supplementary material for: a secondary mutation in braf ... file⚫ supplementary figure s5:...

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.


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

H&E

3, pre

-dabra

fenib

A

pERK

4, astr

ocytic

4, sarc

om

a4, olig

o-lik

e

BRAF - Cen7

B C

F

I

E

H

D

G

J K L

Supplementary Figure S1. Wang et al. Supplemental


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 S4.


Pre-treatment Post-treatment

2

0

-2

Cop

y nu

mbe

r(lo

g ra

tio)

Alle

le-s

peci

fic(lo

g od

ds ra

tio)

Inte

ger

copy

num

ber

420

-2-4

2

0

1

3

6 7 8 9 6 7 8 9

Wang et al. Supplemental

40%35%

0

25%

WT BRAF

BRAF V600E

BRAF L514V

BRAF V600E L514V

40

50

30

20

10

0

Alle

le fr

eque

ncy

of B

RA

F

BRAF 500 NEVGVLR-KTRHVNILLFMGYSTKPQLA-IVTQWCEGSSLYHHLHIIE------------ 545514

299

1180

783

654

573

315 317

1196

798

670

589 595

1202 1203

ERBB2 769 DEAYVMA-GVGSPYVSRLLGICLTSTVQ-LVTQLMPYGCLLDHVRENR------------ 814

ALK 1166 MEALIIS-KFNHQNIVRCIGVSLQSLPRFILLELMAGGDLKSFLRETRPRP--------- 1215

ABL1 285 KEAAVMK-EIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNR----------- 332

KIT 639 SELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHA 698

NTRK1 559 REAELLT-MLQHQHIVRFFGVCTEGRPLLMVFEYMRHGDLNRFLRSHGPDAKLLA----- 612

Pre-treatment

Post-treatment

2

0

-2

Cop

y nu

mbe

r(lo

g ra

tio)

-4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 171819202122


2

0

-2

Cop

y nu

mbe

r(lo

g ra

tio)

-4



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.

Vect

orBR

AFBR

AF V

EBR

AF V

EHBR

AF L

VBR

AF L

VHBR

AF V

ELV

BRAF

VEL

VH

BRAF

ERKpERK

V5pMEK

A. B.

BRAF

ERKpERK

V5pMEK

- + - + - + - + - +

BRAF

BRAF

VE

BRAF

LV

BRAF

VEL

V

BRAF

VEL

VH

dabrafenib

BRAF

MEK1pMEK

dabrafenib

VELVVE

ATP

3 100

10 30 300

0 1 1000

+++++ +++

3 100

10 30 300

0 1 1000

+++++ +++


GFPVEVELV

0 2 4 6 8 10 12 14Time (hr)

rela

tive

pER

K (%

)

0

20

40

60

80

100

120


BRAF

ERKpERK

V5pMEK

NIH-3T3UT VE VELV

- - + + + dabrafenib

Supplementary Figure S6. Wang et al. Supplemental



Supplementary Figure S9. B.

V5pMEK

pERKERK

MEK1

FLAG

V5FLAG

IP: A

nti-V

5In

put

VE-V5

VELV-V5VE-FLAG

VELV-FLAGUT UT UT M

++--

++

--

++--

++

--

++--

++

--

MM

+ lapatinib

BRAF

Cell line

IC90

WT-

V5/F

LAG

p61

VE-V

5/FL

AGVE

-V5/

FLAG

VELV

-V5/

FLAG

IP: A

nti-V

5 In

put

Rel

ativ

e FL

AG (I

P/W

CL)

DMSO

0

0.5

1.0

1.5

2.0

2.5

VEVELV

*FLAG

V5

FLAG

V5

pERKpMEK

MEK1

A.

-

dabrafenib, nM

Cell lineA375_VE

A375_VELV

IC75 IC9011.2189.66

127.07844.08

IC500.999.52


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.


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.



30 1000

100

3000

300

0 10 1000

0

30 1000

100

3000

300

0 10 1000

0

30 1000

100

3000

300

0 10 1000

0

nM

pMEK MEK

DAB

PLX8

394

TAK

BGB3

245

BGB3

290

LY

VEVELV

VEVELV

VEVELV

VEVELV

VEVELV

VEVELV

V5

A.

log [dabrafenib], nM

Rel

ativ

e pE

RK

0

20

40

60

80

100

120

-1 0 321 4

VEVELV

VEVELV

VEVELV

log [PLX8394], nM-1 0 321 4

log [LY3009120], nM-1 0 321 4

Rel

ativ

e pE

RK

0

20

40

60

80

100

120

log [TAK632], nM-1 0 321 4

VEVELV

VEVELV

VEVELV

log [BGB3245], nM-1 0 321 4

log [BGB3290], nM-1 0 321 4

B.

Supplementary Figure S12. pMEK pERK

0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e ph

osph

oryl

atio

n le

vel

dabrafenibtrametinib

30 100 0 30 100- - + + +-

30 100 0 30 100- - + + +-

VEVELV

0 0


VEVELV

0

10

20

30

40

30 100 0 30 100- - + + +

dabrafenibtrametinib

*** ** * * **

Cel

l num

ber v

. DM

SO c

trl (%

)


Cell lineA375_VE

A375_VELV

IC75 IC90IC50

trametinib, nM

2.016.95

31.37115.78

0.130.42


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


densitometric analysis and graphed as a function of compound dose, relative to DMSO

control.

log [SCH772984], nM-3 -2 -1 0 1 2

0

20

40

60

80

100

120

-2 -1 0 1 2

log [Vemurafenib], nM-3 -2 -1 0 1 2

Rel

ativ

e ce

ll vi

abilit

y (%

)

-3 -2 -1 0 1 2

VEVELV

0

20

40

60

80

100

120

Rel

ativ

e ce

ll vi

abilit

y (%

)

-3 -2 -1 0 1 2

0

20

40

60

80

100

120 VEVELV

Rel

ativ

e ce

ll vi

abilit

y (%

)

log [BGB3245], nM-3 -2 -1 0 1 2

VEVELV

log [BGB3290], nM-3 -2 -1 0 1 2


log [PLX8394], nM-3 -2 -1 0 1 2

Rel

ativ

e ce

ll vi

abilit

y (%

)

0

20

40

60

80

100

120

-3log [TAK632], nM

log [Dabrafenib], nM

log [Trametinib], nM

A.

B.


3 3

3 3

3 3

3 3

VEVELV

VEVELV

VEVELV

VEVELV

VEVELV

RatioIC50, nMCompound

VemurafenibDabrafenib

PLX8394TAK-632BGB3245BGB3290Trametinib

SCH772984

A375_VE52.47

18.9545.28

0.99

0.13

24.49127.29

10.21

A375_VELV1222.42

1025.681314.68

9.52

0.42

20.90149.95

7.58

VELV / VE23.30

9.6254.1329.030.851.183.230.74


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.


30 1000

100

3000

300

0 10 1000

0

30 1000

100

3000

300

0 10 1000

0

nMDAB

- dox + dox

BRAFV5pMEKpERKERK

30 1000

100

3000

300

0 10 1000

0

30 1000

100

3000

300

0 10 nMBGB3245

- dox + dox

LGX818

- - - - - - - -

- - - - - - - -

+ + + + + + + +

+ + + + + + + +

A. B.

log [Dabrafenib], nM-1 0 32 41

Rel

ativ

e pE

RK

(%)

0

20

40

60

80

100

120

Rel

ativ

e pE

RK

(%)

0

20

40

60

80

100

120

log [BGB3245], nM-1 0 32 41

VEVELV 2nd site

VEVELV 2nd site


1000

0

Wang et al. Supplemental A. B.

V5

pMEK

pERK

ERK

GAPDH

- + + + + + + + + +UT DMSO

DABBGB32

45

trameti

nib

lapatinbPD03

2590

1

MEK162

SCH7729

84

GDC0994

VELVVE

VELVVE

VELVVE

VELVVE

VELVVE

ERK

V5pMEKpERK

UT

VE VELV

DMSODAB

BGB3245

SCH7729

84

trameti

nib

DMSODAB

BGB3245

SCH7729

84

trameti

nib

BRAFV5pMEKpERKERK

LGX818


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.


Supplementary Tables

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

Gene Alteration

Pre-treatment Post-treatment

Absolute reads

(variant/ total) Percent

Absolute reads

(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

mutation

Secondary

mutation

Cancer

type

Resistant

to

Sensitive to Refs.

BRAF V600E L514V Brain

tumor

Dabrafenib BGB3245 Current

study

ABL1 BCR-ABL V299L CML Imatinib/

Bosutinib/

Dasatinib

Nilotinib (5-7)

ALK EML4-ALK V1180L in vitro

screen

Alectinib/

Crizotinib

Ceritinib/

Brigatinib

(8)

ERBB2 V659E S783P in vitro

screen

Lapatinib Tesevatinib (9)

KIT exon 11

exon 9

V654A GIST Imatinib Sunitinib (10-12)

NTRK1 LMNA-

NTRK1MPRIP-

NTRK1

V573M in vitro

screen

LOXO-101 Entrectinib (13, 14)

Chronic myelogenous leukemia (CML); Gastrointestinal stromal tumor (GIST)


Table S3. BRAF L514V allele frequency determined by ddPCR.

Sample ID Type Droplet

count

Mu

Droplet

count

WT

Total

Detected

(ng)

Ratio AF Sensitivity

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


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)

https://github.com/broadinstitute/mutect

https://github.com/fimad/

http://github.com/mskcc/ngs-filters


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.


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supplementary material for: a secondary mutation in braf ... file⚫ supplementary figure s5:...

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