low high

GSK3

P(S9)GSK3

CD38-APCCD45-Pacific Blue CD123-PEFSC

CD

34

-PE

-Cy7

CD

34

-PE

-Cy7

SS

C

0.3%

A

B

C

50 250200150100(X1000)

50

250

200

150

100

(X1000)

SS

C

50

250

200

150

100

(X1000)

102 105104103 102 105104103

102

103

104

105

102 105104103

102

103

104

105

P(S9) GSK3GSK3

Adh.Sus.

102 105104103

102 105104103 102 105104103

102 105104103102 105104103

102 105104103

FITC

FITC FITC

FITCFITC

FITC

expression

Pre-immune serum

Isotype control

coun

ts40

00

coun

ts25

00

coun

ts 700

0

coun

ts40

00

coun

ts25

00

coun

ts 125

0

low highphosphorylation

Supplementary Figure 1: Representative gating schemes and profiles obtained by flow cytometry of

immature cell populations and of their P(S9)GSK3 and total GSK3content. A- Immature cell

populations (leukemic CD34+38-123+45lo cells and normal CD34+38-45lo cells) were analyzed by flow

cytometry. From each bone marrow sample, total mononuclear (leukemic bulk or normal CD45 lo cells) and

immature cell populations were analyzed. B- P(S9)GSK3 and total GSK3 content of immature cells were

analyzed by flow cytometry and the MFI ratio was calculated. C- P(S9)GSK3 and total GSK3 were

detected by western blotting with the same antibodies used for flow cytometry.

CD

34

-PE

-Cy7

CD

34

-PE

-Cy7

CD38-APC

CD38-APC

CD38-APC

Complex karyotype

Normal karyotype

Normal(healthy donors)

AML

Sus. Adh.

CD38-APC

1,4% 1,5%

15% 14%

0,6% 0,4%

CD38-APC

CD38-APC

CD

34

-PE

-Cy7

CD

34

-PE

-Cy7

CD

34

-PE

-Cy7

CD

34

-PE

-Cy7

100

50

0

BulkAdherent cells

%

AML normal karyotype

Healthy donorsAML complex karyotype

A B

102 105104103

102 105104103

102 105104103

102 105104103

102 105104103

102 105104103

102

105

104

103

102

105

104

103

102

105

104

103

102

105

104

103

102

105

104

103

102

105

104

103

Supplementary Figure 2: Maintenance of immature cell fraction (CD34+38-) upon cell adhesion.

Cells from healthy donors and AML patients (with normal or complex karyotype) were incubated for 1h

in the absence of serum and then allowed to adhere on fibronectin matrix as described in Material and

Methods. After 1h adhesion, the percentage of adhered cells in total mononuclear fraction was quantified

by crystal violet (B, n=3, mean ± S.E.M.). Also, percentages of CD34+38- cells were analyzed before and

after cell adhesion by flow cytometry (A). Data are representative from 16 AML patients with normal

karyotype, 8 AML patients with complex karyotype and 12 healthy donors.

Non-treated control

SB216763

25%

AML CD34+38-123+ cellsMale patient

AML CD34+38-123+ cellsFemale patient

coun

ts60

00

APO2.7-PE-Cy5102 105104103

suspension

coun

ts30

00

APO2.7-PE-Cy5102 105104103

adhesion

32%

coun

ts30

00

APO2.7-PE-Cy5105104103

30%

102

70%

coun

ts50

00

APO2.7-PE-Cy5102 105104103

12% 15%

17%12%

coun

ts30

0

APO2.7-PE-Cy5102 105104103

coun

ts30

0

APO2.7-PE-Cy5102 105104103

APO2.7-PE-Cy5102 105104103

APO2.7-PE-Cy5102 105104103

0

0co

unts

250

0

coun

ts25

00

suspension adhesion

Supplementary Figure 3: Representative P(S9)GSK3, P(S641)GS and APO2.7 flow

cytometry profiles of AML CD34+38-123+ cells in suspension or adhesion. Labelling by

antibodies directed to P(S9)GSK3 and P(S641)GS (glycogen synthase) (a) and by APO2.7-PE-

Cy5 (b) are described in Material and Methods. In a, 2500 cells were counted in average and in b,

apoptotic responses to the GSK3 inhibitor SB216763 are shown.

a-

b-

P(S9)GSK3

P(S641)GS

Sus. Adh.

Supplementary Figure 4: GSK3 modulation and disparity in apoptotic response of immature leukemic cells from male AML patients. After 1h serum starvation and then 1h adhesion on fibronectin, AML samples were treated or not (Ct.) with LY294002 (10 M), Wortmannin (0.1 M), Ara-C (1 M), Etoposide (1 M), NAC (N-acetyl-l-cysteine from Sigma Aldrich, 50 M) or DPI (Diphenyliddonium chloride from Flüka, 25 M). At the end of 1h incubation period, cells were washed, treated for P(S9) GSK3 analysis (a) or incubated in serum-containing medium for 24h for further analysis of P(S9) GSK3(a) andapoptosis (b). P(S9) GSK3 and apoptosis were measured by flow cytometry in immature leukemic fraction (CD34+38-123+ cells) as described in Material and Methods. Results were evaluated as the percentage of apoptotic cells in suspension or in adhesion for each treament. Data are from 16 male AML patients: 12 developed CAM-DR to etoposide (left panel) and 4 to Ara-C displaying also pro-apoptotic response to anti-oxidants NAC and DPI (right panel). Comparison to respective control in suspension or in adhesion, or as indicated: mean ± S.E.M. *P<0.05 **P<0.01 ***P<0.001.

a-

SuspensionAdhesion

Ct. NAC DPI Ara-C Etop.

AML CD34+38-123+ cellsMale patients

70

60

40

30

50

Apoptosis%

20

0

*******

**

*

Ct. NAC DPI Ara-C Etop.

AML CD34+38-123+ cellsMale patients

70

60

40

30

50

Apoptosis%

20

0

*

*

***

b-

102 105104103

FITC

coun

ts0

90

102 105104103

FITCco

unts

09

0102 105104103

FITC

coun

ts0

90

coun

ts

102 105104103

FITC

09

0

Sus. Adh. Adh. + LY294002 Adh. + WortmanninP(S9)GSK3

AML CD34+38-123+ cellsMale patients

LY2940023

1

0

Inhibited GSK3P(S9)GSK3/GSK3

2

1h 24h

Adhesion (n=3) wortmannin

control

MFI ratio

****

***

SuspensionAdhesion

AML CD34+38-123+ cellsFemale patients

Ct.

70

60

40

30

50

Apoptosis%

20

0IGF-1 Testosterone Estradiol

AML CD34+38-123+ cellsMale patients

Ct.

70

60

40

30

50

Apoptosis%

20

0IGF-1 Testosterone Estradiol

Supplementary Figure 5: Hormonal treatment does not modify survival of immature leukemic

cells from female and male AML patients. Serum-starved adherent cells from 4 female and 4 male

AML patients were treated or not (Ct.) with IGF-1 (RD, 3x10-9M), 19-nortestosterone 17-decanoate

(Sigma Aldrich, 10-8M) or 17 estradiol (Sigma Aldrich, 10-8M) for 1h. At the end of the incubation

period, cells were washed and incubated in serum-containing medium for 24h. Measurement of

apoptosis was performed by flow cytometry in immature leukemic fraction (CD34+38-123+

progenitors) using labeling by APO2.7 as described in Materials and Methods. Results were

evaluated as the percentage (mean ± S.E.M.) of apoptotic cells in suspension or in adhesion for each

treatment.

GSK3High level

GSK3Low level P-value

Median age (years)

Gender (%)MaleFemale

CD34+ % (n)

Cytogenetic %nFavorableIntermediateUnfavorable

FAB subtypes (%) M0M1M2M3M4M5

FLT3-ITD % (n)

WBC (x109/L)

Patients % (n) 20 (15) 80 (58)

5447

62533847

070

64147

613342

2513

7262

ns

ns

61 (48)39 (14) <0.05

23 (48)29 (14)

20795114

4703321

Supplementary Table 1: Initial clinico-biological characteristics of 73 AML patients according to

GSK3 expression. The level of GSK3 was analyzed by western blot and groups « high » and

« low » levels were determined by comparison with CD34+ normal cells. FAB, French American

British classification; WBC, White Blood Cell count; CD34+, percentage of CD34+ in the total

mononuclear cell fraction; FLT3-ITD, FLT3 Internal Tandem Duplication; ns, not significant. Patient

characteristics and clinical parameters were compared between different groups of AML patients (high

vs low GSK3 expression and female vs male) using the 2 test.

ns

ns

Supplementary Table 2: Expression of several hormone and adhesion receptors and signaling

molecules in immature leukemic cells from female and male AML patients. Cells from AML patients

were incubated for 1h in the absence of serum and then allowed to adhere on a fibronectin matrix. After 1h

adhesion, protein expression was quantified by flow cytometry in immature fractions (CD34+38-123+ cells) as

described in Material and Methods. Results are expressed as the mean fluorescence intensity (MFI) of stained

samples. Data are from 3 to 9 female and 4 to 16 male AML patients (mean ± S.E.M, ° comparison between

female and male patients P<0.05, *comparison adhesion vs suspension P<0.05). Source of material: Er and

IGF1R polyclonal antibodies (Abcam), 4 monoclonal antibody (Chemicon), Akt and p21 polyclonal

antibodies (Santa Cruz Biotechnology), P(S473)Akt polyclonal antibody (Cell Signaling technology).

suspension adhesion suspension adhesion

ER 3037 ± 247 3381 ± 222 3582 ± 317 4037 ± 420 IGF1R 6683 ± 817 4554 ± 944 6338 ± 1154 5194 ± 918 Integrin 4 20153 ± 4758° 16259 ± 3074 10425 ± 2454 10430 ± 2550 Survivin 2942 ± 344 4088 ± 399 4111 ± 954 3537 ± 616 P21 4782 ± 205 7790 ± 1045* 4554 ± 847 4309 ± 886

Female Male

P(S473)Akt/ Akt 0.29 ± 0.10 0.21 ± 0.08° 1.18 ± 0.20 0.90 ± 0.25 Akt 9494 ± 1581 12915 ± 3281 3892 ± 226 4133 ± 632 P(S473)Akt 2837 ± 300 2790 ± 276 4625 ± 1056 3882 ± 203 P27 5936 ± 802 5347 ± 880 5581 ± 567 4646 ± 588

AML CD34+38-123+ cells

Supplementary Table 3: Discordant sex-related GSK3 phenotypes of one female and one male AML

patients, and U937. Measurement of GSK3 phosphorylation, apoptosis and RACK1 level were performed

as described in Material and methods. Note that AML patients #18 and #28 have no specific clinico-

biological characteristics (not shown).

suspension adhesion suspension adhesion

Male AML patient #28 5.00 2.34 11622 15800

Female AML patient #18 1.21 1.76 2300 1600

U937 2.50 1.90 4000 8000

P(S9)GSK3/GSK3 RACK1 level

AML CD34+38-123+ cells

% apoptosis in adhesion

Cont. Etop. SB21676315 13 35 28 60 27 36 25 60