Supplementary Figure 1. a. b. Relative cell viability. Nature Genetics: doi: /ng SCR shyap1-1 shyap

Transcription

1 Supplementary Figure 1. a. b. p-value for depletion in vehicle (DMSO) 1e-05 1e-03 1e Genes log2 normalized shrna counts in T sh1 shluc log2 normalized shrna counts in DMSO c HCC364 A2058 BRAF mutant cell lines HT29 WiDr KHM-5M HTC/C3 A549 H2347 SW1573 MOR/CPR SK-MEL-2 MM415 HPAF-II PANC RAS mutant cell lines

2 Supplementary Figure 1 Effects of 1 inhibition on cell growth. (a) Primary screen data showing that the 1 gene was not depleted over 10 d of vehicle (DMSO) treatment in HCC364 cells (DMSO versus T0). (b) Primary screening data showing that shrnas targeting 1 were not depleted over 10 d of vehicle (DMSO) treatment in HCC364 cells (DMSO versus T0). (c) Effect of 1 knockdown using two independent shrnas on cell growth in HCC364, A2058, HT29, WiDr, KHM-5M, HTC/C3, A549, H2347, SW1573, MOR/CPR, SK-MEL-2, MM415, HPAF-II and PANC02.03 cells, measured at 3 d by CellTiter-Glo assay.

3 Supplementary Figure 2. a. b. HCC364 (BRAF V600E ) HCC364 (BRAF V600E ) c. IC 50 Vemurafenib (nm) e. IC 50 Vemurafenib (nm) g HCC364 (BRAF V600E ) shtead2-1 shtead HCC364 (BRAF V600E ) shtead4-1 shtead4-2 Pemetrexed ( M) (BRAF V600E HCC364 ) shtead2-1 shtead Vemurafenib (nm) HCC364 (BRAF V600E ) shtead4-1 shtead Vemurafenib (nm) d. f. IC 50 IC Cisplatin (nm) (BRAF V600E HCC364 ) shtead2-1 h. shtead2-2 HCC364 (BRAF V600E ) shtead4-1 shtead4-2 DMSO Vemurafenib HCC364 (BRAF V600E ) shtead2-1 shtead (BRAF V600E HCC364 ) shtead4-1 shtead Trametinib Relative mrna expression TEAD2 shtead2-1 shtead2-2 Relative mrna expression TEAD4 shtead4-1 shtead shtead shtead

4 Supplementary Figure 2 The effects of silencing were specific to targeted inhibition of RAF-MEK signaling and are through transcriptional activity. (a) Effects of 1 knockdown using two independent shrnas on pemetrexed sensitivity in HCC364 lung cancer cells. (b) Effects of 1 knockdown using two independent shrnas on cisplatin sensitivity in HCC364 lung cancer cells. (c) Effects of TEAD2 knockdown using two independent shrnas on vemurafenib sensitivity in HCC364 lung cancer cells (shown are the IC 50 and relative cell viability). (d) Validation of the effects of TEAD2 knockdown on trametinib sensitivity in HCC364 BRAF-mutant lung cancer cells (shown are the IC 50 and cell viability). (e) Effects of TEAD4 knockdown using two independent shrnas on vemurafenib sensitivity in HCC364 lung cancer cells (shown are the IC 50 and relative cell viability). (f) Validation of the effects of TEAD4 knockdown on trametinib sensitivity in HCC364 BRAF-mutant lung cancer cells (shown are the IC 50 and cell viability). (g) mrna expression of TEAD2 and TEAD4 in cells expressing scrambled control shrna or shrna to TEAD2 or TEAD4. (h) Effects of TEAD2 and TEAD4 knockdown on vemurafenib and trametinib sensitivity in HCC364 BRAF-mutant lung cancer cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition).

5 Supplementary Figure 3. a. BRAF V600E lines C N C N C N C N GAPDH (Cytosol marker) PARP (Nuclear marker) HCC364 Lung A2058 HT29 KHM-5M Melanoma Colon Thyroid b. C RAS mutant lines N C N C N C N C N GAPDH (Cytosol marker) Parp (Nuclear marker) A549 H23 MOR/CPR Miapac2 MM415 Lung Pancreas Melanoma

6 Supplementary Figure 3 Nuclear expression in BRAF- and RAS-mutant cancer cell lines. Nuclear/cytoplasmic fractionation and immunoblot analysis of the indicated proteins in (a) BRAF V600E mutant cancer cell lines and (b) RASmutant cancer cell lines. Data represent three independent experiments.

7 Supplementary Figure 4. a. IC 50 Vemurafenib (nm) HCC364 (BRAF V600E ) EV TAZ EV 1 TAZ TAZ EV 1 TAZ HCC364 (BRAF V600E ) Vemurafenib (nm) b. HCC364 (BRAF V600E ) 1000 >1000 >1000 HCC364 (BRAF V600E ) IC EV TAZ EV 1 TAZ

8 Supplementary Figure 4 Exogenous expression of or TAZ promotes resistance to RAF-MEK inhibition. (a) Effects of 1 or TAZ overexpression on vemurafenib sensitivity in HCC364 lung cancer cells (shown are the IC 50 and relative cell viability). (b) Effects of 1 or TAZ overexpression on trametinib sensitivity in HCC364 lung cancer cells (shown are the IC 50 and relative cell viability).

9 Supplementary Figure 5. a. BRAF V600E Melanoma Colon Thyroid % Maximal growth inhibition in trametinib A2058 WM793 HT29 WiDr KHM-5M HTC/C3 b. NSCLC Melanoma Pancreas A549 KRASG12S H23 KRASG12C Calu6 KRASG12C SW1573 KRASG12C MOR/CPR KRASG12C % Maximal growth inhibition in trametinib H2347 NRASQ61R MM415 NRASQ61L SK-MEL-2 NRASQ61R HPAF-II KRASG12D PANC KRASG12D

10 Supplementary Figure 5 Increase in maximal growth inhibition upon trametinib treatment in 1-depleted cells. (a) Effects of 1 knockdown on trametinib sensitivity in the indicated BRAF-mutant cell lines, shown as percentage of maximal growth inhibition (n = 4, +s.e.m. for all cell viability data shown). (b) Effects of 1 knockdown on trametinib sensitivity in the indicated RAS-mutant cell lines, shown as percentage of maximal growth inhibition (n = 4, +s.e.m. for all cell viability data shown).

11 Supplementary Figure 6. a. A2058 BRAFV600E Vemurafenib (nm) A2058 BRAFV600E A c. BRAFV600E A2058 DMSO Vemurafenib Trametinib b. BRAFV600E WM Vemurafenib (nm) sh-1 sh-2 BRAFV600E WM d. BRAFV600E WM793 DMSO Vemurafenib Trametinib e. f. g. h. HT29 BRAFV600E Vemurafenib (nm) BRAFV600E WiDr Vemurafenib (nm) BRAFV600E KHM-5M Vemurafenib (nm) BRAFV600E HTC/C Vemurafenib (nm) HT29 sh-1 sh-2 WiDr KHM-5M HTC/C3 sh-1 sh-2 BRAFV600E HT BRAFV600E WiDr BRAFV600E KHM-5M BRAFV600E HTC/C

12 Supplementary Figure 6 1 knockdown sensitizes cancer cells to RAF-MEK inhibition across multiple tumor types. (a,b) Effects of 1 knockdown using two independent shrnas on vemurafenib and trametinib sensitivity in (a) A2058 and (b) WM793 BRAF V600E mutant melanoma cancer cells. (c,d) Effects of 1 knockdown on vemurafenib and trametinib sensitivity in (c) A2058 and (d) WM793 BRAF V600E mutant melanoma cancer cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition). (e,f) Effects of 1 knockdown using two independent shrnas on vemurafenib and trametinib sensitivity in (e) HT29 and (f) WiDr BRAF V600E mutant colon cancer cells. (g,h) Effects of 1 knockdown using two independent shrnas on vemurafenib and trametinib sensitivity in (g) KHM-5M and (h) HTC/C3 BRAF V600E mutant thyroid cancer cells.

13 Supplementary Figure 7. a. Tumor volumes (mm 3 ) n.s 0 A2058 xenograft model 6 b. PLX4720 c. Fold change in tumor volume sh1 PLX4720 Trametinib sh1 Trametinib Trametinib Vemurafenib sh A2058 Xenograft tumors cl-parp d. -1 A2058 xenograft model e. f. Tumor volumes (mm 3 ) n.s 0 HT29 xenograft model Fold change in tumor volume PLX4720 sh1 PLX4720 HT29 xenograft model Trametinib sh1 Trametinib Trametinib Vemurafenib sh HT29 Xenograft tumors g. h. Fold change in tumor volume DMSO MOR/CPR xenograft model sh1 DMSO Trametinib sh1 Trametinib sh1 Trametinib MOR/CPR Xenograft tumors

14 Supplementary Figure 7 1 inhibition sensitizes tumors to RAF-MEK inhibition in vivo. (a) Average tumor volume of xenografts generated from A2058 cells infected with either scrambled control shrna or 1 shrna (n = 10/group, +s.e.m.). (b) Waterfall plot indicating fold change in tumor volume upon vemurafenib or trametinib treatment of A2058 xenografts infected with either scrambled control shrna or 1 shrna (n = 8 12/group, ±s.e.m.). (c) Immunoblot analysis for each indicated protein in lysates from representative A2058 xenograft tumors. d, Average tumor volume of xenografts generated from HT29 cells infected with either scrambled control shrna or 1 shrna (n = 10/group, +s.e.m.). (e) Waterfall plot indicating fold change in tumor volume upon vemurafenib or trametinib treatment of HT29 xenograft model infected with either scrambled control shrna or 1 shrna (n = 8 12/group, ±s.e.m.). (f) Immunoblot analysis for each indicated protein in lysates from representative HT29 xenograft tumors. (g) Waterfall plot indicating fold change in tumor volume upon trametinib treatment of MOR/CPR xenograft model infected with either scrambled control shrna or 1 shrna (n = 8 12/group, ±s.e.m.). (h) Immunoblot analysis for each indicated protein in lysates from representative MOR/CPR xenograft tumors.

15 Supplementary Figure 8. a. b. A2058 Vemurafenib +Erlotinib Vemurafenib (nm) A2058 Trametinib +Erlotinib c. d. KHM-5M Vemurafenib +Erlotinib Vemurafenib (nm) Trametinib +Erlotinib KHM-5M

16 Supplementary Figure 8 Effect of treatment with the EGFR kinase inhibitor erlotinib on RAF-MEK inhibitor sensitivity. (a,b) Effect of treatment with the EGFR kinase inhibitor erlotinib on (a) vemurafenib and (b) trametinib sensitivity in A2058 BRAF V600E mutant melanoma cells. (c,d) Effect of EGFR kinase inhibitor erlotinib on (c) vemurafenib and (d) trametinib sensitivity in KHM-5M BRAF V600E mutant thyroid cancer cells..

17 Supplementary Figure 9. a. b. NRASQ61L MM415 DMSO MM415 NRASQ61R SK-MEL-2 DMSO SK-MEL-2 c. HPAF-II e. f. NRASQ61L MM415 DMSO DMSO KRASG12D Trametinib d. KRASG12D PANC02.03 NRASQ61R SK-MEL-2 DMSO DMSO Trametinib

18 Supplementary Figure 9 1 knockdown sensitizes RAS-mutant melanoma and pancreatic cancer cells to MEK inhibition. (a,b) Effects of 1 knockdown using two independent shrnas on trametinib sensitivity in (a) MM415 NRAS Q61L mutant, (b) SK-MEL-2 NRAS Q61R mutant melanoma cells. (c,d) Effects of 1 knockdown using two independent shrnas on trametinib sensitivity in (c) HPAF-II KRAS G12D mutant and (d) Panc KRAS G12D mutant pancreatic cancer cells. (e,f) Effects of 1 knockdown using two independent shrnas on trametinib sensitivity in (e) MM415 NRAS Q61L mutant and (f) SK-MEL-2 NRAS Q61R mutant melanoma cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition).

19 Supplementary Figure 10. a. c. A549 KRASG12S sh-1 sh-2 A549 DMSO e. f. KRASG12C Calu-6 DMSO KRASG12C MOR/CPR sh-1 sh-2 Calu-6 MOR/CPR DMSO b. d. KRASG12C H23 sh-1 sh-2 H23 DMSO H2347 KRASG12C KRASG12C g. MOR/CPR h. i. SW1573 DMSO Trametinib DMSO Trametinib NRASQ61R H2347 DMSO SW1573 KRASG12C sh-1 sh-2 DMSO SW1573 NRASQ61R H2347 DMSO Trametinib

20 Supplementary Figure 10 1 knockdown sensitizes RAS-mutant NSCLC cells to MEK inhibition. (a f) Effects of 1 knockdown using two independent shrnas on trametinib sensitivity in (a) A549 KRAS G12S mutant, (b) H23 KRAS G12C mutant, (c) Calu-6 KRAS G12C mutant, (d) H2347 NRAS Q61R mutant, (e) MOR/CPR KRAS G12C mutant and (f) SW1573 KRAS G12C mutant NSCLC cell lines. (g i) Effects of 1 knockdown using two independent shrnas on trametinib sensitivity in (g) MOR/CPR KRAS G12C mutant cells, (h) SW1573 KRAS G12C mutant cells and (i) H2347 NRAS Q61R mutant cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition).

21 Supplementary Figure 11. a. c. Caspase activity/dmso Caspase activity/dmso WiDr (BRAF V600E ) WiDr ) Vemurafenib Trametinib b. d. Caspase activity/dmso Caspase activity/dmso Trametinib Trametinib (BRAF V600E A549 (KRAS G12S ) Calu-1 (KRAS G12C ) e. f. g. h. HCC364 (BRAF V600E ) A2058 (BRAF V600E ) A549 (KRAS G12S ) Calu-1 (KRAS G12C ) P<1 P<1 P<1 P<1 DMSO Vemurafenib Trametinib TW-37 Vemurafenib Trametinib +TW-37 DMSO Vemurafenib Trametinib TW-37 Vemurafenib Trametinib +TW-37 DMSO Trametinib ABT-263 TW-37 ABT-263 TW-37 +Trametinib DMSO Trametinib ABT-263 TW-37 ABT-263 TW-37 +Trametinib

22 Supplementary Figure 11 1 knockdown induces apoptosis upon RAF-MEK inhibition. (a,b) Effects of 1 knockdown on apoptosis induced upon (a) vemurafenib and (b) trametinib treatment in WiDr BRAF-mutant colon cancer cells as measured by caspase-3/7 activation. (c,d) Effects of 1 knockdown on apoptosis induced upon (c) vemurafenib and (d) trametinib treatment in A549 KRAS-mutant lung cancer cells as measured by caspase-3/7 activation. (e,f) Effects of pharmacological inhibition of BCL-xL using TW-37 on vemurafenib or trametinib sensitivity in (e) HCC364 BRAF-mutant lung cancer cells and (f) A2058 BRAF-mutant melanoma cells (n = 3, ±s.e.m. for all cell viability data shown; P values are indicated for statistical analysis). (g,h) Effects of pharmacological inhibition of BCL-xL using ABT-263 or TW-37 on trametinib sensitivity in (g) A549 KRAS G12S mutant and (h) Calu-1 KRAS G12C mutant lung cancer cells (n = 3, ±s.e.m. for all cell viability data shown; P values are indicated for statistical analysis).

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24 Supplementary Figure 12 inhibition plus RAF-MEK inhibition suppresses BCL-xL expression. (a c) Effects of suppression and vemurafenib or trametinib treatment on BCL-xL levels by immunoblot analysis in BRAF V600E mutant (a) A2058 melanoma, (b) HT29 colon and (c) KHM-5M thyroid cancer cells. Results represent three independent experiments.

25 Supplementary Figure 13. a. b. IC 50 Vemurafenib (nm) GFP + control sirna BCL-xL + control sirna HCC364 (BRAF V600E ) GFP + 1 sirna BCL-xL + 1 sirna HCC364 (BRAF V600E ) 860 HCC364 (BRAF V600E ) GFP Control + sirna BCL-xL + Control sirna GFP + 1 sirna-2 BCL-xL + 1 sirna Vemurafenib (nm) HCC364 (BRAF V600E ) IC > GFP Control + sirna BCL-xL + Control sirna GFP + 1 sirna-2 BCL-xL + 1 sirna c. 0 GFP + control sirna Vemurafenib BCL-xL + control sirna 1 sirna GFP + 1 sirna NT sirna + + GFP BCL-xL BCL-xL + 1 sirna HCC364 (BRAF V600E ) BCL-xL Parp cl-parp

26 Supplementary Figure 13 BCL-xL expression rescues 1-depleted cells from growth suppression by RAF-MEK inhibition. (a,b) Effects of BCL-xL overexpression and 1 knockdown on (a) vemurafenib and (b) trametinib sensitivity in HCC364 lung cancer cells (shown are the IC 50 and relative cell viability). (c) Immunoblot analysis for each indicated protein in lysates from HCC364 cells overexpressing either GFP or BCL-xL and treated with either control sirna or 1 sirna.

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28 Supplementary Figure 14 Gene sets enriched among the genes specifically altered by and MEK coinhibition. Pathway analysis indicating enriched gene sets among those genes whose expression was specifically decreased by and MEK coinhibition (Supplementary Table 5). The top six significantly represented pathways among these genes are shown (significance of pathway representation is shown as log q, with the red line indicating q < 5). The genes highlighted in the inset are the significantly altered apoptosis-related genes.

29 Supplementary Figure 15. a. b. staining Pre-treatment P1 Low P2 Intermediate P3 P4 High Acquired resistance

30 Supplementary Figure 15 protein expression in human tumor specimens by immunohistochemistry. (a) Representative images of immunohistochemistry staining of protein in representative human tumor specimens with low, intermediate and high staining (brown). (b) staining by immunohistochemistry in BRAF V600E mutant melanomas obtained before RAF or MEK inhibitor treatment and upon acquired resistance. Shown are the 4 out of 16 cases in which the pretreatment tumor did not harbor high levels of (showing either low or intermediate staining by immunohistochemistry). Bars represent 50 microns.

31 Supplementary Tables Supplementary Table 1. Gene targets included in the pooled shrna screening library. Supplementary Table 2. Primary screening hits, using a p-value of <05 for depletion in vemurafenib-treated cells. Gene Dropout p-value 1 0E-05 PCK1 2.00E-04 UPB1 2.00E-04 CHRNA9 3.00E-04 PLCB3 5.00E-04 SDHC 5.00E-04 WIPI1 6.00E-04 HSF2 8.00E-04 SUZ E-04 TLN1 8.00E-04 GAS6 011 SH2B2 012 NDUFA SLCO1A2 014 SLC13A5 015 HCLS1 018 BIRC5 02 MAP3K3 021 PARP1 025 MVP 028 TNFAIP3 028 NCOR1 029 SFRP2 03 PKMYT1 031 LDB1 033 TOP3B 04 GAB1 042 ST6GALNAC6 046

32 Supplementary Table 3. IC 50 of cell lines to BRAF inhibitor vemurafenib and MEK inhibitor trametinib infected with scramble shrna or 1 shrna. Cell lines Genotypes Vemurafenib IC 50 // Trametinib IC 50 // HCC364 BRAF V600E 800/100/200 10/2.1/4.03 A2058 BRAF V600E >5000/1500/ /1.5/2 WM793 BRAF V600E 3000/800/600 >1000/3/3 HT29 BRAF V600E >10000/6000/ /10/10 WiDr BRAF V600E 2500/200/700 2/0.5/0.5 KHM-5M BRAF V600E >10000/3000/5000 >1000/2.5/25 HTC/C3 BRAF V600E >5000/200/ /2/7 Cal-12T BRAF G466V 723/10.1/15.3 A549 KRAS G12S 75/30/30 H23 KRAS G12C 75/15/25 Calu-6 KRAS G12C 50/15/5 H2347 NRAS Q61R 9/2/3 SW1573 KRAS G12C >1000/200/180 MOR/CPR KRAS G12C >1000/150/150 MM415 NRAS Q61L 1.767/0.77/5 SK-MEL-2 NRAS Q61R 1.55/0.75/1 HPAF-II KRAS G12D 9/3.6/4.3 PANC KRAS G12D 40/6/15

33 Supplementary Table 4. Percentage of maximal growth inhibition upon MEK inhibitor trametinib treatment in cell lines infected with scramble shrna or 1 shrna. Cell lines Genotypes Trametinib maximal growth inhibition // HCC364 BRAF V600E 64.3%/94.6%/94.7% A2058 BRAF V600E 62%/84%/80% WM793 BRAF V600E 50%/76%/73% HT29 BRAF V600E 81%/88%/90% WiDr BRAF V600E 91%/96%/96% KHM-5M BRAF V600E 48%/85%/74% HTC/C3 BRAF V600E 54%/67%/65% Cal-12T BRAF G466V 53%/71%/72% A549 KRAS G12S 60%/82%/77% H23 KRAS G12C 68%/86%/82% Calu-6 KRAS G12C 70%/94%/85% H2347 NRAS Q61R 70%/94%/82% SW1573 KRAS G12C 42%/61%/65% MOR/CPR KRAS G12C 35%/73%/81% MM415 NRAS Q61L 89%/96%/95% SK-MEL-2 NRAS Q61R 92%/99%/95% HPAF-II KRAS G12D 89%/95%/96% PANC KRAS G12D 84%/93%/92%

34 Supplementary Table 5. Differentially expressed genes in BRAF V600E NSCLC cells (HCC364), upon either knockdown alone (by shrna), trametinib treatment alone, and combined knockdown + trametinib treatment. Gene name, fold change in expression (compared to scramble control), and adjusted p-value are listed for each gene that was significantly altered under any of the three conditions using the following significance criteria: adjusted p-value < 5 and at least 40% decreased expression or at least 66% increased expression, compared to the control. Columns H, I, J indicate whether each gene significantly altered under any condition was significantly altered in the other conditions. N = not altered. Y = altered. The genes specifically significantly altered by combined knockdown + trametinib treatment (those with N for both knockdown and trametinib treatment alone and Y for combined knockdown and trametinib) were subjected to pathway analysis (shown in Figure S15).

35 Supplementary Table 6. 1 expression as measured and scored by IHC in the human clinical specimens with complete response (CR) or incomplete response (IR, including partial response/pr and stable disease/sd) to MAPK pathway inhibitor treatment. Patients Treatment Type of responses score Cancer types 1 vemurafenib CR low melanoma 2 vemurafenib CR low melanoma 3 vemurafenib CR intermediate melanoma 4 vemurafenib CR high melanoma 5 dabrafenib + trametinib CR intermediate melanoma 6 vemurafenib CR intermediate melanoma 7 vemurafenib IR (PR) low melanoma 8 dabrafenib IR (PR) low melanoma 9 dabrafenib IR (PR) high melanoma 10 vemurafenib IR (PR) high melanoma 11 dabrafenib IR (PR) high melanoma 12 dabrafenib + trametinib IR (PR) high melanoma 13 dabrafenib IR (PR) high melanoma 14 dabrafenib + trametinib IR (PR) intermediate melanoma 15 dabrafenib + trametinib IR (PR) high melanoma 16 dabrafenib + trametinib IR (PR) high melanoma 17 dabrafenib + trametinib IR (PR) high melanoma 18 dabrafenib + trametinib IR (PR) high melanoma 19 dabrafenib + trametinib IR (PR) high melanoma 20 dabrafenib + trametinib IR (PR) high melanoma 21 dabrafenib + trametinib IR (PR) high melanoma 22 dabrafenib + trametinib IR (PR) high melanoma 23 dabrafenib + trametinib IR (SD) high melanoma 24 dabrafenib + trametinib IR (SD) high melanoma 25 dabrafenib + trametinib IR (SD) high melanoma 26 vemurafenib IR (PR) intermediate melanoma 27 vemurafenib IR (PR) high melanoma 28 vemurafenib IR (PR) high melanoma 29 vemurafenib IR (SD) high melanoma 30 vemurafenib IR (SD) high melanoma 31 LGX818+MEK162 IR (SD) intermediate melanoma 32 LGX818+MEK162 IR (SD) high melanoma 33 LGX818+MEK162 IR (PR) high melanoma 34 LGX818+MEK162 IR (PR) high melanoma 35 LGX818+MEK162 IR (PR) high melanoma 36 dabrafenib IR (PR) intermediate NSCLC 37 dabrafenib IR (PR) high NSCLC 38 dabrafenib IR (PR) high NSCLC 39 dabrafenib IR (PR) high NSCLC 40 dabrafenib IR (PR) high NSCLC

36 Supplementary Table 7. Melanoma patient information for acquired RAF-MEK inhibitor resistance analysis (average TTP of 9.9 months, consistent with prior studies 1-4 ). Patients Treatment score Time to progression Pretreatment Acquired resistance (TTP) P1 vemurafenib Low High 17 months P2 vemurafenib Intermediate High 10 months P3 vemurafenib Intermediate High 21 months P4 dabrafenib + trametinib P5 dabrafenib + trametinib Intermediate High 17 months High High 9 months P6 vemurafenib High High 2 months P7 dabrafenib + trametinib P8 dabrafenib + trametinib High High 3 months High High 2 months P9 vemurafenib High High 5 months P10 dabrafenib + trametinib High High 30 months P11 dabrafenib High High 7 months P12 dabrafenib + trametinib High High 10 months P13 vemurafenib High High 8 months P14 dabrafenib + trametinib P15 dabrafenib + trametinib High Intermediate N/A High Intermediate 3 months

37 P16 vemurafenib High Low 5 months Supplementary references 1. Flaherty, K.T. et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med 367, (2012). 2. Flaherty, K.T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 363, (2010). 3. Flaherty, K.T. et al. Improved survival with MEK inhibition in BRAFmutated melanoma. N Engl J Med 367, (2012). 4. Sosman, J.A. et al. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med 366, (2012).