Supplementary Figure S1 TdF binding assay for SCH772984, Vertex 11e and GSK1120212

ERK + 5uM GSK1120212 ERK + 5uM SCH772984

Tm = -0.15 ± 0.20 Tm = 10.3 ± 0.30

KD = NBidiNo Binding KD = 0240.24 ± 0140.14 n M

Protein + 5uM Compound + 5uM Compound Protein only Protein only

ERK + 5uM Vertex 11e MEK + 5uM GSK1120212

Tm =68= 6.8 ± 131.3 Tm =80= 8.0 ± 0510.51 K = 5.4 ± 2.2 nM D KD = 7.1 ± 2.7 nM

Protein + 5uM Compound Protein + 5uM Compound Protein only Protein only

MEK + 5uM SCH772984 MEK + 5uM Vertex 11e

Tm = -0.17 ± 0.14 Tm = -0.11 ± 0.13

KD = No Binding KD = No Binding

Protein + 5uM Compound Protein only Protein + 5uM Compound Protein only Supplementary Figure S1 (cont.)

p-ERK + 5uM GSK1120212

Tm = 0.23 ± 0.31

KD = No Binding

Protein + 5uM Compound Protein only

P-ERK + 5uM SCH772984

Tm = 9.8 ± 0.23

KD = 0.19 ± 0.12nM

Protein + 5uM Compound Protein only

p-ERK + 5uM Vertex 11e

Tm =10.3 ± 1.3

KD = 0110.11 ± 0. 062nM

Protein + 5uM Compound Protein only Supplementary Figure S2 Inhibition of BRAF inhibitor-induced of ERK by SCH772984 in HCT-116 colorectal and Calu-6 non-small lung cancer lines

Calu-6 HCT-116 PLX4720 10uM 1uM- 10uM 1uM - 10uM 1uM- 10uM 1uM - 772984 --- + + + --- + + + 300nM pERK

tERK

pMEK

tMEK

1 2 3 4 5 6 7 8 9 10 11 12 Supplementary Figure S3 Time course of pRSK & pERK suppression in A375 cells treated with SCH772984 or control (DMSO)

A375 BRAFV600E melanoma

2hrs 6hrs 16hrs 24hrs 36hrs SCH772984 2uM _ + _ + _ + _ + _ + pRSK RSK pERK ERK pCRAF CRAF pMEK MEK pBRAF

BRAF Actin Supplementary Figure S4 Supplementary Figure S5 Flow Cytometry Analysis of BRAF mutant cell lines (LOX) treated with SCH772984

A No drug

24h 48h

M1 = M1 = 0.8% 2 %

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

B SCH772984

24h 48h

M1 = M1 = 20% 9 %

0 50 100 150 200 250 0 50 100 150 200 250 Channels (FL2-A-FL2-Area) Channels (FL2-A-FL2-Area) Supplementary Figure S6 Treatment of BRAF inhibitor resistant RKO colorectal cell lines with SCH772984 or GSK1120212 (MEK inhibitor)

BRAFi-resistant, BRAF-mutant RKO colorectal

RKO RKO RKO RKO BR (Acquired NRASG12C) RKO BR (Acquired NRASG12C) RKO BR (Acquired NRASG12C) 140 140 140 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 Viability (% control)

Viability (% control) 20 20 Viability (% control) 20 0 0 0 0 -9 -8 -7 -6 -5 -4 0 -9 -8 -7 -6 -5 -4 0 -9 -8 -7 -6 -5 -4 PLX4032 log [M] GSK1120212 log [M] SCH772984 log [M] Supplementary Figure S7

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

DUSP6 MYC LIF IL8

121.2 6 hr 141.4 121.2 606.0 1.0 18 hr 1.2 1.0 5.0 24 hr 1.0 0.8 0.8 4.0 0.8 0.6 0.6 3.0 0.6

mRNA expression mRNA 0.4 0.4 2.0

e 040.4 0.2 0.2 0.2 1.0

Relativ 0.0 0.0 0.0 0.0 CH772984 CH772984 CH772984 CH772984 CH772984 CH772984 CH772984 CH772984 untreated untreated untreated untreated 375 DMSO 375 DMSO DMSO 375 DMSO 375 DMSO 375 5 PLX4032 5 PLX4032 5 PLX4032 5 PLX4032 R PLX4032 R PLX4032 R PLX4032 R PLX4032 5 5 5 5 S S S S S S S S 7 7 7 7 A A A A A3 A3 A3 A3 A37 A37 A37 A37 A375 A375 A375 A375 A375 A375B A375B A375B A375B A375BR A375BR A375BR A375BR Supplementary Figure S8 Cell lines overexpressing constitutively active MEK1 respond to SCH772984

A A375 + RFP A375 + MEK1 F129L 120 120

) 100 100

80 80

60 60

40 40 ability (% control ability i 20 20 V

0 0 0 -9 -8 -7 -6 -5 -4 0 -9 -8 -7 -6 -5 -4 PLX4032 log [M] SCH772984 log [M] B A375 + RFP A375 + MEK1 DD 120 120

100 100

80 80 control) 60 60

40 40

20 20 Viability (%

0 0 0 -9 -8 -7 -6 -5 -4 0 -9 -8 -7 -6 -5 -4 PLX4032 log [M] SCH772984 log [M] Supplementary Figure S9 Real-time proliferation assay of RKO dual resistant cell line in the presence of SCH772984

BRAFi + MEKi combination resistance in BRAF-mutant colorectal

RKO RKO A B parental BR + MR RKO parental (IncuCyte) PLX4032 (10 uM) + + + + GSK1120212 (1 uM) + + + + DMSO SCH772984 (1 uM) + ++ pMEK MEK

pERK ERKi ERK BRAFi+MEKi BRAFi+MEKi+ERKi pRSK (light)

pRSK (dark)

RSK RKO BR+MR (I ncuC yt e) GAPDH

BRAFi+MEKi DMSO C IC50 for SCH772984 in 5-day viability assay (ViaLight)

Tumor line SCH772984 IC50 (nM) ERKi RKO parental 11 BRAFi+MEKi+ERKi RKO BR+MR (with 13 PLX4032+GSK1120212)

RKO BR+MR (without 44 PLX4032+GSK1120212)

Supplementary Figure S10 Dual resistant cell lines A101D exhibit increased levels of that include BRAF, CRAF, PDGFRB and phosphorylated AKT Parental A101D A101D BR + MR

BRAF

CRAF IGF1Rβ

PDGFRB

pAKT S473 AKT Actin Supplementary Table S1 Invitro profile of SCH772984 in a panel of 309

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% 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, 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, 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 ( KHS 1), MAPK8 (J NK1), MAPK9 (J NK2), MAPKAPK2, MAPKAPK3, 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, , PLK2, PLK3, PRKACA, PRKCA, PRKCB1, PRKCB2, PRKCD, PRKCE, PRKCG, PRKCH, PRKCI, PRKCN, PRKCQ, 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, *, WNK2*, YES1, ZAK*, ZAP70 *Denotes binding assay (rather than activity assay) Supplementary Table S2

In vitro efficacy of SCH772984 in tumor cell line panel SCH 772984 Cell line Type EC50 (nM) Ras BRAF BRAF Status Malme 3M Melanoma 10 WT V600E Homozygous WM-266-4 Melanoma 20 WT V600D Heterozygous UACC-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 Heterozygous M14 Melanoma 47 WT V600E Heterozygous HT-29 Colon 50 WT V600E Heterozygous 8505C Thyroid 50 WT V600E Heterozygous WM-115 Melanoma 60 WT V600D Heterozygous HT-144 Melanoma 60 WT V600E Homozygous SK-Mel-5 Melanoma 66 WT V600E Heterozygous A375-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 Heterozygous K1 Thyroid 130 WT V600E Heterozygous Hs-695T Melanoma 165 WT V600E Heterozygous 8305C Thyroid 170 WT V600E Homozygous BHT-101 Thyroid 300 WT V600E Heterozygous RPMI-7951 Melanoma 344 WT V600E Heterozygous A2058 Melanoma 360 WT V600E Heterozygous SK-Hep-1 Liver 1422 WT V600E Heterozygous A673 Rhabdomyosarcoma 3001 WT V600E Heterozygous DBTRG-05MG CNS 3001 WT V600E Hetero SW-626 Ovarian 33 Kras G12V WT LoVo Colon 47 Kras G13D WT MiaPaCa Pancreas 53 Kras G12C WT SW-620 Colon 104 Kras G12V WT CAPAN-1 Pancreas 104 Kras G12V WT SW-527 Breast 121 Kras G12V WT HCT-116 Colon 128 Kras G13D WT SW-480 Colon 165 Kras G12V WT HPAC Pancreas 170 Kras G12D WT OVCAR-5 Ovarian 208 Kras G12V WT AsPc-1 Pancreas 270 Kras G12D WT A549 NSCLC 326 KG12SKras G12S WT SNU-1 Gastric 354 Kras G12D WT HOP62 NSCLC 676 Kras G12C WT H23 NSCLC 1000 Kras G12C WT Cal 62 Thyroid 1000 Kras G12R WT MB-231 Breast 1000 Kras G13D G464V SU.86.86 Pancreas 1001 Kras G12D WT CFPAC-1 Pancreas 1001 Kras G12V WT A427 NSCLC 1433 Kras G12D WT MDAH-2774 Ovarian 2657 Kras G12V WT NCI-H157 Lung 3000 Kras G12R WT HTB-177 NSCLC 3000 Kras Q61H WT UM-UC-3 Bladder 3001 Kras G12C WT HCT-8 Colon 3001 Kras G12D WT Panc-1 Pancreas 3001 Kras G12D WT DLD-1 Colon 3001 Kras G13D WT HCT-15 Colon 3001 Kras G13D WT HL-60 Leukemia 30 Nras Q61L WT SK-Mel-2 Melanoma 34 Nras Q61R WT RD Rhabdomyosarcoma 123 Nras Q61H WT HT-1197 Bladder 316 Nras Q61R WT Molt-3 Leukemia 600 Nras G12C - PA-1 Ovarian 1001 Nras G12D WT Molt-4 Leukemia 3001 Nras G12C WT Supplementary Table S2 (continued) Hs 700T panc/intest 50 - WT NCI-H292 Lung 90 WT WT A2780 Ovarian 143 WT WT IGROV-1 Ovarian 146 WT WT SK-N-SH CNS 150 WT WT BxPc- 3 Pancreas 184 WT WT N-87 Gastric 307 WT WT H322 NSCLC 325 WT WT H716 Colon 334 WT WT TT Thyroid 406 WT WT Caki-1 Renal 450 WT WT 5637 Bladder 610 WT WT MB-453 Breast 672 WT WT RT-4 Bladder 810 WT WT HOP92 NSCLC 820 WT WT KG-1 Leukemia 900 WT WT Hs-294T Melanoma 945 WT WT SF-539 CNS 1000 WT WT U-251 CNS 1000 WT WT MB-468 Breast 1000 WT WT HS746T Gastric 1000 - WT SCABER Bladder 1000 - WT MCF- 7 Breast 1001 WT WT CHL-1 Melanoma 1460 WT WT U87MG CNS 2000 WT WT SJCRH30 Rhabdomyosarcoma 2002 WT WT ES-2 Ovarian 2659 - WT2 HT-1376 Bladder 2800 WT WT A172 CNS 3000 WT WT 769P Renal 3000 WT WT NCI- H520 LngLung 3000 WT WT DU145 Prostate 3000 WT WT K562 Leukemia 3000 WT WT U-937 Leukemia 3000 WT WT A204 Rhabdomyosarcoma 3001 WT WT DAOY CNS 3001 WT WT SF-268 CNS 3001 WT WT SF-295 CNS 3001 WT WT SNB- 19 CNS 3001 WT WT SNB-75 CNS 3001 WT WT U373-MG CNS 3001 WT WT 786-O Renal 3001 WT WT A498 Renal 3001 WT WT ACHN Renal 3001 WT WT EKVX NSCLC 3001 WT WT H226 NSCLC 3001 WT WT H522 NSCLC 3001 WT WT HeLa Cervix 3001 WT WT SK-OV-3 Ovarian 3001 WT WT Ln Cap Prostate 3001 WT WT PC3 Prostate 3001 WT WT SNU-16 Gastric 3001 WT WT FTC-133 Thyroid 3001 WT WT Ro82-W-1 Thyroid 3001 WT WT Daudi Leukemia 3001 WT WT Jijoye Leukemia 3001 WT WT Jurkat Leukemia 3001 WT WT J-82 Bladder 3001 WT WT TCC-SUP Bladder 3001 WT WT BT-474 Breast 3001 WT WT ZR-75-1 Breast 3001 WT