Bastian et al. 2012 SUPPLEMENTARY TABLES Synergistic activity of bortezomib and HDACis in preclinical models of B-cell precursor acute lymphoblastic leukemia via modulation of p53, PI3K/AKT and NF-B Lorenz Bastian, Jana Hof, Madlen Pfau, Iduna Fichtner, Cornelia Eckert, Günter Henze, Javier Prada, Arend von Stackelberg, Karl Seeger and Shabnam Shalapour 0 Bastian et al. 2012 Supplementary Table S1. Fractional Product of Webb calculations for concomitant combinations of HDACis (VPA, SAHA) and bortezomib or chemotherapeutic agents in Nalm6 cells. VPA SAHA bortezomib 6 nM 2.73 3.47 8 nM 1.23 1.27 idarubicin 10 nM 1.56 1.51 20 nM 1.19 1.22 cytarabine 20 nM 1.53 1.29 80 nM 0.85 0.83 vincristine 3 nM 1.53 1.89 5 nM 0.90 0.99 Nalm6 cells were treated for 72 h with either BTZ or chemotherapeutic agents alone or in combination with HDACis (VPA, SAHA). BTZ and chemotherapeutics were applied at moderate and submaximal effective dose levels as indicated. Apoptosis was assessed by FACS analysis of annexin-V/PI stained cells. Results are shown in Supplementary Figure S2A. Fractional product method of Webb relates a measured effect of combined treatment to an expected effect calculated from the effects of treatment with single drugs. Values <1 indicate antagonism, ~ 1 indicate additivity, > 1 indicate synergism. Abbreviations: VPA, valproic acid; SAHA, suberoylanilide hydroxamic acid. 1 Bastian et al. 2012 Supplementary Table S2. Sensitivities of Reh, Nalm6 and 697 cells to treatments with VPA and SAHA. VPA (mM) SAHA (μM) Reh Ic25 3.6 1.3 Ic50 > 6 > 2.4 Nalm6 Ic25 2.1 1.6 Ic50 > 3 2.2 697 Ic25 1.5 0.9 Ic50 2.1 1.1 Cell lines were treated with serial dilutions of VPA or SAHA. After 72 h apoptosis was measured by FACS analysis of annexin-V / PI stained cells, as indicated in Supplementary Figure S1B-D. The 25% and 50% effective concentrations were determined by linear regression analysis. 2 Bastian et al. 2012 Supplementary Table S3. Fractional Product of Webb calculations for concomitant combinations of BTZ and VPA in 697, SD-1 and SEM cells at three different time points. VPA (24 h) VPA (48 h) VPA (72 h) 697 BTZ (10 nM) 4.21 5.34 12.36 BTZ (14 nM) 2.74 2.86 4.30 SD-1 BTZ (10 nM) 1.67 2.54 36.91 BTZ (12 nM) 1.12 1.01 1.19 SEM BTZ (2 nM) 1.20 1.51 1.60 BTZ (4 nM) 1.61 1.31 1.20 697, SD-1 and SEM cells were exposed to BTZ at two concentrations, combined concomitantly with or without VPA. After 24 h, 48 h and 72 h of treatment, cytotoxicity was assessed by FACS analysis of PI stained cells. The IC 50 concentrations determined for BTZ single treatments after 48h were 18.2 nM, 10.5 nM and 4.0 nM for 697, SD-1 and SEM respectively. Fractional product method of Webb relates a measured effect of combined treatment to an expected effect calculated from the effects of treatment with single drugs. Values <1 indicate antagonism, ~ 1 indicate additivity, > 1 indicate synergism. Abbreviations: BTZ, bortezomib. VPA, valproic acid. 3 Bastian et al. 2012 Supplementary Table S4. Fractional Product of Webb calculations for concomitant and sequential combinations of BTZ and HDACis (VPA, SAHA) in Reh and Nalm6 cells. BTZ + HDACi BTZ + HDACi BTZ b HDACi HDACi b BTZ HDACi (36 h) (72 h) Reh VPA 5.36 5.82 1.28 0.30 SAHA 4.94 4.44 1.12 0.88 Nalm6 VPA n.d. 3.79 0.89 0.59 SAHA n.d. 3.17 1.31 0.99 Reh and Nalm6 cells were treated with BTZ for 36 h, washed and resuspended in fresh media containing HDACis (VPA, SAHA) for further 36 h or the reversed order of drug application. As controls were used: treatments with single drugs for 36 h, after which cells were washed and resuspended in fresh media without drugs for further 36 h or treatments with the combination of HDACI/BTZ for 36 h, after which cells were washed and resuspended again for further 36 h in media containing the HDACI/BTZ combination. After 72 h cells were stained with Annexin-V/PI and the proportions of apoptotic cells were determined by FACS. Apoptosis rates in Reh cells after 36 h of treatment with VPA/BTZ and SAHA/BTZ were 89.8% (±0.4) and 72.9% (± 3.6) respectively. Representative results are shown in Figure 1C. Fractional product method of Webb relates a measured effect of combined treatment to an expected effect calculated from the effects of treatment with single drugs. Values <1 indicate antagonism, ~ 1 indicate additivity, > 1 indicate synergism. Abbreviations: BTZ, bortezomib; VPA, valproic acid; SAHA, suberoylanilide hydroxamic acid; n.d. not determined. 4 Bastian et al. 2012 Supplementary Table S5. NF-B subunits gene expression in leukemia cells in correlation with the clinical and biological characteristics of the BCP-ALL relapse patient cohort. Parameter Total RelA RelB NFKB2 NFKB1 Total N median (min;max) p-value median (min;max) p-value median (min;max) p-value median (min;max) p-value gender 0.16 0.36 0.24 0.25 male 36 8.25(7.40; 8.97) 7.57(6.39;9.4) 7.42(6.66;8.50) 9(8.25;10.61) female 16 8.18 (7.56;8.56) 7.37(6.17;8.75) 7.05(6.51;8.81) 8.75(7.64;10.61) age at relapse 0.80 0.28 0.38 0.09 < 5 years 7 8.34(7.84;8.62) 7.79(6.88;8.75) 7.03(6.86;8.50) 8.63(8.35;9.62) 5 and < 10 years 22 8.12(7.40;8.96) 7.6(6.17;9.4) 7.49(6.61;8.81) 9.2(8.43;10.61) 10 years 23 8.27(7.56;8.97) 7.33(6.57;8.32) 7.24(6.51;8.40) 8.97(7.64;9.99) timepoint of relapse 0.03 0.05 0.03 0.05 very early 14 8.15(7.46;8.55) 7.22(6.57;8.42) 6.9(6.6;8.5) 8.75(8.25;9.62) early 10 8.05(7.40;8.62) 7.46(6.39;8.4) 7.25(6.81;8.45) 8.87(7.64;10.69) late 28 8.35(7.56;8.97) 7.65(6.17;9.4) 7.5(6.51;8.81) 9.12(8.35;10.61) relapse on/off initial treatment 0.006 0.01 0.008 0.03 on treatment 18 8.05(7.46;8.62) 7.22(6.57;8.42) 7.02(6.60;8.50) 8.82(7.64;10.59) off treatment 34 8.35(7.40;8.97) 7.65(6.17;9.4) 7.5(6.51;8.81) 9.08(8.35;10.61) site of relapse 0.24 0.19 0.19 0.43 BM isolated 45 8.21(7.40;8.96) 7.44(6.17;9.4) 7.26(6.51;8.81) 8.96(7.64;10.61) BM combined 7 8.36(7.99;8.97) 7.88(7.43;8.4) 7.44(7.07;8.49) 9.11(8.76;9.89) immunophenotype 0.60 0.18 0.35 0.57 Pro–B-cell ALL 4 8.44(7.93;8.69) 7.85(7.58;8.4) 7.53(6.97;8.49) 9.71(8.35;9.89) Common ALL 29 8.17(7.46;8.97) 7.44(6.17;9.4) 7.14(6.51;8.48) 8.9(7.64;10.61) Pre-B-cell ALL 15 8.3(7.40;8.84) 7.43(6.39;8.59) 7.43(6.74;8.81) 9.01(8.25;10.61) BCP-ALL not specified 4 response to treatment 0.44 0.22 0.04 0.86 early/normal 31 8.27(7.40;8.97) 7.58(6.17;9.4) 7.53(6.51;8.81) 8.93(8.25;10.61) late/non-response 18 8.18(7.76;8.61) 7.33(6.7;8.9) 7.05(6.61;8.48) 8.99(7.64;10.69) (no data) 3 Outcome 0.63 0.59 0.48 0.23 CCR 13 8.16(7.40;8.97) 7.9(6.17;9.4) 7.26(6.51;8.47) 8.81(8.4;9.96) death in CR 3 8.61(8.07;8.72) 7.01(6.9;7.37) 7.05(6.74;7.77) 8.82(8.59;9.06) second relapse 24 8.32(7.46;8.94) 7.62(6.57;8.59) 7.5(6.61;8.81) 9.12(7.64;10.61) non-response 7 8.27(7.85;8.42) 7.42(6.7;8.9) 7.24(6.9;8.48) 9.17(8.38;9.77) induction death 1 7.93(-;-) 7.12(-;-) 6.6(-;-) 8.37(-;-) (non protocol) 4 second event 0.67 0.79 0.70 0.20 no 13 8.16(7.40;8.97) 7.9(6.17;9.4) 7.26(6.51;8.47) 8.81(8.4;9.96) yes 35 8.31(7.46;8.94) 7.48(6.57;8.9) 7.4(6.6;8.81) 9.06(7.64;10.61) (non protocol) 4 Abbreviations: BCP, B-cell precursor; ALL, acute lymphoblastic leukemia; BM, bone marrow; CR, complete remission; CCR, continuous complete remission.