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 Protein + 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 phosphorylation of ERK by SCH772984 in HCT-116 colorectal and Calu-6 non-small cell 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] Relative mRNA expression 0.0 0.2 0.4 0.6 0.8 1.0 121 . 2 By SCH772984 in BRAF-mutan in SCH772984 By A375 untreated Transcriptional inhibitionof several ERK target genes Transcriptional DUSP6 A375 DMSO A375 PLX4032 A375 SCH772984 A375BR PLX4032 24 hr 18 hr 6 hr A375BR SCH772984 Supplementary Figure S7 0.0 0.2 040 0.6 0.8 1.0 1.2 141 . 4 4 A375 untreated A375 DMSO MYC A375 PLX4032 A375 SCH772984 A375BR PLX4032 A375BR SCH772984 t melanomacellline(A375) 121 0.0 0.2 0.4 0.6 0.8 1.0 . 2 A375 untreated A375 DMSO LIF A375 PLX4032 A375 SCH772984 A375BR PLX4032 A375BR SCH772984 606 0.0 1.0 2.0 3.0 4.0 5.0 . 0 A375 untreated A375 DMSO IL8 A375 PLX4032 A375 SCH772984 A375BR PLX4032 A375BR SCH772984 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 proteins 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 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% 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, PLK1, 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, WEE1*, 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