Supplementary Figure S1 - Cancer Discovery · Supplementary Figure S2 Inhibition of BRAF...
Transcript of Supplementary Figure S1 - Cancer Discovery · Supplementary Figure S2 Inhibition of BRAF...
ERK + 5uM GSK1120212 ERK + 5uM SCH772984
Supplementary Figure S1TdF binding assay for SCH772984, Vertex 11e and GSK1120212
Tm = -0.15 ± 0.20 K N Bi di
Tm = 10.3 ± 0.30K 0 24 ± 0 14 MKD = No Binding
Protein + 5uM CompoundProtein only
KD = 0.24 ± 0.14 nM
Protein + 5uM CompoundProtein only
ERK + 5uM Vertex 11e MEK + 5uM GSK1120212ERK + 5uM Vertex 11e
Tm = 6 8 ± 1 3
MEK + 5uM GSK1120212
Tm = 8 0 ± 0 51Tm = 6.8 ± 1.3 KD = 5.4 ± 2.2 nM
Protein + 5uM CompoundProtein only
Tm = 8.0 ± 0.51KD = 7.1 ± 2.7 nM
Protein + 5uM CompoundProtein only
MEK + 5uM SCH772984 MEK + 5uM Vertex 11e
Tm = -0.17 ± 0.14KD = No Binding
Protein + 5uM CompoundProtein only
Tm = -0.11 ± 0.13 KD = No Binding
Protein + 5uM CompoundProtein only
Supplementary Figure S1 (cont.)
p-ERK + 5uM GSK1120212
Tm = 0.23 ± 0.31 KD = No Binding
Protein + 5uM CompoundProtein only
P-ERK + 5uM SCH772984
Tm = 9.8 ± 0.23 KD = 0.19 ± 0.12nM
Protein + 5uM CompoundProtein only
p-ERK + 5uM Vertex 11e
Tm =10.3 ± 1.3 KD = 0 11 ± 0 062nMKD 0.11 ± 0.062nM
Protein + 5uM CompoundProtein only
Supplementary Figure S2Inhibition of BRAF inhibitor-induced phosphorylation of ERK by SCH772984 in HCT-116 colorectal and Calu-6 non-small cell lung cancer lines
PLX4720772984
10uM 1uM 10uM 1uM
+- --- + +
- 10uM
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+- --- + +
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772984
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+- --- + +
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772984
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+- --- + +
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+- --- + +
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HCT-1161uM 10uM 1uM
+- --- + +
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772984 + + +300nM
+ + +
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772984 + + +300nM
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772984 + + +300nM
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pMEK
tMEK
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tMEKtMEK
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Supplementary Figure S3Time course of pRSK & pERK suppression in A375 cells treated withSCH772984 or control (DMSO)
A375 BRAFV600E l
pRSK
pERK
RSK
2hrs 6hrs 16hrs 24hrs 36hrs_ _ _ _ _+ + + + +SCH772984 2uM
A375 BRAFV600E melanoma
pERK
pCRAF
pMEK
ERK
CRAF
MEK
Actin
pBRAF
BRAF
Supplementary Figure S4
Supplementary Figure S5Flow Cytometry Analysis of BRAF mutant cell lines (LOX) treated with SCH772984
A No drug
24h 48h
No drug
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A
M1 =0.8%
M1 =2 %
M1 =0.8%M1 =0.8%
M1 =2 %M1 =2 %
Channels (FL2-A-FL2-Area)0 50 100 150 200 250
Channels (FL2-A-FL2-Area)0 50 100 150 200 250
SCH772984
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Channels (FL2-A-FL2-Area)0 50 100 150 200 250
SCH772984B
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M1 =20%M1 =20%M1 =
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20%M1 =9 %M1 =9 %
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Channels (FL2-A-FL2-Area)0 50 100 150 200 250 Channels (FL2-A-FL2-Area)
0 50 100 150 200 250
Channels (FL2-A-FL2-Area)0 50 100 150 200 250 Channels (FL2-A-FL2-Area)
0 50 100 150 200 250
Supplementary Figure S6Treatment of BRAF inhibitor resistant RKO colorectal cell lines with SCH772984or GSK1120212 (MEK inhibitor)
BRAFi-resistant, BRAF-mutant RKO colorectal
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RKO BR
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Transcriptional inhibition of several ERK target genesBy SCH772984 in BRAF-mutant melanoma cell line (A375)
Supplementary Figure S7
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Supplementary Figure S8Cell lines overexpressing constitutively active MEK1 respond to SCH772984
120120
A375 + RFPA375 + MEK1 F129L
)
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Supplementary Figure S9Real-time proliferation assay of RKO dual resistant cell line in the presence of SCH772984
BRAFi + MEKi combination resistance in BRAF-mutant colorectal
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IC50 for SCH772984 in 5-day viability assay (ViaLight) Tumor line SCH772984 IC50 (nM)
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RKO parental 11
RKO BR+MR (with PLX4032+GSK1120212)
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RKO BR+MR (without PLX4032+GSK1120212)
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Supplementary Figure S10Dual resistant cell lines A101D exhibit increased levels of proteins that include BRAF, CRAF, PDGFRB and phosphorylated AKT
Pare
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Supplementary Table S1Invitro Kinase profile of SCH772984 in a panel of 309 kinases
Kinases with ≥50% inhibition at 1 uM SCH772984 (% inhibition)
CLK2 (65%) FLT4 (VEGFR3) 60% GSG2 (Haspin) 51%
MAP4K4 (HGK) 71% MAPK1 (ERK2) 100%
MINK1 66% PRKD1 (PKC mu) 50%
TTK* 62%TTK* 62%
Kinases with <50% inhibition at 1 uM SCH772984 ABL1, ABL1 E225K, ABL1 G250E, ABL1 T315I, ABL1 Y253F, ABL2, ACVR1*, ACVR1B, ADRBK1, ADRBK2, AKT1, AKT2, AKT3, ALK, AMPKA1/B1/G1, AMPKA2/B1/G1, AURKA, AURKB, AURKC, AXL, BLK, BMPR1A*, BMX, BRAF*, BRAFV599E*, BRSK1, BTK, CAMK1, CAMK1D, CAMK2A, CAMK2B, CAMK2D, CAMK4, CAMKK1*, CAMKK2*, CDC42BPA, CDC42BPB, CDK1/cycB, CDK2/cycA, CDK5/p25, CDK5/p35,CAMKK2 , CDC42BPA, CDC42BPB, CDK1/cycB, CDK2/cycA, CDK5/p25, CDK5/p35, CDK4/cycH/MNAT1, CDK8/cycC*, CDK9/cycK*, CDK9/cycT1, CHEK1, CHEK2, CHUK, CLK1, CLK3, cRAF, CSF1R, CSK, CSNK1A1, CSNK1D, CSNK1E, CSNK1G1, CSNK1G2, CSNK1G3, CSNK2A1, CSNK2A2, DAPK1, DAPK3, DCAMKL2, DDR2*, DMPK*, DNA-PK, DYRK1A, DYRK1B, DYRK3, DYRK4, EEF2K, ERBB1, ERBB1 L858R, ERBB1 L861Q, ERBB1 T790M, ERBB1 T790M L858R, EPHA1, EPHA2, EPHA4, EPHA5, EPHA7*, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, ERBB2, ERBB4, FER, FES (FPS), FGFR1, FGFR2, FGFR3, FGFR3 K650E, FGFR4, FGR, FLT1, FLT3, FLT3 D835Y, FRAP1, FRK, FYN, GRK4, GRK5, GRK6 GRK7 GSK3A GSK3B HCK HIPK1 HIPK2 HIPK4 IGF1R IKBKB IKBKE INSRGRK6, GRK7, GSK3A, GSK3B, HCK, HIPK1, HIPK2, HIPK4, IGF1R, IKBKB, IKBKE, INSR, INSRR, IRAK1, IRAK4, ITK, JAK1, JAK2, JAK2 JH1 JH2, JAK2 JH1 JH2 V617F, JAK3, KDR, KIT, KIT T670I, KIT V654A*, LCK, LIMK1*, LIMK2*, LTK, LYNA, LYNB, MAP2K1 (MEK1)*, MAP2K1 S218D S222D*, MAP2K2 (MEK2)*, MAP2K3 (MEK3)*, MAP2K6 (MKK6), MAP2K6 (MKK6)*, MAP2K6 (MKK6) S207E T211E*, MAP3K10 (MLK2)*, MAP3K11 (MLK3)*, MAP3K14 (NIK)*, MAP3K2 (MEKK2)*, MAP3K3 (MEKK3)*, MAP3K5 (ASK1)*, MAP3K7/MAP3K7IP1 (TAK1-TAB1)*, MAP3K9 (MLK1), MAP4K2 (GCK), MAP45 (KHS1), MAPK8 (JNK1), MAPK9 (JNK2), MAPKAPK2, MAPKAPK3, (GC ), 5 ( S ), 8 (J ), 9 (J ), , 3,MAPKAPK5 (PRAK), MARK1, MARK2, MARK3, MARK4, MATK, MELK, MERTK, MET, MET M1250T, MKNK1, MLCK*, MST1R, MST4, MUSK, MYLK*, MYLK2, NEK1, NEK2, NEK4, NEK6, NEK7, NEK9, NLK*, NTRK1, NTRK2, NTRK3, NUAK1, PAK1, PAK2, PAK3, PAK4, PAK6, PAK7, PASK, PDGFRA, PDGFRA D842V, PDGFRA T574I, PDGFRB, PDK1, PDK1 Direct, PHKG1, PHKG2, PI4KA, PI4KB, PIK3C2A, PIK3C2B, PIK3C3, PIK3CA/PIK3R1, PIK3CD/PIK3R1, PIK3CG, PIM1, PIM2, PKN1, PLK1, PLK2, PLK3, PRKACA, PRKCA, PRKCB1, PRKCB2, PRKCD, PRKCE, PRKCG, PRKCH, PRKCI, PRKCN, PRKCQ PRKCZ PRKD2 PRKG1 PRKG2 PRKX PTK2 PTK2B PTK6 RAF1 Y340DPRKCQ, PRKCZ, PRKD2, PRKG1, PRKG2, PRKX, PTK2, PTK2B, PTK6, RAF1 Y340D Y341D*, RET, RET V804L, RET Y791F, RIPK2*, ROCK1, ROCK2, ROS1, RPS6KA1, RPS6KA2, RPS6KA3, RPS6KA4, RPS6KA5, RPS6KA6, RPS6KB1, SGK, SGK2, SGKL, SLK*, SNF1LK2, SPHK1, SPHK2, SRC, SRC N1, SRMS, SRPK1, SRPK2, STK16*, STK17A*, STK22B, STK22D, STK23, STK24, STK25, STK3, STK33*, STK4, SYK, TAOK2, TAOK3*, TBK1, TEC*, TEK, TGFBR1*, TNK2*, TXK, TYK2, TYRO3, WEE1*, WNK2*, YES1, ZAK*, ZAP70 *Denotes binding assay (rather than activity assay)Denotes binding assay (rather than activity assay)
Supplementary Table S2
In vitro efficacy of SCH772984 in tumor cell line panel
Cell line TypeSCH 772984 EC50 (nM) Ras BRAF BRAF Status
Malme 3M Melanoma 10 WT V600E HomozygousygWM-266-4 Melanoma 20 WT V600D HeterozygousUACC-62 Melanoma 30 WT V600E Homozygous Colo-205 Colon 36 WT V600E Homozygous SK-Mel-1 Melanoma 37 WT V600E Heterozygous
WiDr Colon 39 WT V600E HeterozygousM14 Melanoma 47 WT V600E Heterozygous
HT-29 Colon 50 WT V600E Heterozygous8505C Thyroid 50 WT V600E Heterozygous
WM-115 Melanoma 60 WT V600D HeterozygousWM 115 Melanoma 60 WT V600D HeterozygousHT-144 Melanoma 60 WT V600E Homozygous
SK-Mel-5 Melanoma 66 WT V600E HeterozygousA375-SM Melanoma 75 WT V600E Homozygous
SK-Mel-28 Melanoma 85 WT V600E Homozygous LOX Melanoma 100 WT V600E Homozygous
SK-Mel-3 melanoma 118 WT V600E HeterozygousK1 Thyroid 130 WT V600E Heterozygous
Hs-695T Melanoma 165 WT V600E Heterozygous8305C Thyroid 170 WT V600E Homozygous8305C Thyroid 170 WT V600E Homozygous
BHT-101 Thyroid 300 WT V600E HeterozygousRPMI-7951 Melanoma 344 WT V600E Heterozygous
A2058 Melanoma 360 WT V600E HeterozygousSK-Hep-1 Liver 1422 WT V600E Heterozygous
A673 Rhabdomyosarcoma 3001 WT V600E HeterozygousDBTRG-05MG CNS 3001 WT V600E Hetero
SW-626 Ovarian 33 Kras G12V WTLoVo Colon 47 Kras G13D WT
MiaPaCa Pancreas 53 Kras G12C WTMiaPaCa Pancreas 53 Kras G12C WTSW-620 Colon 104 Kras G12V WT
CAPAN-1 Pancreas 104 Kras G12V WTSW-527 Breast 121 Kras G12V WTHCT-116 Colon 128 Kras G13D WTSW-480 Colon 165 Kras G12V WTHPAC Pancreas 170 Kras G12D WT
OVCAR-5 Ovarian 208 Kras G12V WTAsPc-1 Pancreas 270 Kras G12D WTA549 NSCLC 326 K G12S WTA549 NSCLC 326 Kras G12S WT
SNU-1 Gastric 354 Kras G12D WTHOP62 NSCLC 676 Kras G12C WT
H23 NSCLC 1000 Kras G12C WTCal 62 Thyroid 1000 Kras G12R WT
MB-231 Breast 1000 Kras G13D G464VSU.86.86 Pancreas 1001 Kras G12D WTCFPAC-1 Pancreas 1001 Kras G12V WT
A427 NSCLC 1433 Kras G12D WTMDAH-2774 Ovarian 2657 Kras G12V WTNCI-H157 Lung 3000 Kras G12R WTHTB-177 NSCLC 3000 Kras Q61H WTUM-UC-3 Bladder 3001 Kras G12C WT
HCT-8 Colon 3001 Kras G12D WTPanc-1 Pancreas 3001 Kras G12D WTDLD-1 Colon 3001 Kras G13D WT
HCT-15 Colon 3001 Kras G13D WTHL-60 Leukemia 30 Nras Q61L WT
SK-Mel-2 Melanoma 34 Nras Q61R WTRD Rhabdomyosarcoma 123 Nras Q61H WT
HT-1197 Bladder 316 Nras Q61R WTMolt-3 Leukemia 600 Nras G12C -PA-1 Ovarian 1001 Nras G12D WTMolt-4 Leukemia 3001 Nras G12C WT
Supplementary Table S2 (continued)Hs 700T panc/intest 50 - WT
NCI-H292 Lung 90 WT WTA2780 Ovarian 143 WT WT
IGROV-1 Ovarian 146 WT WTSK-N-SH CNS 150 WT WTBxPc-3 Pancreas 184 WT WTBxPc 3 Pancreas 184 WT WTN-87 Gastric 307 WT WTH322 NSCLC 325 WT WTH716 Colon 334 WT WT
TT Thyroid 406 WT WTCaki-1 Renal 450 WT WT5637 Bladder 610 WT WT
MB-453 Breast 672 WT WTRT-4 Bladder 810 WT WT
HOP92 NSCLC 820 WT WTKG-1 Leukemia 900 WT WT
Hs-294T Melanoma 945 WT WTSF-539 CNS 1000 WT WTU-251 CNS 1000 WT WT
MB-468 Breast 1000 WT WTHS746T Gastric 1000 - WTSCABER Bladder 1000 - WTMCF 7 B 1001 WT WTMCF-7 Breast 1001 WT WTCHL-1 Melanoma 1460 WT WTU87MG CNS 2000 WT WT
SJCRH30 Rhabdomyosarcoma 2002 WT WTES-2 Ovarian 2659 - WT2
HT-1376 Bladder 2800 WT WTA172 CNS 3000 WT WT769P Renal 3000 WT WT
NCI H520 L ng 3000 WT WTNCI-H520 Lung 3000 WT WTDU145 Prostate 3000 WT WTK562 Leukemia 3000 WT WTU-937 Leukemia 3000 WT WTA204 Rhabdomyosarcoma 3001 WT WTDAOY CNS 3001 WT WTSF-268 CNS 3001 WT WTSF-295 CNS 3001 WT WTSNB-19 CNS 3001 WT WTSNB 19 CNS 3001 WT WTSNB-75 CNS 3001 WT WT
U373-MG CNS 3001 WT WT786-O Renal 3001 WT WTA498 Renal 3001 WT WTACHN Renal 3001 WT WTEKVX NSCLC 3001 WT WTH226 NSCLC 3001 WT WTH522 NSCLC 3001 WT WTHeLa Cervix 3001 WT WT
SK-OV-3 Ovarian 3001 WT WTLn Cap Prostate 3001 WT WT
PC3 Prostate 3001 WT WTSNU-16 Gastric 3001 WT WTFTC-133 Thyroid 3001 WT WT
Ro82-W-1 Thyroid 3001 WT WTDaudi Leukemia 3001 WT WTJijoye Leukemia 3001 WT WTJurkat Leukemia 3001 WT WTJ-82 Bladder 3001 WT WT
TCC-SUP Bladder 3001 WT WTBT-474 Breast 3001 WT WTZR-75-1 Breast 3001 WT