lowhigh GSK3  P(S9)GSK3  CD38-APC CD45-Pacific BlueCD123-PE FSC CD34-PE-Cy7 SSC 0.3% A B C (X1000) (X1000) SSC (X1000) P(S9) GSK3  GSK3  Adh. Sus FITC expression Pre-immune serum Isotype control counts counts counts counts counts counts lowhigh phosphorylation Supplementary Figure 1: Representative gating schemes and profiles obtained by flow cytometry of immature cell populations and of their P(S9)GSK3  and total GSK3  content. A- Immature cell populations (leukemic CD lo cells and normal CD lo 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.

CD34-PE-Cy7 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 CD34-PE-Cy Bulk Adherent cells % AML normal karyotype Healthy donors AML complex karyotype A B Supplementary Figure 2: Maintenance of immature cell fraction (CD ) 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 CD 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.

Supplementary Figure 3: Representative P(S9)GSK3 , P(S641)GS and APO2.7 flow cytometry profiles of AML CD 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 SB 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 LY (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) and  apoptosis (b). P(S9) GSK3  and apoptosis were measured by flow cytometry in immature leukemic fraction (CD 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< a FITC counts FITC counts FITC counts 0 90 counts FITC 0 90 Sus. Adh. Adh. + LY Adh. + Wortmannin P(S9)GSK3  AML CD cells Male patients LY Inhibited GSK3  P(S9)GSK3  /GSK3  2 1h24h Adhesion (n=3) wortmannin control MFI ratio ** ***

Suspension Adhesion AML CD cells Female patients Ct Apoptosis % 20 0 IGF-1 TestosteroneEstradiol AML CD cells Male patients Ct Apoptosis % 20 0 IGF-1 TestosteroneEstradiol 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 -9 M), 19-nortestosterone 17-decanoate (Sigma Aldrich, M) or 17  estradiol (Sigma Aldrich, M) 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 (CD 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.

GSK3  High level GSK3  Low levelP-value Median age (years) Gender (%) Male Female CD34 + % (n) Cytogenetic % n Favorable Intermediate Unfavorable FAB subtypes (%) M0 M1 M2 M3 M4 M5 FLT3-ITD % (n) WBC (x10 9 /L) Patients % (n)20 (15)80 (58) ns 61 (48)39 (14)< (48)29 (14) 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

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 (CD 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). suspensionadhesionsuspensionadhesion ER  3037 ± ± ± ± 420 IGF1R 6683 ± ± ± ± 918 Integrin  ± 4758°16259 ± ± ± 2550 Survivin 2942 ± ± ± ± 616 P ± ± 1045*4554 ± ± 886 FemaleMale P(S473)Akt/ Akt 0.29 ± ± 0.08°1.18 ± ± 0.25 Akt 9494 ± ± ± ± 632 P(S473)Akt 2837 ± ± ± ± 203 P ± ± ± ± 588 AML CD 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). suspensionadhesionsuspensionadhesion Male AML patient # Female AML patient # U P(S9)GSK3  /GSK3  RACK1 level AML CD cells % apoptosis in adhesion Cont.Etop.SB