-9 -8 -7 -6 -5 -4 -3Log [M-110] (M)

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Supplementary Figure 1. (A) Dose response curves of PIM-1 inhibition by M-110 were performed in the presence of varying concentrations of ATP. (B) Double reciprocal plots of the results in (A)show increased slopes as a function of increasing ATP concentrations.

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Supplementary Figure 2. DU-145 cells were treated with 10 µM M-110 or SGI-1776 and analyzed for pSTAT3-tyr705 expression after 8 hr and 18 hrs. Results are the mean of 3 exps+/- 1 s.e.m. The difference in pSTAT3-tyr705 expression in DU-145 cells treated for 8 hr or 18 hr is significant ( P = 0.036)

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Supplementary Figure 3. MiaPaca2 cells were incubated with 10 µM M-110, SGI-1776 or M-142 for 18 hr. An equal number of untreated cell equivalents (lane 1) and treated cell equivalents (lanes 4-6) were analyzed by Western blotting for expression of pSTAT3-tyr705 or of STAT3. Quantitation was done as in Figure 4. Results are presented as a percentage of expression in untreated cells and are normalized for the expression of STAT3. The bars represent the mean of three experiments +/- 1 sem. **, P = < 0.01. *, P = <0.05.

Supplementary Figure 4. 22RV1 cells were treated with 10 µM M-110 or SGI-1776 and analyzed for pSTAT3-tyr705 expression after 18 hrs. Quantitation was done as in Figure 4. Results are the mean of 3 exps +/- 1 s.e.m.

% Enzyme Activity % Enzyme Activity % Enzyme Activity % Enzyme ActivityM-110 M-110 M-110 M-110 M-110 M-110 M-110 M-110

Kinase: Data 1 Data 2 Kinase: Data 1 Data 2 Kinase: Data 1 Data 2 Kinase: Data 1 Data 2ABL1 97.30 93.58 DCAMKL2 93.61 97.86 JAK3 103.27 101.11 PIM2 96.38 97.12ABL2/ARG 99.69 97.24 DDR2 98.97 100.61 JNK1α1 102.09 99.13 PKA 99.83 101.40AKT1 94.59 94.51 DMPK 100.75 102.92 JNK2α2 93.03 91.79 PKCα 102.29 96.52AKT2/PKBβ 92.37 97.87 DRAK1 95.08 97.91 JNK3 103.24 102.43 PKCβI 96.96 91.86AKT3 95.19 96.23 DYRK1/DYRK1A 91.84 93.79 KDR/VEGFR2 104.09 105.89 PKCβII 93.25 99.12ALK 92.94 96.72 DYRK1B 95.64 96.42 LCK 93.82 92.77 PKCδ 90.76 97.77ALK4/ACVR1B 92.53 94.47 DYRK2 88.90 87.82 LIMK1 95.43 97.35 PKCε 95.99 97.10ALK5/TGFB-R1 93.00 95.22 DYRK3 103.00 103.67 LKB1/STK11 89.43 90.21 PKCeta 91.77 101.92ARAF 93.56 93.20 DYRK4 104.10 103.92 LOK/STK10 91.76 91.22 PKCγ 94.16 103.33ARK5 86.49 87.65 EEF2K 99.69 96.05 LRRK2 100.94 99.18 PKCiota 95.10 92.07ASK1/MAP3K5 85.95 80.77 EGFR 85.32 87.99 LYN 89.33 90.61 PKCmu/PKD1 94.33 95.31Aurora A 88.50 84.15 EPHA1 86.96 85.91 LYNB 82.13 84.94 PKCnu/PKD3 89.25 94.04Aurora B 84.46 80.90 EPHA2 101.45 102.36 MAPKAPK2 98.93 98.41 PKCtheta 89.70 88.34Aurora C 90.07 91.06 EPHA3 84.88 84.84 MAPKAPK3 98.41 98.41 PKCζ 96.61 92.36AXL 91.42 92.01 EPHA4 91.30 92.82 MAPKAPK5/PRAK 95.78 95.29 PKD2/PRKD2 92.89 88.29BLK 94.77 94.85 EPHA5 95.80 95.84 MARK1 96.95 96.53 PKG1α 99.22 101.02BMX/ETK 86.57 87.25 EPHA7 95.33 95.23 MARK2/PAR-1Bα 100.67 94.63 PKG1β 102.33 102.12BRAF 95.10 97.66 EPHA8 92.33 93.41 MARK3 101.53 101.12 PKG2/PRKG2 94.24 90.69BRK 89.03 86.54 EPHB1 86.48 85.51 MEK1 95.78 99.81 PKN2/PRK2 101.57 102.63BRSK1 91.84 94.22 EPHB2/HEK5 94.99 95.80 MEK2 103.09 104.11 PLK1 103.46 103.04BRSK2 94.16 93.05 EPHB3 100.23 96.99 MELK 87.31 91.68 PLK2 101.35 108.18BTK 87.12 88.77 EPHB4 103.18 100.81 MINK 80.88 81.52 PLK3 99.56 97.52CAMK1α 93.86 93.68 ErbB2/HER2 94.63 93.56 MLCK/MYLK 97.43 99.50 PRKX 97.72 98.04CAMK1δ 95.76 96.05 ErbB4/HER4 93.10 92.35 MLCK2/MYLK2 97.65 97.54 PYK2/FAK2 105.29 104.60CAMK2α 93.67 94.31 ERK1 82.26 89.23 MLK1/MAP3K9 83.50 85.26 RAF1 95.43 94.51CAMK2β 90.38 86.82 ERK2/MAPK2 78.54 83.44 MNK2 90.03 89.63 RET 98.54 97.57CAMK2δ 102.26 100.33 FAK 97.39 99.27 MRCKα/CDC42BPA 91.74 97.58 RIPK2 92.56 95.09CAMK2γ 96.59 95.48 FER 86.66 90.35 MRCKβB/CDC42BPB 88.55 87.86 ROCK1 100.89 100.40CAMK4 68.42 68.15 Fes/FPS 94.34 93.65 MSK1 /RPS6KA5 89.13 89.49 ROCK2 93.80 92.50CAMKK2 95.48 94.47 FGFR1 97.57 97.63 MSK2 /RPS6KA4 81.14 81.18 RON/MST1R 92.67 92.16CDK1/cyclinB 88.32 84.76 FGFR2 96.65 99.50 MSSK1/STK23 102.99 102.77 ROS/ROS1 94.15 91.60CDK2/cyclinA 99.74 100.53 FGFR3 96.68 98.16 MST1/STK4 101.93 104.95 RSK1 90.39 92.89CDK2/cyclinE 103.63 103.97 FGFR4 89.97 91.40 MST2/STK3 100.27 99.55 RSK2 90.65 89.27CDK3/cyclinE 97.58 97.85 FGR 96.52 95.07 MST3/STK24 111.24 111.06 RSK3 90.94 94.21CDK4/Cyclin D1 86.56 87.37 FLT1 63.65 77.79 MST4 87.14 89.48 RSK4/RPS6KA6 105.37 105.99CDK5/p25 103.79 101.31 FLT3 99.17 94.54 mTOR/FRAP1 95.75 92.16 SGK1 94.19 92.58CDK5/p35 96.01 97.92 FLT4 89.24 89.90 MUSK 84.55 96.43 SGK2 95.94 97.80CDK6/cyclinD1 96.13 95.89 FMS 94.10 94.65 NEK1 73.58 76.39 SGK3/SGKL 103.01 103.16CDK6/cyclinD3 95.56 95.63 FRK/PTK5 97.73 99.46 NEK2 84.98 92.31 SIK2/SNF1LK2 103.88 101.32CDK7/cyclinH/MNAT1 94.44 94.62 FYN 97.83 97.45 NEK3 76.84 77.49 SRPK1 88.73 86.47CDK9/cyclin K 89.33 91.29 GCK/MAP4K2 98.73 95.03 NEK4 93.81 98.52 SRPK2 94.03 90.85CDK9/cyclin T1 90.73 91.13 GRK2/ADRBK1 98.70 97.81 NEK6 90.38 90.41 STK22D 96.23 94.17CHK1 98.54 100.88 GRK3 92.12 90.94 NEK7 100.51 105.48 STK33 95.60 95.35CHK2 89.89 92.29 GRK4 102.85 102.58 NEK9 93.56 94.20 SYK 100.17 95.26CK1α1 95.53 99.20 GRK5 100.32 99.02 NEK11 79.98 88.85 TAK1-TAB1/MAP3K7 98.64 102.07CK1δ 95.50 100.67 GRK6 101.18 103.17 NIK/MAP3K14 104.63 95.86 TAOK2/TAO1 84.82 95.28CK1ε 95.54 88.19 GRK7 105.03 104.95 NLK 86.80 91.04 TAOK3/JIK 89.80 87.91CK1γ1/CSNK1G1 96.52 100.94 GSK3a 89.87 88.43 P38α/MAPK14 84.30 77.68 TBK1 84.27 78.58CK1γ2/CSNK1G2 99.73 98.88 GSK3β 96.53 96.91 P38β/MAPK11 96.92 97.45 TEC 73.90 78.70CK1γ3/CSNK1G3 100.04 104.06 HCK 95.44 97.51 P38δ/SAPK4 73.07 73.28 TIE2/TEK 83.18 81.63CK2α 105.14 107.65 HGK/MAP4K4 87.72 87.80 P38γ 86.28 85.31 TRKA/NTRK1 93.81 91.45CK2α2 50.80 51.34 HIPK1 72.74 75.68 P70S6K 99.83 99.99 TRKB/NTRK2 91.32 89.91c-Kit 96.37 97.08 HIPK2 82.30 82.40 PAK1 94.88 94.51 TRKC/NTRK3 96.70 92.52CLK1 76.32 72.25 HIPK3 73.38 73.46 PAK2 102.11 101.35 TSSK2 100.56 96.69CLK2 89.22 85.31 HIPK4 93.71 97.12 PAK3 97.41 94.56 TTK 100.06 103.12CLK3 73.08 73.66 IGF1R 99.80 101.98 PAK4 97.06 95.15 TYK1/LTK 99.10 99.28CLK4 97.69 94.76 IKKα/CHUK 95.88 95.05 PAK5 90.61 94.33 TYK2 98.62 100.20c-MER 86.12 86.17 IKKβ 98.96 100.13 PAK6 90.87 93.82 TYRO3/SKY 86.36 78.69c-Met 87.11 89.81 IR 97.05 98.23 PASK 97.83 99.16 VRK1 116.17 108.62COT1/MAP3K8 95.29 93.35 IRAK1 96.49 93.73 PBK/TOPK 95.74 92.24 WEE1 101.67 99.31CSK 92.42 93.56 IRAK4 104.54 100.45 PDGFRα 101.80 100.34 WNK2 61.58 64.23c-Src 93.05 96.76 IRR/INSRR 96.94 100.78 PDGFRβ 95.10 98.78 WNK3 84.00 74.52CTK/MATK/HYL 89.43 85.52 ITK 99.16 103.68 PDK1/PDPK1 95.81 99.72 YES 100.57 102.20DAPK1 93.72 99.15 JAK1 96.30 96.66 PHKγ2 85.82 89.72 ZAK/MLTK 91.17 98.10DAPK2 103.19 102.82 JAK2 103.51 100.88 PIM1 15.44 14.08 ZAP70 90.65 86.25

ZIPK/DAPK3 96.02 93.67

Supplementary Table 1 List of all 261 kinases tested for inhibition by M-110 at 5 µM. Kinases were tested in duplicate and the enzyme activities in the presence of M-110 are expressed as the % activity in the presence of DMSO only.