Supplementary Data: Mortensen, Et Al. CC-223, a Potent and Selective Inhibitor of Mtor Kinase
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Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Information CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.
Deborah S. Mortensen1, Kimberly E. Fultz1, Shuichan Xu1, Weiming Xu1, Garrick Packard1, Godrej Khambatta1, James Gamez1, Jim Leisten1, Jingjing Zhao1, Julius Apuy1, Kamran Ghoreishi1, Matt Hickman1, Rama Krishna Narla1, Rene Bissonette1, Samantha Richardson1, Sophie X. Peng1, Sophie Perrin-Ninkovic1, Tam Tran1, Tao Shi1, Wen Qing Yang1, Zeen Tong2, Brian E. Cathers1, Mehran F. Moghaddam1, Stacie S. Canan1, Peter Worland1, Sabita Sankar1 and Heather K. Raymon1
Celgene Corporation, 1San Diego, California and 2Summit, New Jersey.
Contents: Suppl. Figure S1: Prolif. Curves, PC3 Cell Cycle & solid tumor apoptosis page S2 Suppl. Figure S2: PK/PD Model Performance and eIF4E In Vivo Biomarker page S3 Suppl. Figure S3: HCT-116, MBA MD231 & A549 Efficacy Studies page S4 Suppl. Table S1: Kinase Selectivity page S5-S8 Suppl. Table S2: Cellular Biomarker with SEM page S9 Suppl. Table S3: Cellular Proliferation with SEM page S10 Suppl. Table S4: Hematological Panel page S11-12 Suppl. Table S5: IRF4 Gene and Protein Data page S13 Suppl. Table S6: PK/PD Data PK Data page S14 Suppl. Table S7: In Vivo Efficacy Across Tumor Types page S15
S1 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
A. Rapamycin, 72 hour treatment B. CC-223, 72 hour treatment PC-3 Cells PCHCT-3 Cells-116 Cells HCT-116 Cells
plateau ~75% plateau ~30% sensitive insensitive
PC-3 Cells PCHCT-3 Cells-116 Cells HCT-116 Cells plateau ~75% plateau ~30% sensitive insensitive
C. Cell Cycle Analysis PC-3
PC-3 PC-3 PC-3 HeLa Untreated PC-3 Untreated DMSO Control 0.5 µM CC-223 5 µM CC-223
G1
S
G2/M
HeLa Untreated PC3 Untreated PC3 DMSO 0.5 μM CC-223 5 μM CC-223
D. Caspase Induction
Caspase 3/7 Induction at 24 Hours Supplementary Figure S1. (A) Rapamycin effects on 10 proliferation plateau in many cell lines, PC-3 cells with a WSU-DLCL2 8 A549 plateau of ~75% inhibition were classified as sensitive e g
n and HCT-116 cells were classified as insensitive with a
a 6 h
C inhibitory plateau of ~30%. (B) Proliferation dose d
l 4
o response curves for PC-3 and HCT-116 cells following F 2 treatment with CC-223. (C) Cell Cycle analysis in PC- 3. (D) Induction of Caspase 3/7 by CC-223 in DLBCL 0 0.001 0.1 10 line, WSU-DLCL2, and not in NSCLC line, A549. [conc] uM
S2 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
A.
Performance of Model for pS6RP Performance of Model for pAkt
pS6RP pAkt 100 100
) 80 ) 80 % % ( (
n n o o i i 60 60 t t i i b b i i h h n n i i
40 40 d d e e t t c c i i d d 20 20 e e r r p p 0 0 0 20 40 60 80 100 0 20 40 60 80 100 observed inhibition (%) observed inhibition (%)
B.
Supplementary Figure S2. A. Performance correlation of PK/PD model for predicted and observed biomarker inhibition in the PC-3 xenograft model. B. Inhibition of p4EPB1 by CC-223 in mice with PC-3 tumors.
S3 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
A. B.
C.
Supplementary Figure S3. A. MDA-MB-231 breast model with BID dosing of 1, 5 or 10 mg/kg. B. HCT 116 colon xenograft model with BID dosing at 1, 5 or 10 mg/kg, daily dosing (qd) or every second day (q2d) dosing at 25 mg/kg. C. A549 lung model with 1, 5, or 10 mg/kg BID dosing.
S4 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Table S1. Invitrogen Selectivity Data. The selectivity profile of CC-223 at a concentration of 10 μM was determined using a panel of 246 kinases from Invitrogen’s SelectScreen™ profiling service. The data are reported as the percent activity remaining as compared with the no inhibitor control wells. Kinases with more than 80% inhibition at 10 µM were considered as hits. CC-223 10 µM % Activity Kinase Remaining Abl 60 Abl_E255K 67 Abl_G250E 72 Abl_T315I 63 Abl_Y253F 51 ALK 82 ALK4 93 AMPK 85 AMPK_A2_B1_G1 72 Arg 76 Aurora-A 43 Aurora-B 71 Aurora-C 73 Axl 67 Blk 72 Bmx 84 BRAF 64 BRAF V599E 60 BRK 94 BrSK1 63 BTK 82 CaMKI 96 CaMKI-delta 94 CaMKII-alpha 92 CaMKII-beta 86 CaMKII-delta 93 CaMKIV 95 CDK1_cyclinB 70 CDK2_cyclinA 63 CDK5_p25 66 CDK5_p35 67 CDK7_cyclinH_MAT1 98 CDK9_cyclinT1 59 CHK1 89 CHK2 83
S5 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
CC-223 10 µM % Activity Kinase Remaining CK1-alpha1 94 CK1-delta 81 CK1-epsilon 78 CK1-gamma1 95 CK1-gamma2 79 CK1-gamma3 88 CK2 95 CK2-alpha2 86 cKit 88 cKit_T670I 86 CLK1 78 CLK2 34 CLK3 66 c-RAF 74 CSK 93 cSRC 71 cSRC-N1 65 DAPK1 108 DCAMKL2 93 DYR1A 90 DYRK1B 92 DYRK3 72 DYRK4 100 EEF2K 94 EGFR 98 EGFR_L858R 101 EGFR_L861Q 98 EGFR_T790M 99 EGFR_T790M,L858R 97 EphA1 77 EphA2 85 EphA3 96 EphA4 86 EphA5 86 EphA8 90 EphB1 74 EphB2 86 EphB3 4 EphB4 93 ErbB2 104
S6 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
CC-223 10 µM % Activity Kinase Remaining ErbB4 86 FAK 86 Fer 82 Fes 85 FGFR1 67 FGFR2 51 FGFR3 75 FGFR3_K650E 51 FGFR4 75 Fgr 57 Flt1 72 Flt3 52 Flt3_D835Y 45 Flt4 14 Fms 2 Fyn 74 Gck 80 GRK2 101 GRK3 99 GRK4 96 GRK5 99 GRK6 101 GRK7 96 GSK3-alpha 39 GSK3-beta 51 Hck 72 Hgk 69 HIPK1 93 HIPK4 80 IGF-1R 92 IKK1 105 IKK-beta 84 IKK-epsilon 87 IR 87 IRAK4 100 IRR 92 Itk 98 JAK1 87 JAK2 79 JAK2-JH1-JH2 83
S7 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
CC-223 10 µM % Activity Kinase Remaining JAK2-JH1-JH2_V617F 83 JAK3 61 JNK1-alpha1 72 JNK2-alpha2 103 JNK3 92 KDR 22 Khs_1 62 Lck 63 LRRK2 71 lRRK2_G2019S 50 Ltk 94 LYN-A 68 LYN-B 57 MAP3K8 79 MAPK1 93 MAPK3 90 MAPKAP-K2 101 MAPKAP-K3 90 MARK1 83 MARK2 81 MARK3 83 MARK4 82 MATK 97 MEK1 87 MEK2 77 MELK 54 Mer 82 Met 91 Met_M1250T 91 MINK 91 MKK6 88 MLK1 84 MRCK-alpha 95 MRCK-beta 93 MSK1 90 MSK2 87 MSSK1 96 MST1 27 MST2 48 MST3 52
S8 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
CC-223 10 µM % Activity Kinase Remaining MST4 44 mTOR 0 MuSK 79 MYLK2 84 NEK1 200 NEK2 94 NEK4 89 NEK6 91 NEK7 97 NEK9 91 p38-alpha 102 p38-beta 87 p38-delta 91 p38-gamma 93 p70S6K 94 PAK2 97 PAK3 88 PAK4 88 PAK6 92 PAK7 88 PASK 87 PDGFR-alpha 41 PDGFR-alpha_D842V 46 PDGFR-alpha_T674I 83 PDGFR-alpha_V561D 24 PDGFR-beta 83 PDK1 82 PhK-gamma1 95 PhK-gamma2 90 Pim-1 96 Pim-2 102 PKA 98 PKB-alpha 98 PKB-beta 91 PKB-gamma 90 PKC-alpha 93 PKC-betaI 97 PKC-betaII 83 PKC-delta 94 PKC-epsilon 90
S9 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
CC-223 10 µM % Activity Kinase Remaining PKC-eta 94 PKC-gamma 85 PKC-iota 96 PKC-mu 68 PKC-theta 95 PKC-zeta 87 PKD2 73 PKD3 59 Plk1 96 Plk2 91 Plk3 94 PRAK 91 PRK1 91 PRKG1 65 PRKG2 93 PrKX 91 PTK2B 86 PTK5 68 Ret 23 Ret_V804L 49 Ret_Y791F 30 ROCK-I 94 ROCK-II 93 Ron 94 Ros 82 Rse 72 Rsk1 80 Rsk2 78 Rsk3 59 Rsk4 65 SGK 91 SGK2 91 SGK3 95 SNF1LK2 56 SRMS 78 SRPK1 89 SRPK2 98 STK25 59 Syk 77 TAO1 86
S10 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
CC-223 10 µM % Activity Kinase Remaining TBK1 56 Tie2 94 TrkA 77 TrkB 79 TrkC 70 TSSK1 80 TSSK2 94 Tyk2 73 Yes 65 ZAP-70 103 ZIPK 98
S11 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Table S2. TORC1 and TORC2 cellular biomarker inhibition data across a panel of cancer cell lines with SEM included. pS6RP p4EBP1 pAkt(S473) Cell Line Tumor Type mean ± SEM mean ± SEM mean ± SEM PC-3 Prostate 0.031 ± 0.002 0.405 ± 0.047 0.011 ± 0.010 MDA-MB-231 Breast 0.027 ± 0.010 0.120 (n=1) 0.036 ± 0.006
A549 Lung 0.036 (n=1) 0.330 (n=1) 0.117 ± 0.018
NCI-H23 Lung 0.071 ± 0.028 0.120 (n=1) 0.094 ± 0.023
HCT 116 Colon 0.081 ± 0.009 0.392 ± 0.033 0.099 ± 0.023
U-87 MG Glioma 0.184 ± 0.030 1.05 ± 0.115 0.150 ± 0.048
S12 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Table S3. Cellular growth inhibition for CC-223 across a panel of cell lines classified by rapamycin sensitivity including SEM.
Rapamycin Antiproliferative IC (µM) Cell Line Tumor Type 50 Sensitivity mean ± SEM PC-3 prostate sensitive 0.114 ± 0.015
CAL-51 breast sensitive 0.140 ± 0.010
A549 lung sensitive 0.208 ± 0.007
T47D breast partially 0.092 ± 0.024
NCI-H460 lung partially 0.200 ± 0.004
HepG2 HCC partially 0.321 ± 0.074
AU565 breast partially 0.329 ± 0.117
Hep3B HCC partially 0.338 ± 0.066
U-87 MG glioma partially 0.555 ± 0.115
HCT 116 colon insensitive 0.371 ± 0.053
MDA-MB-231 breast insensitive 0.669 ± 0.056
NCI-H23 lung insensitive 1.039 ± 0.215
S13 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Table S4. Summary of data for proliferation and apoptosis in the hematological cell panel. Proliferation (n ≥ 3) Apoptosis (n = 1) CC-223 GI50 CC-223 CC-223 Rapamyci Cell Line Disease Subtype (μM) IC50 (μM) CalX (μM) n CalX (μM) KARPAS-422 NHL DLBCL 0.04 ± 0.02 0.07 ± 0.02 6.95 9.3963 Toledo NHL DLBCL 0.07 ± 0.03 1.19 ± 0.09 > 10 > 10 NU-DHL-1 NHL DLBCL 0.08 ± 0.05 0.11 ± 0.07 2.5 > 10 Pfeiffer NHL DLBCL 0.08 ± 0.05 0.24 ± 0.1 > 10 > 10 RC-K8 NHL DLBCL 0.13 ± 0.01 0.28 ± 0.04 > 10 > 10 SU-DHL-8 NHL DLBCL 0.14 ± 0.08 0.18 ± 0.07 0.66 4.4945 WSU-DLCL2 NHL DLBCL 0.16 ± 0.03 0.17 ± 0.03 0.43 2.7005 KARPAS-1106P NHL DLBCL 0.16 ± 0.04 0.16 ± 0.04 1.04 7.0143 HT NHL DLBCL 0.17 ± 0.06 0.35 ± 0.02 > 10 > 10 RIVA NHL DLBCL 0.24 ± 0.08 1.34 ± 0.18 > 10 > 10 U-2940 NHL DLBCL 0.24 ± 0.17 0.43 ± 0.27 2.47 7.9192 SU-DHL-4 NHL DLBCL 0.27 ± 0.08 0.28 ± 0.08 5.33 6.0389 SU-DHL-6 NHL DLBCL 0.33 ± 0.14 0.44 ± 0.24 > 10 4.1442 OCI-LY-3 NHL DLBCL 0.35 ± 0.14 0.52 ± 0.17 2.99 > 10 DB NHL DLBCL 0.37 ± 0.17 0.45 ± 0.15 > 10 > 10 SU-DHL-16 NHL DLBCL 0.38 ± 0.08 0.39 ± 0.08 1.07 0.2977 OCI-LY-19 NHL DLBCL 0.47 ± 0.15 0.94 ± 0.19 > 10 > 10 OCI-LY-10 NHL DLBCL 0.47 ± 0.28 0.71 ± 0.37 > 10 > 10 SU-DHL-10 NHL DLBCL 0.54 ± 0.22 0.55 ± 0.22 1.43 8.6406 SU-DHL-5 NHL DLBCL 0.54 ± 0.31 0.58 ± 0.29 1.9 7.9066 OCI-LY-7 NHL DLBCL 0.65 ± 0.16 0.77 ± 0.16 1.1 0.0108 Farage NHL DLBCL 1.36 ± 0.5 1.49 ± 0.56 6.25 5.8901 U-2932 NHL DLBCL 1.61 ± 0.21 3.51 ± 0.42 > 10 > 10 WSU-NHL NHL FL 0.07 ± 0.02 0.11 ± 0.02 0.37 0.0088 WSU-FSCCL NHL FL 0.22 ± 0.12 0.36 ± 0.24 1.48 5.5646 DOHH-2 NHL FL 0.24 ± 0.08 0.31 ± 0.12 0.19 0.2341 SC-1 NHL FL 0.6 ± 0.39 0.82 ± 0.44 > 10 > 10 JEKO-1 NHL MCL 0.09 ± 0.05 0.12 ± 0.05 6.14 > 10 JVM-2 NHL MCL 0.32 ± 0.23 1.07 ± 0.73 > 10 > 10 REC-1 NHL MCL 1.08 ± 0.36 2.44 ± 1.61 8.36 > 10 Granta-519 NHL MCL 1.69 ± 0.91 8.01 ± 1.74 > 10 > 10 Mino NHL MCL 3.23 ± 0.76 3.99 ± 0.89 > 10 9.8826 JVM-13 NHL MCL 4.78 4.56 10 ± 0 > 10 9.4223 Leukemi KG-1 AML 0.02 ± 0.02 0.32 ± 0.16 > 10 > 10 a Leukemi KASUMI-1 AML 0.08 ± 0.02 0.17 ± 0.03 > 10 > 10 a Leukemi MOLM-13 AML 0.14 ± 0.03 0.15 ± 0.03 7.85 > 10 a THP-1 Leukemi AML 0.21 ± 0.09 0.64 ± 0.23 > 10 > 10
S14 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Proliferation (n ≥ 3) Apoptosis (n = 1) CC-223 GI50 CC-223 CC-223 Rapamyci Cell Line Disease Subtype (μM) IC50 (μM) CalX (μM) n CalX (μM) a Leukemi HL-60 AML 0.46 ± 0.17 0.7 ± 0.29 > 10 > 10 a KARPAS-299 ALCL T-cell 0.52 ± 0.22 0.93 ± 0.4 > 10 > 10 SU-DHL-1 ALCL T-cell 1.36 ± 0.49 2.18 ± 0.92 > 10 > 10
S15 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Table S5. Protein expression levels of IRF4 in the 40 hematological cancer cell lines. IRF4 protein expression was measured by RPPA array and is presented in log2 scale.
IRF4 Cell Line Protein Expression DB -0.241 HT -0.373 KASUMI-1 0.226 JEKO-1 -0.126 NU-DHL-1 -0.025 WSU-DLCL2 -0.209 KARPAS-422 -0.111 SU-DHL-4 -0.207 SU-DHL-16 -0.321 KG-1 -0.272 WSU-NHL -0.170 HL-60 0.170 THP-1 0.006 WSU-FSCCL 0.051 SU-DHL-5 -0.323 MOLM-13 0.469 SU-DHL-10 -0.151 OCI-LY-10 -0.271 SU-DHL-6 0.110 RIVA NA DOHH-2 0.962 OCI-LY-19 0.362 OCI-LY-7 1.594 KARPAS-1106P 0.093 U-2932 NA SU-DHL-8 0.348 Pfeiffer 0.233 REC-1 0.228 SU-DHL-1 1.281 RC-K8 0.491 Farage -0.001 SC-1 0.259 U-2940 NA Mino 0.384 KARPAS-299 1.823 Granta-519 0.581
S16 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
IRF4 Cell Line Protein Expression Toledo 0.438 JVM-13 2.120 OCI-LY-3 1.213 JVM-2 1.880
S17 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Table S6. In Vivo PD biomarker inhibition and plasma concentrations of CC- 223 following a single oral dose of 1, 10 or 25 mg/kg.
Sample PD Biomarker Dose Collection Time Plasma Concentration pS6 pAkt mg/kg hour mean ± SD (µM) Percent Inhibition 25 1 10.51 ± 4.09 87.9 89.1 25 3 3.89 ± 1.89 95.1 73.6 25 8 2.14 ± 1.08 92.3 71.6 25 16 3.86 ± 0.82 92.1 71.7 25 24 0.78 ± 0.34 83.7 34.5 25 48 0.03 ± 0.03 47.2 40.5 25 72 BLQ 68.3 44.1
10 1 3.17 ± 0.7 79.5 88.6 10 3 1.11 ± 0.4 91.9 79.2 10 8 1.12 ± 0.3 91.2 64.1 10 16 0.17 ± 0.09 32.4 41.7 10 24 0.02 ± 0.01 32.7 46.1 10 48 BLQ 39.5 41.4 10 72 BLQ 47.9 38.6
1 0.5 0.56 ± 0.02 80.8 72.1 1 2 0.31 ± 0.1 82.8 66.7 1 4 0.19 ± 0.1 78.6 66.5 1 6 0.09 ± 0.02 53.1 39.2 1 10 0.06 ± 0.04 14.4 52.3 1 24 0.009 ± 0.002 20.6 55.9
S18 Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”
Supplementary Table S7. In Vivo Efficacy Across Tumor Types.
Maximum Efficacy with Minimum Efficacious Dose Tumor Tolerated Dose Cell Line Type Dose Dose TVR* (%) TVR* (%) (mg/kg) (mg/kg) PC-3 Prostate 5 bid 64.9 25 qd 86.7
U-87 MG Glioma 1 qd 64.7 5 bid 89.2
MDA-MB-231 Breast 10 bid 55.2 10 bid# 55.2
A549 Lung 1 bid 69.6 10 bid 95.2
HCT116 Colon 10 bid 48.8 10 bid 48.8
ST140 PDX Lung 10 qd 47 10 qd# 47
*TVR=Tumor Volume Reduction = Vehicle – Treated x 100% Vehicle # highest dose tested
S19