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

Calu-61uM 10uM 1uM

+- --- + +

-

HCT-116PLX4720

772984

10uM 1uM 10uM 1uM

+- --- + +

- 10uM

Calu-61uM 10uM 1uM

+- --- + +

-

HCT-116PLX4720

772984

10uM 1uM 10uM 1uM

+- --- + +

- 10uM

Calu-61uM 10uM 1uM

+- --- + +

-

HCT-1161uM 10uM 1uM

+- --- + +

-

HCT-116

772984 + + +300nM

+ + +

pERK

tERK

pMEK

772984 + + +300nM

+ + +

pERK

tERK

pMEK

772984 + + +300nM

+ + +

pERK

tERK

pMEK

+ + +

pERK

tERK

pMEK

tMEK

1 2 3 4 5 6 7 8 9 10 11 12

tMEK

1 2 3 4 5 6 7 8 9 10 11 12

tMEKtMEK

1 2 3 4 5 6 7 8 9 10 11 12

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

24h 48h

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

Channels (FL2-A-FL2-Area)0 50 100 150 200 250

Channels (FL2-A-FL2-Area)0 50 100 150 200 250

SCH772984B

24h 48h

M1 =20%

24h 48h

M1 =20%M1 =20%M1 =

9 %

20%M1 =9 %M1 =9 %

20%20%

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

140

RKO BR

RKO

140

RKO BR

RKO

140

RKO BR

RKO

(Acquired NRASG12C) (Acquired NRASG12C) (Acquired NRASG12C)

20

40

60

80

100

120V

iab

ility

(%

co

ntr

ol)

20

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Via

bili

ty (

% c

on

tro

l)

20

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60

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100

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Via

bili

ty (

% c

on

tro

l)

0-9 -8 -7 -6 -5 -4

SCH772984 log [M]0

0-9 -8 -7 -6 -5 -4

GSK1120212 log [M]

00

-9 -8 -7 -6 -5 -4

PLX4032 log [M]

0

Transcriptional inhibition of several ERK target genesBy SCH772984 in BRAF-mutant melanoma cell line (A375)

Supplementary Figure S7

DUSP6

1 2

MYC

1 4

LIF

1 2

IL8

6 0

DUSP6

1 2

MYC

1 4

LIF

1 2

IL8

6 0

0.4

0.6

0.8

1.0

1.2 6 hr18 hr24 hr

e m

RN

A e

xpre

ssio

n

0 4

0.6

0.8

1.0

1.2

1.4

0.4

0.6

0.8

1.0

1.2

2.0

3.0

4.0

5.0

6.0

0.4

0.6

0.8

1.0

1.2 6 hr18 hr24 hr

e m

RN

A e

xpre

ssio

n

0 4

0.6

0.8

1.0

1.2

1.4

0.4

0.6

0.8

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1.2

2.0

3.0

4.0

5.0

6.0

5 u

ntr

ea

ted

A37

5 D

MS

O

75

PL

X4

03

2

SC

H7

72

98

4

R P

LX

40

32

SC

H7

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4

0.0

0.2

Rel

ativ

e

5 u

ntr

eate

d

A3

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MS

O

75

PL

X4

03

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SC

H77

298

4

R P

LX

40

32

SC

H77

298

4

0.0

0.2

0.4

5 u

ntr

eate

d

A3

75 D

MS

O

75

PL

X4

03

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SC

H77

298

4

R P

LX

40

32

SC

H77

298

4

0.0

0.2

5 u

ntr

eate

d

A3

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MS

O

75

PL

X4

03

2

SC

H77

298

4

R P

LX

40

32

SC

H77

298

4

0.0

1.0

5 u

ntr

ea

ted

A37

5 D

MS

O

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PL

X4

03

2

SC

H7

72

98

4

R P

LX

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32

SC

H7

72

98

4

0.0

0.2

Rel

ativ

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5 u

ntr

eate

d

A3

75 D

MS

O

75

PL

X4

03

2

SC

H77

298

4

R P

LX

40

32

SC

H77

298

4

0.0

0.2

0.4

5 u

ntr

eate

d

A3

75 D

MS

O

75

PL

X4

03

2

SC

H77

298

4

R P

LX

40

32

SC

H77

298

4

0.0

0.2

5 u

ntr

eate

d

A3

75 D

MS

O

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PL

X4

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SC

H77

298

4

R P

LX

40

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SC

H77

298

4

0.0

1.0

A3

75 A

A3

7

A3

75

S

A3

75

B

A3

75

BR

S

A3

75 A

A37

A37

5 S

A3

75B

A3

75B

R S

A3

75 A

A37

A37

5 S

A3

75B

A3

75B

R S

A3

75 A

A37

A37

5 S

A3

75B

A3

75B

R S

A3

75 A

A3

7

A3

75

S

A3

75

B

A3

75

BR

S

A3

75 A

A37

A37

5 S

A3

75B

A3

75B

R S

A3

75 A

A37

A37

5 S

A3

75B

A3

75B

R S

A3

75 A

A37

A37

5 S

A3

75B

A3

75B

R S

Supplementary Figure S8Cell lines overexpressing constitutively active MEK1 respond to SCH772984

120120

A375 + RFPA375 + MEK1 F129L

)

A

20

40

60

80

100

20

40

60

80

100

iab

ility

(%

co

ntr

ol)

B

0

20

-9 -8 -7 -6 -5 -40

SCH772984 log [M]

0

20

-9 -8 -7 -6 -5 -40

PLX4032 log [M]

Vi

80

100

120A375 + MEK1 DD

A375 + RFP

con

tro

l)

80

100

120

0

20

40

60

-9 -8 -7 -6 -5 -40

Via

bili

ty (

%

0

20

40

60

-9 -8 -7 -6 -5 -40PLX4032 log [M]

0

SCH772984 log [M]

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

BA RKOparental

RKOBR + MRBA

RKO parental (IncuCyte)

DMSO

pMEK

MEK

PLX4032 (10 uM)

GSK1120212 (1 uM)

SCH772984 (1 uM)

++

++

+

++

+ +

++

parental BR + MR

ERKi

BRAFi+MEKi+ERKiBRAFi+MEKi

pERK

ERK

pRSK (light)

pRSK (dark)

RSK

GAPDHRKO BR+MR (I C t )

CDMSO

ERKi

BRAFi+MEKi

GAPDHRKO BR+MR (IncuCyte)

IC50 for SCH772984 in 5-day viability assay (ViaLight) Tumor line SCH772984 IC50 (nM)

RKO parental 11BRAFi+MEKi+ERKi

RKO parental 11

RKO BR+MR (with PLX4032+GSK1120212)

13

RKO BR+MR (without PLX4032+GSK1120212)

44

Supplementary Figure S10Dual resistant cell lines A101D exhibit increased levels of proteins that include BRAF, CRAF, PDGFRB and phosphorylated AKT

Pare

ntal

A10

1D

A10

1D B

R +

MR

PDGFRB

BRAF

IGF1RβCRAF

pAKT S473

AKT

Actin

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