary human AML blasts (n=7) and normal CB-CD34+ cells (n=4). (B)
Heatmap of the transcripts whose expression was down-regulated at least 8 fold (p < 0.00005) in
AML blasts compared to normal CB-CD34+ cells.
Fig. S18.
Fig. S18. Effects of the addition of recombinant human IL-6 on normal CB-CD34+
progenitors undergoing erythroid differentiation. Recombinant human IL-6 (0-500 ng/ml)
was added to triplicate liquid cultures containing normal human CD34+ progenitors (n=3 CB-
CD34+ cells from independent donors) in EPO, IL-3, and SCF. The number of CD71
+GPA
+ cells
was determined by flow cytometry on day 6 of culture.
0
2
4
6
Num
ber
of N
orm
obla
sts
(10
6)
**
******
**
- 50 100 500
Recombinant IL-6 (ng/ml)
Fig. S19.
Fig. S19. Siltuximab treatment does not decrease leukemic burden in NSGspln-
mice
engrafted with human AML. NSGspln-
mice were engrafted with indicated human AML
samples. Control antibody and siltuximab treatment began on day 3 after engraftment and
continued every 72 hours until the end of the experiment. Human AML disease burden was
examined at week 6-8 in the BM of NSGspln-
mice engrafted with (A) SU540, (B) SU575, (C)
SU555, (D) SU351. AML burden was assessed in the PB of NSGspln-
mice engrafted with (E)
SU555 and (F) SU351. N.S. = not significant; n = 5 for all experiments above.
Isot
ype
Siltux
imab
30
40
50
60
70B
M E
ngra
ftm
ent %
N.S.
Isot
ype
Siltux
imab
30
40
50
60
BM
Engra
ftm
ent %
N.S.
Isot
ype
Siltux
imab
30
40
50
60
BM
Engra
ftm
ent %
N.S.
Isot
ype
Siltux
imab
40
50
60
70
BM
Engra
ftm
ent %
N.S.
SU540 SU575 SU555 SU351
Isot
ype
Siltux
imab
0
2
4
6
8
10
PB
Engra
ftm
ent %
N.S.
Isot
ype
Siltux
imab
0
5
10
PB
Engra
ftm
ent %
N.S.
SU555 SU351
A B C D
E F
Fig. S20.
Fig. S20. Siltuximab treatment does not decrease leukemic burden in various organs of
NSGspln-
mice engrafted with human AML. Representative IHC stain of human CD45 (brown)
in the lung, kidney, liver, and heart to evaluate organ infiltration by human AML in NSGspln-
-
PDX (SU540) mice treated with isotype control (n=5) or siltuximab (n=5) as described. Scale
bars represent 100 µm (10x top panel) and 50 µm (40x bottom panel).
Isotype Control Siltuximab Isotype Control Siltuximab
10X
40X
10X
40X
Lung
Liv
er
Kid
ney
Heart
Fig. S21.
Fig. S21. Siltuximab treatment initiated after establishment of disease also improves
anemia and overall survival in human AML xenografts. NSGspln-
-PDX mice engrafted with
SU540 were treated with control antibody or siltuximab (20 mg/kg) every 3 days starting on day
21. (A) The decline in hemoglobin was delayed in the siltuximab treatment group (**p<0.01,
***p<0.001, unpaired t-test). (B) BM leukemic burden (hCD45+CD33
+) was similar between
treatment groups. N.S.= not significant. (C) Overall survival was improved with siltuximab
treatment (11.5 weeks vs 14.5 weeks, **p=0.003, log rank test).
0 2 4 6 8 10 12 14 160
5
10
15
Time (weeks)
Hem
oglo
bin
g/d
LSU540 + Isotype Control (n=4)
SU540 + Siltuximab (n = 6)
**
***
**
0 5 10 15 200
50
100
Weeks
Perc
ent surv
ival
**
A. B.
C.
Iso
typ
e
Silt
uxim
ab
88
90
92
94
96
98
Hum
an A
ML e
ngra
ftm
ent % N.S.
Fig. S22.
A.
B.
Fig. S22. IL-1β and CCL3 production in NSGspln--PDX mice and AML conditioned medium.
(A) IL-1 concentrations in the BM plasma of splenectomized NSG mice engrafted with SU540
and SU575 determined by multiplex protein array (n = 15) compared to similar mice engrafted
with CB-CD34+ cells (n=5) or irradiated only control mice (n=4). **p<0.01 unpaired t-test. (B)
CCL3 production by purified primary AML blasts (n = 10) in culture determined by multiplex
protein array compared to CB-CD34+ cells (n=5) and medium only control (n=4) N.S.=not
significant.
0
50
100
150
200pg/m
lIrradiation only
CB-CD34+
SU540
SU575
IL-1b
**
0
5000
10000
15000
20000
25000
pg/m
l
CCL3Media Only
CB-CD34+ Conditioned Media
AML-Conditioned Media
N.S.
Table S1. Summary of characteristics of patients whose clinical data were analyzed in Fig.
1 (A to D). ELN: European Leukemia Net risk category.
Stanford Cancer Center
Number of Patients 293
Time of Diagnosis 2011-2014
Age (yrs)
Mean (SD)
Median
63 (16)
67 (21-89)
Sex
Male
Female
169
124
Risk Category (2017 ELN)
Favorable
Intermediate
Adverse
14
153
126
Table S2. Summary of characteristics of patients whose clinical data were analyzed in figs.
S1 and S2. ELN: European Leukemia Net risk category.
Campbell et al.
Number of Patients 1,376
Time of Diagnosis 2005-2015
Age (yrs)
Mean (SD)
Median
47.8 (12.3)
49 (18-84)
Sex
Male
Female
740
636
Risk Category (2017 ELN)
Favorable
Intermediate
Adverse
195
867
204
Table S3. Patient- and disease-specific characteristics of human AML samples investigated
here. ELN: European Leukemia Net risk category; FAB: French-American-British AML
classification; CG: cytogenetics; WBC: white blood cell (103/µl); HGB: hemoglobin (g/dL); PLT:
platelet (103/µL).
Sample Sex Age Type ELN FAB CG Somatic
Mutation
WBC HGB PLT
SU209 F 66 de novo INT M2 46XY NPM1, FLT3,
NOTCH2
166 8.8 133
SU266 M 65 de novo Adverse - Inv(3) SETD2, TET2,
AK2, ASXL1,
FLT3-ITD
374 9.9 36
SU351 M 75 secondary
MDS-
AML
Adverse M5 Complex IDH2, NRAS,
BCORL1,
SETD
3.2 9.7 26
SU353 M 66 de novo INT M4 46XY NPM1, FLT3-
ITD,
DMNT3A,
111 9.6 73
SU456 M 55 secondary
MDS-
AML
Adverse - Complex NRAS, TP53 1.8 7.6 146
SU480 F 64 de novo INT M1 46XX CEPBA, FLT3-
ITD,
DNMT3A,
TET2, BCOR,
ZRSR2, TET2
16.9 7.4 112
SU496 M 24 de novo INT - (+)8 TP53, IDH2,
DNMT3A,
STAG2,
BCOR,
ZRSR2, TET2
35.4 10.3 44
SU540 F 68 de novo INT - 46XX DNMT3A,
MYC, CEBPA,
NPM1, FLT3-
ITD, TET2
94.4 5.6 189
SU555 F 27 de novo INT - - PTPN11,
SETD2,
MTND5
103.2 7.5 28
SU575 M 72 secondary
MDS-
AML
INT - 46XY NPM1, FLT3-
ITD, DMNT2,
ZRSR2, JAK3
28.4 8.3 23
Table S4. Human AML conditioned medium suppresses mouse progenitor colony
formation. Numbers of mouse erythroid and myeloid colonies formed in the presence of control
or AML-CM as presented in Figure 4B. All numbers are average values of technical triplicates.
GM M G GEMM BFU
Media 14 15 21 23 69
CB-CD34+ 21 8 44 22 83
CB-CD34+ 20 5 39 21 55
CB-CD34+ 10 5 29 14 65
CB-CD34+ 15 5 27 16 65
CB-CD34+ 16 4 35 19 69
CB-CD34+ 17 6 33 19 72
CB-CD34+ 13 4 43 20 59
SU540 6 5 36 16 35
SU575 9 12 38 16 37
SU555 10 6 35 17 19
SU209 8 16 33 16 35
SU480 11 14 28 15 32
SU266 11 11 32 15 38
SU353 9 8 27 15 43
SU351 8 11 64 18 37
SU496 9 9 33 17 29
SU456 8 7 31 15 42
Table S5. Human AML conditioned medium suppresses human progenitor colony
formation. Numbers of human erythroid and myeloid colonies formed in the presence of
control or AML-CM as presented in Figure 4D. All numbers are average values of technical
triplicates.
GM M G GEMM BFU
Media 14 15 21 23 69
CB-CD34+ 21 8 44 22 83
CB-CD34+ 20 5 39 21 55
CB-CD34+ 10 5 29 14 65
SU540 6 5 36 16 37
SU575 9 12 38 16 37
SU555 10 6 35 17 19
SU209 8 16 33 16 35
SU480 11 14 28 15 38
SU369 11 11 32 15 38
SU456 9 8 27 15 43
SU351 8 11 64 18 37