mct.aacrjournals.orgmct.aacrjournals.org/.../142079_1_supp_2898612_nl4pqx.docx · web...

Supplementary Data: Mortensen, et al. “CC-223, a Potent and Selective Inhibitor of mTOR Kinase”

Supplementary InformationCC-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 S2Suppl. Figure S2: PK/PD Model Performance and eIF4E In Vivo Biomarker page S3Suppl. Figure S3: HCT-116, MBA MD231 & A549 Efficacy Studies page S4Suppl. Table S1: Kinase Selectivity page S5-S8Suppl. Table S2: Cellular Biomarker with SEM page S9Suppl. Table S3: Cellular Proliferation with SEM page S10 Suppl. Table S4: Hematological Panel page S11-12Suppl. Table S5: IRF4 Gene and Protein Data page S13Suppl. Table S6: PK/PD Data PK Data page S14Suppl. Table S7: In Vivo Efficacy Across Tumor Types page S15

S1


A. Rapamycin, 72 hour treatment B. CC-223, 72 hour treatment

C. Cell Cycle Analysis PC-3

HeLa Untreated PC3 Untreated PC3 DMSO 0.5 μM CC-223 5 μM CC-223

D. Caspase Induction

Supplementary Figure S1. (A) Rapamycin effects on proliferation plateau in many cell lines, PC-3 cells with a plateau of ~75% inhibition were classified as sensitive and HCT-116 cells were classified as insensitive with a inhibitory plateau of ~30%. (B) Proliferation dose response curves for PC-3 and HCT-116 cells following treatment with CC-223. (C) Cell Cycle analysis in PC-3. (D) Induction of Caspase 3/7 by CC-223 in DLBCL line, WSU-DLCL2, and not in NSCLC line, A549.

S2

plateau ~75%sensitive

plateau ~30%insensitive

PC-3 Cells HCT-116 CellsPC-3 Cells HCT-116 Cells

plateau ~75%sensitive

plateau ~30%insensitive

PC-3 Cells HCT-116 CellsPC-3 Cells HCT-116 Cells

Caspase 3/7 Induction at 24 Hours

[conc] uM0.001 0.1 10

Fold

Cha

nge

0

2

4

6

8

10WSU-DLCL2A549

HeLa Untreated PC-3 Untreated

G1

G2/M

S

DMSO ControlPC-3

0.5 µM CC-223 5 µM CC-223PC-3 PC-3


A.

Performance of Model for pS6RP Performance of Model for pAkt

0

20

40

60

80

100

0 20 40 60 80 100

pred

icte

d in

hibi

tion

(%)

observed inhibition (%)

pAkt

0

20

40

60

80

100

0 20 40 60 80 100

pred

icte

d in

hibi

tion

(%)

observed inhibition (%)

pS6RP

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


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 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

Kinase % Activity Remaining

Abl 60Abl_E255K 67Abl_G250E 72Abl_T315I 63Abl_Y253F 51

ALK 82ALK4 93AMPK 85

AMPK_A2_B1_G1 72Arg 76

Aurora-A 43Aurora-B 71Aurora-C 73

Axl 67Blk 72

Bmx 84BRAF 64

BRAF V599E 60BRK 94

BrSK1 63BTK 82

CaMKI 96CaMKI-delta 94

CaMKII-alpha 92CaMKII-beta 86CaMKII-delta 93

CaMKIV 95CDK1_cyclinB 70CDK2_cyclinA 63

CDK5_p25 66CDK5_p35 67

CDK7_cyclinH_MAT1 98CDK9_cyclinT1 59

CHK1 89CHK2 83

CC-223 10 µM


CK1-alpha1 94CK1-delta 81

CK1-epsilon 78CK1-gamma1 95CK1-gamma2 79CK1-gamma3 88

CK2 95CK2-alpha2 86

cKit 88cKit_T670I 86

CLK1 78CLK2 34CLK3 66c-RAF 74CSK 93

cSRC 71cSRC-N1 65DAPK1 108

DCAMKL2 93DYR1A 90

DYRK1B 92DYRK3 72DYRK4 100EEF2K 94EGFR 98

EGFR_L858R 101EGFR_L861Q 98EGFR_T790M 99

EGFR_T790M,L858R 97EphA1 77EphA2 85EphA3 96EphA4 86EphA5 86EphA8 90

S5


CC-223 10 µM


EphB1 74EphB2 86EphB3 4EphB4 93ErbB2 104ErbB4 86FAK 86Fer 82Fes 85

FGFR1 67FGFR2 51FGFR3 75

FGFR3_K650E 51FGFR4 75

Fgr 57Flt1 72Flt3 52

Flt3_D835Y 45Flt4 14Fms 2Fyn 74Gck 80

GRK2 101GRK3 99GRK4 96GRK5 99GRK6 101GRK7 96

GSK3-alpha 39GSK3-beta 51

Hck 72Hgk 69

HIPK1 93HIPK4 80IGF-1R 92IKK1 105

IKK-beta 84IKK-epsilon 87

IR 87IRAK4 100

CC-223 10 µM


IRR 92Itk 98

JAK1 87JAK2 79

JAK2-JH1-JH2 83JAK2-JH1-JH2_V617F 83

JAK3 61JNK1-alpha1 72JNK2-alpha2 103

JNK3 92KDR 22

Khs_1 62Lck 63

LRRK2 71lRRK2_G2019S 50

Ltk 94LYN-A 68LYN-B 57

MAP3K8 79MAPK1 93MAPK3 90

MAPKAP-K2 101MAPKAP-K3 90

MARK1 83MARK2 81MARK3 83MARK4 82MATK 97MEK1 87MEK2 77MELK 54Mer 82Met 91

Met_M1250T 91MINK 91MKK6 88MLK1 84

MRCK-alpha 95MRCK-beta 93

MSK1 90

S6


CC-223 10 µM


MSK2 87MSSK1 96MST1 27MST2 48MST3 52MST4 44mTOR 0MuSK 79MYLK2 84NEK1 200NEK2 94NEK4 89NEK6 91NEK7 97NEK9 91

p38-alpha 102p38-beta 87p38-delta 91

p38-gamma 93p70S6K 94PAK2 97PAK3 88PAK4 88PAK6 92PAK7 88PASK 87

PDGFR-alpha 41PDGFR-alpha_D842V 46PDGFR-alpha_T674I 83PDGFR-alpha_V561D 24

PDGFR-beta 83PDK1 82

PhK-gamma1 95PhK-gamma2 90

Pim-1 96Pim-2 102PKA 98

PKB-alpha 98PKB-beta 91

PKB-gamma 90

CC-223 10 µM


PKC-alpha 93PKC-betaI 97PKC-betaII 83PKC-delta 94

PKC-epsilon 90PKC-eta 94

PKC-gamma 85PKC-iota 96PKC-mu 68

PKC-theta 95PKC-zeta 87

PKD2 73PKD3 59Plk1 96Plk2 91Plk3 94

PRAK 91PRK1 91

PRKG1 65PRKG2 93PrKX 91

PTK2B 86PTK5 68Ret 23

Ret_V804L 49Ret_Y791F 30

ROCK-I 94ROCK-II 93

Ron 94Ros 82Rse 72Rsk1 80Rsk2 78Rsk3 59Rsk4 65SGK 91

SGK2 91SGK3 95

SNF1LK2 56SRMS 78

S7


CC-223 10 µM


SRPK1 89SRPK2 98STK25 59

Syk 77TAO1 86TBK1 56Tie2 94TrkA 77TrkB 79

CC-223 10 µM


TrkC 70TSSK1 80TSSK2 94Tyk2 73Yes 65

ZAP-70 103ZIPK 98

S8


Supplementary Table S2. TORC1 and TORC2 cellular biomarker inhibition data across a panel of cancer cell lines with SEM included.

Cell Line Tumor Type pS6RP p4EBP1 pAkt(S473)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

S9


Supplementary Table S3. Cellular growth inhibition for CC-223 across a panel of cell lines classified by rapamycin sensitivity including SEM.

Cell Line Tumor Type Rapamycin Sensitivity

Antiproliferative IC50 (µM) 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

S10


Supplementary Table S4. Summary of data for proliferation and apoptosis in the hematological cell panel.

Cell Line DiseaseSubtyp

e

Proliferation (n ≥ 3) Apoptosis (n = 1)CC-223 GI50

(μM)CC-223

IC50 (μM)CC-223

CalX (μM)Rapamycin CalX (μM)

KARPAS-422 NHL DLBCL 0.04 ± 0.02 0.07 ± 0.02 6.95 9.3963Toledo 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 > 10Pfeiffer NHL DLBCL 0.08 ± 0.05 0.24 ± 0.1 > 10 > 10RC-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 4.4945WSU-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 7.0143HT NHL DLBCL 0.17 ± 0.06 0.35 ± 0.02 > 10 > 10

RIVA NHL DLBCL 0.24 ± 0.08 1.34 ± 0.18 > 10 > 10U-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 6.0389SU-DHL-6 NHL DLBCL 0.33 ± 0.14 0.44 ± 0.24 > 10 4.1442OCI-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 > 10SU-DHL-16 NHL DLBCL 0.38 ± 0.08 0.39 ± 0.08 1.07 0.2977OCI-LY-19 NHL DLBCL 0.47 ± 0.15 0.94 ± 0.19 > 10 > 10OCI-LY-10 NHL DLBCL 0.47 ± 0.28 0.71 ± 0.37 > 10 > 10SU-DHL-10 NHL DLBCL 0.54 ± 0.22 0.55 ± 0.22 1.43 8.6406SU-DHL-5 NHL DLBCL 0.54 ± 0.31 0.58 ± 0.29 1.9 7.9066OCI-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 5.8901U-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 0.0088WSU-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 0.2341SC-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 > 10JVM-2 NHL MCL 0.32 ± 0.23 1.07 ± 0.73 > 10 > 10REC-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 > 10Mino 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

KG-1 Leukemia AML 0.02 ± 0.02 0.32 ± 0.16 > 10 > 10

KASUMI-1 Leukemia AML 0.08 ± 0.02 0.17 ± 0.03 > 10 > 10

MOLM-13 Leukemia AML 0.14 ± 0.03 0.15 ± 0.03 7.85 > 10

THP-1 Leukemi AML 0.21 ± 0.09 0.64 ± 0.23 > 10 > 10

S11


Cell Line Disease Subtype

Proliferation (n ≥ 3) Apoptosis (n = 1)CC-223 GI50

(μM)CC-223

IC50 (μM)CC-223

CalX (μM)Rapamycin CalX (μM)

a

HL-60 Leukemia AML 0.46 ± 0.17 0.7 ± 0.29 > 10 > 10

KARPAS-299 ALCL T-cell 0.52 ± 0.22 0.93 ± 0.4 > 10 > 10SU-DHL-1 ALCL T-cell 1.36 ± 0.49 2.18 ± 0.92 > 10 > 10

S12


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.

Cell LineIRF4

Protein Expression

DB -0.241HT -0.373

KASUMI-1 0.226JEKO-1 -0.126

NU-DHL-1 -0.025WSU-DLCL2 -0.209KARPAS-422 -0.111

SU-DHL-4 -0.207SU-DHL-16 -0.321

KG-1 -0.272WSU-NHL -0.170

HL-60 0.170THP-1 0.006

WSU-FSCCL 0.051SU-DHL-5 -0.323MOLM-13 0.469

SU-DHL-10 -0.151OCI-LY-10 -0.271SU-DHL-6 0.110

RIVA NADOHH-2 0.962

OCI-LY-19 0.362OCI-LY-7 1.594

KARPAS-1106P 0.093U-2932 NA

SU-DHL-8 0.348Pfeiffer 0.233REC-1 0.228

SU-DHL-1 1.281RC-K8 0.491Farage -0.001SC-1 0.259

U-2940 NAMino 0.384

KARPAS-299 1.823Granta-519 0.581

Toledo 0.438JVM-13 2.120

OCI-LY-3 1.213JVM-2 1.880

S13


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.

DoseSample

Collection Time Plasma ConcentrationPD Biomarker

pS6 pAkt

mg/kg hour mean ± SD (µM) Percent Inhibition

25 1 10.51 ± 4.09 87.9 89.125 3 3.89 ± 1.89 95.1 73.625 8 2.14 ± 1.08 92.3 71.625 16 3.86 ± 0.82 92.1 71.725 24 0.78 ± 0.34 83.7 34.525 48 0.03 ± 0.03 47.2 40.525 72 BLQ 68.3 44.1

10 1 3.17 ± 0.7 79.5 88.610 3 1.11 ± 0.4 91.9 79.210 8 1.12 ± 0.3 91.2 64.110 16 0.17 ± 0.09 32.4 41.710 24 0.02 ± 0.01 32.7 46.110 48 BLQ 39.5 41.410 72 BLQ 47.9 38.6

1 0.5 0.56 ± 0.02 80.8 72.11 2 0.31 ± 0.1 82.8 66.71 4 0.19 ± 0.1 78.6 66.51 6 0.09 ± 0.02 53.1 39.21 10 0.06 ± 0.04 14.4 52.31 24 0.009 ± 0.002 20.6 55.9

S14


Supplementary Table S7. In Vivo Efficacy Across Tumor Types.

*TVR=Tumor Volume Reduction = Vehicle – Treated x 100%Vehicle

# highest dose tested

S15

Cell Line Tumor Type

Minimum Efficacious Dose Maximum Efficacy with Tolerated Dose

Dose(mg/kg) TVR* (%) Dose

(mg/kg) TVR* (%)

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

mct.aacrjournals.orgmct.aacrjournals.org/.../142079_1_supp_2898612_nl4pqx.docx · web...

Documents