Supplementary Material For

Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Supplementary Material for:

A secondary mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor

Jiawan Wang, Zhan Yao, Philip Jonsson, Amy N. Allen, Alice Can Ran Qin, Sharmeen Uddin, Ira J. Dunkel, Mary Petriccione, Katia Manova, Sofia Haque, Marc K. Rosenblum, David J. Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas

Supplementary Figures

⚫ Supplementary Figure S1: Representative H&E, phospho-ERK and FISH images of pre-dabrafenib (3) and post-dabrafenib (4) tumors. ⚫ Supplementary Figure S2: WES analysis of copy number variation in pre- and post- dabrafenib tumors. ⚫ Supplementary Figure S3: Whole copy number profiles from WES of pre- and post- treatment tumors. ⚫ Supplementary Figure S4: Homology alignment of BRAF p. L514 with residues in other tyrosine kinases. ⚫ Supplementary Figure S5: Relative frequency of BRAF variant alleles in SK-BT-DR cells determined by individual clone sequencing. ⚫ Supplementary Figure S6: BRAF V600E/L514V reduces dabrafenib sensitivity in NIH- 3T3 cells, related to Figure 2. ⚫ Supplementary Figure S7: BRAF V600E/L514V confers biochemical resistance to dabrafenib over a time course, related to Figure 2G. ⚫ Supplementary Figure S8: Comparison of IC50, IC75 and IC90 of dabrafenib against A375 cells expressing BRAF V600E and BRAF V600E/L514V, related to Figure 2H. ⚫ Supplementary Figure S9: The BRAF V600E/L514V double mutant promotes homodimerization, related to Figure 3A and B. ⚫ Supplementary Figure S10: BRAF L514V alone is hypoactive and associated with decreased ERK signaling that is not sensitive to dabrafenib. ⚫ Supplementary Figure S11: BRAF V600E/L514V is inhibited by dabrafenib in a purified kinase assay, indicating that it is not a gatekeeper mutation. ⚫ Supplementary Figure S12: Quantitation of p-MEK and p-ERK immunoblots, related to Figure 4B. ⚫ Supplementary Figure S13: Comparison of IC50, IC75 and IC90 of trametinib against A375 expressing BRAF V600E and BRAF V600E/L514V, related to Figure 4C. Wang et al. Supplemental: Acquired resistance mutation in BRAF.

 Supplementary Figure S14: The BRAF V600E/L514V mutant mediates resistance to dabrafenib that cannot be completely overcome by trametinib or dabrafenib plus trametinib, related to Figure 4D.  Supplementary Figure S15: V5, p-MEK, total MEK immunoblots, and quantitation of p-ERK immunoblots, related to Figure 5A.  Supplementary Figure S16: Novel RAF dimer inhibitors, MEK inhibitor and ERK inhibitor equipotently inhibit cell growth in BRAF V600E and V600E/L514V expressing cells.  Supplementary Figure S17: Novel RAF dimer inhibitor, MEK inhibitor and ERK inhibitor equipotently inhibit ERK signaling in BRAF V600E and V600E/L514V expressing cells.  Supplementary Figure S18: BGB3245 binds mutant BRAF V600E monomer and second site of V600E/L514V dimer with similar affinity.

Supplementary Tables

 Supplementary Table S1: Mutations identified by WES of pre-dabrafenib and post-

dabrafenib tumors.

 Supplementary Table S2: Secondary mutations associated with acquired resistance

and occurring in residues homologous with L514 in BRAF.

 Supplementary Table S3: BRAF L514V allele frequency determined by ddPCR.

Supplementary Methods

Supplementary References

Supplementary Figure S1. Wang et al. Supplemental

H&E pERK BRAF - Cen7 A B C 3, pre-dabrafenib

D E F 4, astrocytic

G H I 4, sarcoma

JK L 4, oligo-like Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Fig. S1. Representative H&E, phospho-ERK and FISH images of pre-dabrafenib (3) and post-dabrafenib (4) tumors.

The post-dabrafenib tumor was comprised of three distinct histologic areas, including astrocytic, rhabdomyosarcomatous, and oligo-like, shown separately. A, D, G and J. H&E images. B, E, H and K. Representative images from immunohistochemical detection of phospho-ERK. C, F, I and L. 3-color BRAF/Cen7 fluorescence in situ hybridization (FISH) detection of BRAF. 3’ BRAF = Green; 5’ BRAF = Red. A centromeric repeat plasmid for chromosome 7 served as the control (Blue). Scale bars = 30 μm. Supplementary Figure S2. Wang et al. Supplemental Pre-treatment Post-treatment 2

(log ratio) -2 Copy number 4 2 0 -2

Allele-specific -4 (log odds ratio) 3 2 1 Integer 0 copy number 6 7 8 9 6 7 8 9 Supplementary Figure S3. Pre-treatment 2 0 -2 (log ratio)

Copy number -4 Post-treatment 2 0 -2 (log ratio)

Copy number -4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 171819202122 Supplementary Figure S4. 514 BRAF 500 NEVGVLR-KTRHVNILLFMGYSTKPQLA-IVTQWCEGSSLYHHLHIIE------545 299 315 317 ABL1 285 KEAAVMK-EIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNR------332 1180 1196 1202 1203 ALK 1166 MEALIIS-KFNHQNIVRCIGVSLQSLPRFILLELMAGGDLKSFLRETRPRP------1215 783 798 ERBB2 769 DEAYVMA-GVGSPYVSRLLGICLTSTVQ-LVTQLMPYGCLLDHVRENR------814 654 670 KIT 639 SELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHA 698 573 589 595 NTRK1 559 REAELLT-MLQHQHIVRFFGVCTEGRPLLMVFEYMRHGDLNRFLRSHGPDAKLLA----- 612 Supplementary Figure S5. 50

40% 40 35% BRAF

30 25%

10 Allele frequency of

0 0 WT BRAF BRAF BRAF BRAF V600E L514V V600E L514V Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Fig. S2. WES analysis of copy number variation in pre- and post-dabrafenib tumors.

Total, allele-specific, and integer copy number data (top, middle, and bottom) from pre- and post-treatment WES data as indicated (left and right, respectively), showing chromosomes 6-9. The BRAF locus is indicated with a green line.

Fig. S3. Whole copy number profiles from WES of pre- and post- treatment tumors.

Total copy number data from pre- (top) and post- (bottom) treatment tumors, showing chromosomes 1 – 22.

Fig. S4. Homology alignment of BRAF p. L514 with residues in other tyrosine kinases.

Alignment of the relevant BRAF sequence with that of five other tyrosine kinase targets in which secondary mutations occur as major mechanisms of acquired resistance. L514 is homologous to the amino acid changes associated with acquired resistance as shown.

References: ABL1: (5-7); ALK: (8); ErbB2: (9); KIT: (10-12); NTRK1: (13, 14).

Fig. S5. Relative frequency of BRAF variant alleles in SK-BT-DR cells determined by individual clone sequencing.

Twenty individual clones were sequenced. The frequency of wild-type BRAF and variant alleles (V600E, L514V, or V600E/L514V) is shown as a percent of total.

Supplementary Supplementary S6. Supplementary Figure FigureS11.Supplementary Supplementary A. Input IP: Anti-V5 A. + - A375_VELV 0 UT

Vector WT-V5/FLAG A375_VE Cell line + Cell line + 1 BRAF p61 VE-V5/FLAG NIH-3T3 - + 3 BRAF VE VE-V5/FLAG VE + VE 10 BRAF VEH VELV-V5/FLAG + VELV - MEK1 pERK pMEK V5 FLAG V5 FLAG + 30 BRAF LV + 100 BRAF LVH + FigureS9. FigureS10. ERK pERK pMEK V5 BRAF dabrafenib + 300 FigureS8. IC50 BRAF VELV 9.52 0.99 + 1000 Relative FLAG (IP/WCL)

BRAF VELVH 0.5 1.0 1.5 2.0 2.5 ERK pERK pMEK V5 BRAF +

0 0

+ 1

VE dabrafenib, nM DMSO +

VELV 3 *

+ VELV IC90 89.66 11.21

10 IC75 + 30 + 100

+ 300 B. B. Supplementary

+ 1000 relative pERK (%) 100 120 MEK1 pMEK BRAF dabrafenib ATP 20 40 60 80 844.08 127.07 0 Input IP: Anti-V5 IC90 0 UT - +

BRAF + + - - 2 M - + + - - + lapatinib UT

+ BRAF VE 4 - + + FigureS7. - - M Time (hr) + + + 6 BRAF LV - - UT - 8 + + +

BRAF VELV - - M - + + - - 10 BRAF VELV-FLAG VELV-V5 VE-FLAG VE-V5 ERK pERK MEK1 pMEK V5 FLAG V5 FLAG + BRAF VELVH dabrafenib ERK pERK pMEK V5 BRAF 12 Wang etal.Supplemental VELV VE GFP 14 Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Fig. S6. BRAF V600E/L514V reduces dabrafenib sensitivity in NIH-3T3 cells, related to Figure 2.

NIH-3T3 cells were transfected with BRAF mutants (V600E or V600E/L514V) for 24 hr, followed by treatment with dabrafenib (300 nM, or DMSO as control) for 1 hr.

Untransfected (UT). The indicated proteins were assessed by immunoblot.

Fig. S7. BRAF V600E/L514V confers biochemical resistance to dabrafenib over a time course, related to Figure 2G.

A375 cells expressing doxycycline-inducible BRAF V600E or BRAF V600E/ L514V (or

GFP as control) were treated with dabrafenib (100nM) over a time course as shown.

Phospho-ERK (p-ERK) (detected by immunoblot, Figure 2G) was quantitated by densitometry using Image J, and is shown as a percent of baseline (time = 0 hr) as a function of time.

Fig. S8. Comparison of IC50, IC75 and IC90 of dabrafenib against A375 cells expressing BRAF V600E and BRAF V600E/L514V, related to Figure 2H.

IC50, IC75 and IC90 values were calculated by CompuSyn software.

Fig. S9. The BRAF V600E/L514V double mutant promotes homodimerization, related to Figure 3A and B.

SKBR3 cells expressing WT BRAF and indicated tagged BRAF mutants were treated with

DMSO (A) or lapatinib (B) for 1 hr. The interaction between V5-tagged and FLAG-tagged

BRAF mutants were determined by immunoprecipitation (IP), followed by immunoblotting for the indicated proteins. Untransfected (UT); Whole cell lysate (WCL); Medium (M) indicates medium level of tagged construct expression, as shown in Figure 3B. Relative

FLAG signal (IP/WCL) for BRAF V600E and V600E/L514V mutants in S9A was quantitated by Image J and shown as bar graph with statistical analysis to indicate replicate experiments. Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Fig. S10. BRAF L514V alone is hypoactive and associated with decreased ERK signaling that is not sensitive to dabrafenib.

A-B. SKBR3 cells transiently transfected with the indicated plasmids were treated with lapatinib (1 μM) for 1 hr (A and B) and then treated with dabrafenib (200nM) for 1 hr (B only). Proteins as indicated were detected by immunoblotting. BRAF V600E (VE);

V600E/R509H (VEH); L514V (LV); L514V/R509H (LVH); V600E/L514V (VELV);

V600E/L514V/R509H (VELVH).

Fig. S11. BRAF V600E/L514V is inhibited by dabrafenib in a purified kinase assay, indicating that it is not a gatekeeper mutation.

293H cells expressing V5-tagged BRAF V600E or V600E L514V were treated with dabrafenib, doses as shown. BRAF mutants were immunoprecipitated from whole-cell lysates and subjected to a kinase assay using an inactive MEK1 (K97R, kinase-dead) as the substrate in the presence of ATP Kinase activity of BRAF mutants was determined by immunoblotting with a phospho-MEK antibody.

Supplementary Figure S12. Wang et al. Supplemental pMEK pERK 1.2 VE 1.0 VELV 0.8 0.6 0.4 0.2

Relative phosphorylation level 0 0 30 100 0 30 100 0 30 100 0 30 100 dabrafenib - - - + + + - - - + + + trametinib Supplementary Figure S13. Supplementary Figure S14.

trametinib, nM 40 VE Cell line IC50 IC75 IC90 VELV A375_VE 0.13 2.01 31.37 30 *** ** * * ** A375_VELV 0.42 6.95 115.78 20 Supplementary Figure S15. 10 A. 0 nM DMSO ctrl (%) Cell number v. 0 10 30 100 300 1000 3000 10000 0 10 30 100 300 1000 3000 10000 0 10 30 100 300 1000 3000 10000 30 100 0 30 100 dabrafenib VE - - + + + trametinib

VELV DAB VE

VELV PLX8394 VE

VELV LY VE

VELV TAK VE

VELV BGB3245 VE

VELV BGB3290 V5 pMEK MEK B. 120 VE VE VE 100 VELV VELV VELV 80 60 40 Relative pERK 20 0 -1 0 1 2 3 4 -1 0 1 2 3 4 -1 0 1 2 3 4 log [dabrafenib], nM log [PLX8394], nM log [LY3009120], nM 120 VE VE VE 100 VELV VELV VELV 80 60 40 Relative pERK 20 0 -1 0 1 2 3 4 -1 0 1 2 3 4 -1 0 1 2 3 4 log [TAK632], nM log [BGB3245], nM log [BGB3290], nM Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Fig. S12. Quantitation of p-MEK and p-ERK immunoblots, related to Figure 4B. p-MEK and p-ERK signal intensity in Figure 4B was quantitated using densitometric analysis, relative to DMSO control.

Fig. S13. Comparison of IC50, IC75 and IC90 of trametinib against A375 expressing

BRAF V600E and BRAF V600E/L514V, related to Figure 4C.

IC50, IC75 and IC90 values were calculated by CompuSyn software.

Fig. S14. The BRAF V600E/L514V mutant mediates resistance to dabrafenib that cannot be completely overcome by trametinib or dabrafenib plus trametinib, related to Figure 4D.

A375 transduced with BRAF VE or VELV were exposed to drug or to DMSO, and cell counts were measured at 72 hr. Cells were seeded at a density of 60,000 cells/well in 6- well plates for 24 hr in doxycycline-containing medium and then treated with the indicated drugs for 72 hr, in triplicate for each condition shown. Doxycycline was replenished at 50% every 24 hr. Cells were collected by trypsinization and counted using a BioRad TC20

Automated Cell Counter. Average number of viable cells at 72 hr is expressed as percent relative to DMSO control for each cell line (data represent mean ± SEM, unpaired

Student’s t-test).

Fig. S15. V5, p-MEK, total MEK immunoblots, and quantitation of p-ERK immunoblots, related to Figure 5A.

A. A375 cells expressing BRAF mutants (V600E or V600E/L514V) were treated with the indicated compounds for 1 hr, doses as shown, then the cells were lysed and subjected to immunoblotting. V5, phospho-MEK, and total MEK blots are shown, and correspond to phospho-ERK and total ERK blots shown in Figure 5A. Dabrafenib (DAB); LY3009120

(LY); TAK-632 (TAK). B. p-ERK signal intensity in Figure 5A was quantitated using Wang et al. Supplemental: Acquired resistance mutation in BRAF. densitometric analysis and graphed as a function of compound dose, relative to DMSO control.

Supplementary Figure S16. Wang et al. Supplemental A. 120 VE VE 100 VELV VELV 80 60 40 20 Relative cell viability (%) 0 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 log [Vemurafenib], nM log [Dabrafenib], nM

120 VE VE VELV VELV 100 80 60 40 20

Relative cell viability (%) 0 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 log [PLX8394], nM log [TAK632], nM

120 VE VE 100 VELV VELV 80 60 40 20 Relative cell viability (%) 0 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 log [BGB3245], nM log [BGB3290], nM

120 VE VE 100 VELV VELV 80 60 40 20 Relative cell viability (%) 0 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 log [Trametinib], nM log [SCH772984], nM

B. IC50, nM Ratio Compound A375_VE A375_VELV VELV / VE Vemurafenib 52.47 1222.42 23.30 Dabrafenib 0.99 9.52 9.62 PLX8394 18.95 1025.68 54.13 TAK-632 45.28 1314.68 29.03 BGB3245 24.49 20.90 0.85 BGB3290 127.29 149.95 1.18 Trametinib 0.13 0.42 3.23 SCH772984 10.21 7.58 0.74 Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Fig. S16. Novel RAF dimer inhibitors, MEK inhibitor and ERK inhibitor equipotently inhibit cell growth in BRAF V600E and V600E/L514V expressing cells.

A. A375 cells expressing doxycycline-inducible BRAF mutants (V600E or V600E/L514V) were seeded in 96-well plates at a density of 1500 cells/well and cultured in medium containing doxycycline (200 ng/ml and 150 ng/ml, respectively) for 24 hr. The following day, the cells were treated with the indicated compounds at concentrations shown for another 72 hr. The relative cell viability was quantitated using cell counting kit 8 (CCK-8) on day 3, and are expressed relative to DMSO controls. Doxycycline was replenished at

50% every 24 hr. Data represent mean ± SEM. B. IC50 values of compounds against VE and VELV cell growth were determined by CompuSyn software and are expressed as a ratio of VELV / VE.

Supplementary Figure S17. Wang et al. Supplemental A. B.

UT DMSODABBGB3245trametinibPD0325901MEK162SCHGDC0994772984 - + + + + + + + + + lapatinb UT DMSODABBGB3245trametinibSCH772DMSO984DABBGB3245trametinibSCH772984 VE V5 V5 VELV pMEK VE pERK pMEK VELV ERK VE pERK VE VELV VELV VE ERK VELV VE GAPDH VELV

Supplementary Figure S18. A. B. - dox + dox 120 VE VELV 2nd site 0 10 30 100 300 1000 3000 10000 0 10 30 100 300 1000 3000 10000DAB nM 100 ------+ + + + + + + + LGX818 80 BRAF V5 60 pMEK 40 pERK 20 Relative pERK (%) ERK 0 -1 0 1 2 3 4 - dox + dox log [Dabrafenib], nM 120 VE VELV 2nd site 0 10 30 100 300 1000 3000 10000 0 10 30 100 300 1000 3000 10000BGB3245 nM 100 ------+ + + + + + + + LGX818 80 BRAF V5 60 pMEK 40 pERK 20 Relative pERK (%) ERK 0 -1 0 1 2 3 4 log [BGB3245], nM Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Fig. S17. Novel RAF dimer inhibitor, MEK inhibitor and ERK inhibitor equipotently inhibit ERK signaling in BRAF V600E and V600E/L514V expressing cells.

A. Transiently transfected SKBR3 cells expressing BRAF VE or VELV mutants were pretreated with lapatinib for 1 hr and then treated with DMSO, RAF inhibitors (dabrafenib and BGB3245), MEK inhibitors (trametinib, PD0325901 and MEK162) and ERK inhibitors

(SCH772984 and GDC-0994) for 1 hr. Untransfected (UT) cells treated with or without lapatinib served as control. The indicated proteins were detected by immunoblotting. B.

DBTRG-05MG cells transfected with V5-tagged BRAF VE or VELV were treated with

DMSO or the indicated compounds for 1 hr, with untransfected (UT) cells as control. The indicated proteins were evaluated by immunoblotting.

Fig. S18. BGB3245 binds mutant BRAF V600E monomer and second site of

V600E/L514V dimer with similar affinity.

A. A375 cells expressing doxycycline-inducible BRAF V600E/L514V, either with or without exposure to doxycycline, were pretreated with 1 μM LGX818 (encorafenib) for 1 hr followed by three washes with fresh drug-free medium, and then treated with increasing doses of dabrafenib or BGB3245 for 1 hr. The indicated proteins were assessed by immunoblot. B. p-ERK was quantitated by densitometry using Image J, and is shown as a percent of baseline (DMSO control) as a function of drug dose.

Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Supplementary Tables

Table S1. Mutations identified by WES of pre-dabrafenib and post-dabrafenib tumors.

Pre-treatment Post-treatment Gene Alteration Absolute reads Absolute reads (variant/ total) Percent (variant/ total) Percent BRAF V600E 40/103 38.8% 47/78 60.3%

BRAF L514V 0/151 0% 37/140 26.4%

SMAD4 G510R 58/167 34.7% 63/156 40.4%

FLT1 G1009S 11/55 20.0% 28/60 46.7%

CDK8 R40G 73/182 40.1% 79/184 42.9%

ATM E1428Kfs*23 25/43 58.1% 22/29 75.9%

ABL1 A366D 36/80 45% 29/66 43.9%

Table S2. Secondary mutations associated with acquired resistance and occurring in residues homologous with L514 in BRAF.

Gene Primary Secondary Cancer Resistant Sensitive to Refs. mutation mutation type to BRAF V600E L514V Brain Dabrafenib BGB3245 Current tumor study ABL1 BCR-ABL V299L CML Imatinib/ Nilotinib (5-7) Bosutinib/ Dasatinib ALK EML4-ALK V1180L in vitro Alectinib/ Ceritinib/ (8) screen Crizotinib Brigatinib ERBB2 V659E S783P in vitro Lapatinib Tesevatinib (9) screen KIT exon 11 V654A GIST Imatinib Sunitinib (10-12) exon 9 NTRK1 LMNA- V573M in vitro LOXO-101 Entrectinib (13, 14) NTRK1MPRIP- screen NTRK1 Chronic myelogenous leukemia (CML); Gastrointestinal stromal tumor (GIST) Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Table S3. BRAF L514V allele frequency determined by ddPCR.

Sample ID Type Droplet Droplet Total Ratio AF Sensitivity count count Detected Mu WT (ng)

pre-dabrafenib Pre-treatment 0 6075 30.162 0.0000 0.0000 0.016461 post-dabrafenib Post-treatment 1881 4259 31.746 0.4417 0.3064 0.016287

Blood Normal blood 0 198 0.8712 0.0000 0.0000 0.505051

SK-BT-DR Cell line 1624 5239 32.7492 0.3100 0.2366 0.014571

Water -- 0 0 0 ------

gDNA -- 0 1972 8.844 0.0000 0.0000 0.050710

Wang et al. Supplemental: Acquired resistance mutation in BRAF.

Supplementary Methods

Whole-exome sequencing. Sample preparation, processing, read alignment and variant calling, were performed as previously described (1). Exome capture was performed using

SureSelect XT Human All Exon V4 (Agilent Technologies). Mean target coverage was in the range of 119-139X for all sequenced specimens. Reads were aligned to the human genome (hg19) with Burrows-Wheeler Aligner (BWA-MEM, version 0.7.15) (2) and subsequently processed using the Picard and GATK suites for de-duplication and base recalibration. Single-nucleotide variants and indels were detected using MuTect (version

2.7.1, https://github.com/broadinstitute/mutect) and scalpel (version 0.2.1, https://github.com/fimad/scalpel), respectively. Putative artifacts were removed using filters adapted to our sequencing pipeline (http://github.com/mskcc/ngs-filters). Allele- specific DNA copy number analysis was performed using FACETS (3). Tumor purity and ploidy estimates from FACETS analysis were used to infer the cancer cell fraction (CCF) of mutations using a maximum-likelihood, as previously described (4).

Immunohistochemistry. The immunohistochemical detection of phospho-ERK was performed by the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer

Center using Discovery XT processor (Ventana Medical Systems, Roche). Tissue sections were blocked for 30 minutes in 10% normal goat serum. Slides were incubated with primary antibody (rabbit monoclonal phospho-ERK antibody (Cell Signaling, #4370) at 1 μg/ml in PBS with 2% BSA for 3 hours, followed by 60 minutes incubation with 5.75

μg/mL biotinylated goat anti-rabbit IgG (Vector Laboratories, cat# PK6101). Detection was performed with Streptavidin-HRP D (DABMap kit, Ventana Medical Systems, Roche).

Fluorescence in situ hybridization. The short-term culture SK-BT-DR was harvested and fixed in methanol: acetic acid (3:1) as per standard procedures. FISH was performed on fixed single cell suspension and formalin fixed paraffin embedded (FFPE) sections using an in-house 3-color BRAF/Cen7 probe. Bacterial artificial chromosome (BAC) Wang et al. Supplemental: Acquired resistance mutation in BRAF. clones were selected from the University of California–Santa Cruz genome browser and purchased through Roswell Park Cancer Institute (RPCI). BAC clones mapping to 3’BRAF

(RP11-759K14, RP11-788O6) were labeled with Green dUTP and 5’BRAF (RP11-715H9,

RP11-133N19) were labeled with Red dUTP. A centromeric repeat plasmid for chromosome 7 served as the control (clone p7t1; labeled with DEAC dUTP). Probe labeling, tissue processing, hybridization, post-hybridization washing, and fluorescence detection were performed according to standard laboratory procedures. Slides were scanned using a Zeiss Axioplan 2i epifluorescence microscope equipped with a megapixel

CCD camera (CV-M4+CL, JAI) controlled by Isis 5.5.9 imaging software (MetaSystems

Group Inc). For the short-term cell culture SK-BT-DR, the entire hybridized area was scanned through 63X or 100X objective, representative cells/regions imaged and a minimum of 50 discrete nuclei and 20 metaphases analyzed. For paraffin tissue, marked region(s) within the section was scanned under 63X or 100X objective and representative regions imaged through the depth of the tissue (compressed/merged stack of 12 z-section images taken at 0.5-micron intervals). At least five images per representative region were captured and a minimum of 50 discrete nuclei analyzed. Amplification was defined as a

BRAF/Cen7 (control) ratio of ≥2.0, >10 copies of BRAF (independent of control locus) or at least one small cluster of BRAF (≥4 signals resulting from tandem repeat/duplication).

Cells with 3~5 and 6~10 discrete copies of BRAF/Cen7 were considered to be polysomic and high-polysomic, respectively.

Mutation status and allelic distribution of BRAF in SK-BT-DR cells by individual clone sequencing. RNA was extracted from SK-BT-DR cells, and reverse transcribed per standard protocols. BRAF coding sequences were amplified from the cDNA and the

PCR products were inserted into T-vector by TA cloning. Bacteria competent cells

(Agilent) were transformed by the plasmids carrying BRAF gene. 20 random individual colonies were isolated and sequenced. Wang et al. Supplemental: Acquired resistance mutation in BRAF.

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