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


Perc

ent S

urv

ival %

NSGspln--SU456

NSGsham-SU456

*p = 0.02

0 10 20 30

0

50

100


Perc

ent S

urv

ival %

NSGspln--SU351

NSGsham-SU351

*p = 0.02

0 10 20 30 40

0

50

100


Perc

ent S

urv

ival %

NSGspln--SU353

NSGsham-SU353

*p = 0.01

0 10 20 30

0

50

100


Perc

ent S

urv

ival %

NSGspln--SU266

NSGsham-SU266

*p = 0.01

0 10 20 30

0

50

100


Perc

ent S

urv

ival %

NSGspln--SU480

NSGsham-SU480

*p = 0.01

0 10 20 30 40

0

50

100


Perc

ent S

urv

ival %

NSGspln--SU209

NSGsham-SU209

*p = 0.01

0 10 20 30 40

0

50

100


Perc

ent S

urv

ival %

NSGspln--SU555

NSGsham-SU555

*p = 0.01

0 10 20 30

0

50

100


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


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


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

supplementary materials for · 2020. 4. 6. · table s3. patient- and disease-specific...

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