supplementary materials for - science translational …...2012/05/25 · sen and peng, supplemental...

www.sciencetranslationalmedicine.org/cgi/content/full/4/136/136ra70/DC1

Supplementary Materials for

Kinase-Impaired BRAF Mutations in Lung Cancer Confer Sensitivity to Dasatinib

Banibrata Sen, Shaohua Peng, Ximing Tang, Heidi S. Erickson, Hector Galindo, Tuhina

Mazumdar, David J. Stewart, Ignacio Wistuba, Faye M. Johnson*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 30 May 2012, Sci. Transl. Med. 4, 136ra70 (2012) DOI: 10.1126/scitranslmed.3003513

The PDF file includes:

Methods Fig. S1. Radiographic images of PX’s primary lung tumor and paraspinal metastasis and overall survival. Fig. S2. Gene copy number variation assessed across all chromosomes in PX’s metastatic lymph node. Fig. S3. Sequencing chromatograms demonstrate BRAF mutations. Fig. S4. Alignment of the human BRAF amino acid sequence. Fig. S5. NSCLC cells with kinase-inactive BRAF mutations did not undergo apoptosis. Fig. S6. Cells with inactive BRAF undergo senescence in the presence of dasatinib. Fig. S7. H1666 cells undergo irreversible senescence in the presence of dasatinib at 72 hours. Fig. S8. H661 cells transfected with inactivating BRAF mutations show increased sensitivity to dasatinib. Fig. S9. Transfection with mutant or wild-type BRAF does not affect cell number. Fig. S10. NSCLC cells with an inactivating BRAF mutation are sensitive to CRAF knockdown. Fig. S11. Kinase inhibitor–induced RAF dimerization does not result in drug sensitivity or senescence. Fig. S12. Dasatinib does not have any BRAF mutation–specific changes in BAD or JNK phosphorylation. Fig. S13. Inhibition of c-Src does not affect cell viability in NSCLC harboring a kinase-inactive BRAF mutation. Fig. S14. Dasatinib enhances the cytotoxicity of sorafenib in cancer cells resistant to dasatinib. Table S1. Immunohistochemistry scores for PX’s tumor.

Table S2. Genes analyzed using mass spectroscopy single-nucleotide polymorphism analysis. Table S3. Copy number variation for genes associated with dasatinib targeting and/or NSCLC. Table S4. Mutational statuses of patients with NSCLC treated with dasatinib.

Sen and Peng, Supplemental Tables and Figure Legends, page 1

Supplemental Methods

Cell Culture

Cal12T cells were purchased from Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH. All other human NSCLC cell lines were obtained from Drs. John

Heymach (The University of Texas MD Anderson Cancer Center) and John Minna (The

University of Texas Southwestern Medical Center) and maintained using standard cell

culture techniques (30). COS7 monkey kidney cells were obtained from Dr. Ho-Young

Lee (MD Anderson). All cell lines were validated by cross-comparing their allelic short

tandem repeat patterns generated using the PowerPlex 1.2 system (PromegI) with the

American Type Culture Collection repository database. BRAF mutations were confirmed

using Sanger sequencing of exons 11 and 15.

Immunofluorescence microscopy

Cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with

0.5% NP40 for 10 min at room temperature. Cover slips were blocked in 10% normal

goat serum in PBS for 30 min, and incubated with the anti-HP1γ antibody (1:200

dilution). Cells were then washed and incubated with the anti-Alexa Fluor 594 antibody.

After washing, cells were incubated with DAPI for 30 min, washed again and then the

cover slips were mounted on the slides. Confocal microscopy was performed using an

Olympus IX-81 Spinning Disc Confocal microscope using a 60x water immersion 1.2 NA

objective and 3I Slidebook 5.0 software.

Copy-Number-Variation Analysis


Copy-number-variation analysis of PX’s tumor was conducted using a human genome

CGH microarray kit (244A; Agilent Technologies). This platform uses 240,000 unique

60-mer oligonucleotide probes across the genome, with tighter coverage in the regions

of RefSeq genes. Array hybridization was performed using 2.5 µg of gDNA from PX’s

tissue specimen and a control human gDNA (Promega) according to the array

manufacturer’s protocol. Briefly, hybridization lasted for 40 h at 65°C, and hybridized

arrays were scanned using an Agilent Technologies dual-laser-based scanner. Data

transformation was performed using the Feature Extraction CGH-v4_91 software

program (Agilent Technologies). Statistical aberration detection was conducted using

the Nexus Copy Number software program (version 5.0; BioDiscovery, El Segundo, CA).

MassARRAY Mutation Analysis

gDNA from the specimens was analyzed using polymerase chain reaction (PCR)

amplification followed by matrix-assisted laser desorption/ionization–time-of-flight mass

spectroscopy single-nucleotide polymorphism analysis (MassARRAY; Sequenom, Inc.,

San Diego, CA) for 40 genes with 240 mutations (Table S1). PCR products were

classified as wt or mutant according to their molecular weights. Detected mutations

were then Sanger sequenced.

Primers for Mutational Analysis of BRAF and c-Src

The primers used for c-Src were CTT CTC CTT TCC TCC CTC CTT (forward) and CAG

GAG AGG CAC TCT GCAC (reverse) for exon 7, AGC CAT ATC CAG GGA GAA GC

(forward) and ACA CCC AGC TCA AAC CAC TC (reverse) for exon 8, CCT TCC CTC

CAA TGT CAG G (forward) and AGT CTG CAG CTG AGG CTT TG (reverse) for exon


9, and AGA AGA CCC GCC TAA CTG CT (forward) and ATC CAG CAG AGG CAG

CTA AAG (reverse) for exon 10. The primers used for BRAF were TCC CTC TCA GGC

ATA AGG TAA (forward) and CGA ACA GTG AAT ATT TCC TTT GAT (reverse) for

exon 11 and TCA TAA TGC TTG CTC TGA TAG GA (forward) and GGC CAA AAA TTT

AAT CAG TGG A (reverse) for exon 15.

Mutational Analysis of BRAF and c-Src

Intron-based PCR was used to sequence B-Raf exons 11 and 15 and c-Src exons 7-10

as described previously. FFPE tumor tissue was microdissected, and about 200 cells

were used in each amplification. DNA extracted from either microdissected tissue or cell

pellets was subjected to PCR, and PCR products were directly sequenced using a

PRISM dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). The

primers are listed in the supplemental methods. Sequence variants were confirmed

using independent PCR amplifications and sequenced in both directions

.

DDR2 Sequencing

DDR2 mutations within coding exons were screened from genomic DNA obtained from

patient’s tumor sample using Sanger sequencing method as described (6). The coding

exons were designated based on mRNA transcript variant 2 from DDR2 gene (NM_

006182). Mutations( based on SNP locations) were verified manually with comparison

made to the matched normal sequence.

Fluorescence In Situ Hybridization


A fluorescence in situ hybridization (FISH) assay following a standard protocol was

performed to determine the presence of an ALK gene rearrangement using the LSI ALK

dual-color break-apart probe (Abbott Molecular, Abbott Park, IL). PX’s tissue slides

were incubated for 4 h at 56°C, deparaffinized in Citri-Solv (Fisher), and washed in

100% ethanol for 5 min. The slides were pretreated using a Dako histology FISH

accessory kit (K5599) in a pressure cooker as follows: at 121°C for 1 min, cool-down to

90°C, then at room temperature for 15 min. Excess l iquid was removed and the slides

were incubated in Dako pepsin at 37°C for 2 h. Next , the slides were washed in Dako

wash buffer and dehydrated in ethanol. The probe sets were applied to the tissue which

was covered with a coverslip and sealed with rubber cement. The specimen was

denatured for 8 min at 80°C and incubated at 37°C for 48 h. Posthybridization washes

were performed using a wash buffer (1.5 M urea in 0.1× standard sodium citrate). The

slides were then washed in 2x standard sodium citrate for 2 min and dehydrated in

ethanol. Chromatin was counterstained with DAPI (0.3 µg/mL in Vectashield; Vector

Laboratories, Burlingame, CA). Analysis was performed using a fluorescence

microscope with single interference filter sets for green (SpectrumGreen), orange

(SpectrumOrange), and blue (DAPI) bandpass filters. Monochromatic images were

captured and merged using a CytoVision workstation (Applied Imaging, San Jose, CA).

Cells with single or split signals were defined as those with gene rearrangement. Those

with both signals close or overlapping were negative for rearrangement.

Genomic DNA Preparation

Total nucleic acids were extracted from five tissue sections using an SPRI-TE total

nucleic acid extractor (Beckman Coulter). Each section was individually extracted and


gDNA isolated. Briefly, sections were incubated in 200 µL of lysis buffer for 1 h at 85°C

followed by 30 µL of proteinase K for 1 h at 55°C, producing a tota l nucleic acid fraction.

Next, gDNA was isolated from the sections and cleaned for array comparative genomic

hybridization (aCGH) and matrix-assisted laser desorption/ionization–time-of-flight mass

spectroscopy single-nucleotide polymorphism analysis using a standard column

cleanup with RNase treatment to remove RNA from the total nucleic acid fraction

(DNeasy blood and tissue kit; QIAGEN) and adapted from an FFPE DNA preparation

for oligonucleotide array-based CGH for a gDNA analysis protocol (Agilent

Technologies). gDNA quantity and purity were assessed using a NanoDrop 2000

spectrophotometer (Thermo Scientific). The mean gDNA quantity recovered per tissue

section was 10,340 ng. The mean sample purity (1.81) as assessed using a 260:280

wavelength ratio was within accepted DNA-purity levels (28). Individual gDNA

preparations from each section were then pooled for use.

Immunohistochemistry

An automated stainer (Dako) was used for immunohistochemical staining of tumor

sections. Five-micrometer sections of FFPE tumor were deparaffinized and hydrated,

and antigen retrieval was done (pH 6.0, Dako). Cell pellets from NSCLC cell lines with

high and low protein expression levels according to Western blotting relative to the

levels in a panel of eight NSCLC cell lines were used as positive and negative controls

for the staining. Peroxide blocking was performed using 3% methanol and hydrogen

peroxide for 15 min. Upon blocking with 10% fetal bovine serum, tissue slides were

incubated with the primary antibodies phospho-PDGFRα (Y754, 1:500), total PDGFRα

(1:500), c-Kit (1:200), EphA2 (1:500), phospho-EphA2 (Tyr594, 1:500), phospho-c-Kit


(Y719, 1:150), and p53 (1:400) at room temperature for 1 h. After washing in Tris-

buffered saline and Tween 20, slides were incubated with Dako EnVision+ Dual Link

reagent for 30 min at room temperature followed by a Dako chromogen substrate for 5

min and were then counterstained with hematoxylin for 5 min. Slides were examined for

the staining intensity by a blinded observer using a light microscope with a ×20

magnification objective. Cytoplasmic staining was scored based on both staining

intensity (0-3 scale: 0, below the level of detection; 1, weak; 2, moderate; 3, strong) and

the percentage of cells stained at each intensity level (0-100%). The final score was

calculated by multiplying the intensity score by the percentage, producing a scoring

range of 0-300.


Supplemental Figures and Legends

Figure S1. (A) Radiographic images of PX’s primary lung tumor and paraspinal

metastasis, demonstrating that only a residual lung nodule remains over 3 years after

the completion of treatment with dasatinib alone for 12 weeks. (B) Kaplan-Meier overall

survival and progression free survival curves for the 34 patients enrolled on the

dasatinib phase 2 study at a median of 36 months of follow up.

A B

Fig. S1


Figure S2. Gene copy number variation assessed across all chromosomes in PX’s

metastatic lymph node showing regions of gain (green) and loss (red) of copy numbers.

Regions harboring dasatinib-targeted genes are identified by arrowheads. Gain: EPHA3

(3p11.2), ARG (ABL2; 1q25.2), EGFR (7 p12), HCK (20q11-q12), DDR1 (6p21.3),

ERBB2 (17q21.1), and AKT2 (19q13.1-q13.2); loss: BLK (8p23-p22) and CDKN2A

(9p21).

Fig. S2


Figure S3. Sequencing chromatograms demonstrate BRAF mutations. Sequencing

chromatograms show the heterozygous Y472CBRAF mutation in PX’s tumor tissue (A),

the mutated Y472CBRAF (B), and G466VBRAF constructs (C) that were created using site-

directed mutagenesis.

A B C

Fig. S3


Figure S4. Alignment of the human BRAF amino acid sequence with that of mice (Mus),

rats (Rattus), chickens (Gallus), and frogs (Xenopus). The tyrosine residue marked in

red represents the Y472 site in humans, which is conserved in all tested evolutionary

distant organisms.

Figure S5. NSCLC cells with kinase-inactive BRAF mutations did not undergo

apoptosis when exposed to 150nM dasatinib for 72 hours as measured by TUNEL

staining.

Fig. S4

0.00.20.40.60.81.01.2

Control Dasatinib Control Dasatinib

Cal-12T H1666

Apo

ptos

is (%

tota

l)

Fig. S5


Figure S6. Cells with inactive BRAF undergo senescence in the presence of dasatinib.

Immunofluorescence microscopy was done with Cal12T, H1666 (A), H661 and H2987

(B) cell lines treated with vehicle or 150 nM dasatinib for 72 hours. Cells were labeled

with senescence-associated heterochromatin foci marker HP1-γ and counterstained

with DAPI. Induction of HP1-γ in Dasatinib treated cells is significantly higher in Cal12-T

and H1666. (C) Fold change in HP1-γ expressing cells in dasatinib treated group

compared to vehicle control in three independent experiments.

Fig. S6 A

B

C


Figure S7. H1666 cells were incubated with 100 nM dasatinib for the indicated times

followed by replacement with fresh media (schedule per left panel) and senescence was

quantitated with β-galactosidase staining using light microscopy. (*P value <0.05)

Fig. S7


Figure S8. H661 cells transfected with inactivating BRAF mutations show increased

sensitivity to dasatinib. H661 cells were transfected with the noted BRAF constructs and

incubated with increasing doses of dasatinib for 72 h. Their viability was estimated using

an MTT assay. These data were used to generate the data presented in Figure 3A.

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

10 100 1000 10000

Cel

l via

bilit

y (f

old

cont

rol)

[Dasatinib] nM

Mock

Control

WT

V600E

Y472C

G466V

Fig. S8


Figure S9. Transfection with mutant or wild-type BRAF does not affect cell number.

H1666 and H661 cells were transfected with the noted BRAF constructs and cell

number was estimated 72 h later using the MTT assay.

Fig. S9


Figure S10. NSCLC cells with an inactivating BRAF mutation are sensitive to CRAF

knockdown (KD). H661 cells expressing the noted BRAF proteins were incubated with

CRAF siRNA, and their viability was estimated using an MTT assay. In all cases,

Western blotting was used to confirm CRAF knockdown.

Fig. S10


Figure S11. Kinase inhibitor-induced RAF dimerization does not result in drug

sensitivity or senescence. NSCLC cells were incubated with the noted drugs (dasatinib:

150 nM, nilotinib: 2 µM, bosutinib: 1.5 µM and AZD0530: 5 µM respectively) for 72 h

followed by immuoprecipitation with CRAF from Cal12T cells (A), MTT assay (B), or β-

galactosidase staining (C).

Figure S12. Dasatinib does not have any BRAF mutation-specific changes in BAD or

JNK phosphorylation. The noted NSCLC cell lines were incubated with 150 nM

dasatinib for 72 h followed by Western blotting with the noted antibodies.

Fig. S12


Figure S13. Inhibition of c-Src does not affect cell viability in NSCLC harboring a

kinase-inactive BRAF mutation. H1666 cells were incubated with AZD0530, c-Src

siRNA, or controls. (A and B) Western blotting confirmed c-Src knock down or

inhibition . (C) Neither c-Src siRNA nor controls affect cell viability 72 hours following

transfection. (note: The MTT assay for AZD0530 is included in Figure S11B)

C A B

Fig. S13


Figure S14. Dasatinib enhances the cytotoxicity of sorafenib in cancer cells resistant to

dasatinib. Dasatinib-resistant head and neck cancer (top four panels) and NSCLC

(bottom four panels) cells were incubated with 100 nM dasatinib and 1-10 µM sorafenib

for 72 h, and their viability was estimated using an MTT assay.

Fig. S14


Table S1. Immunohistochemistry scores for PX’s tumor.

Tissue

pEphA2

cytoplasmic

score

Total EphA2

cytoplasmic

score

p-c-Kit

cytoplasmic

score

Total c-Kit

cytoplasmic

score

pPDGFRαααα

cytoplasmic

score

Total

PDGFRαααα

cytoplasmic

score

PX 70 120 40 200 140 70

Positive

control

70 130 30 20 100 200

Negative

control

0 0 0 0 0 0


Table S2. Genes analyzed using mass spectroscopy single-nucleotide polymorphism analysis

Gene Amino acid

position

Nucleic acid change tested

ABCB1 F893I,V G2677TA AKT1 E17K G49A AKT1 G173R G517C AKT1 K179M A536T AKT2 E17K G49A AKT2 G175R G523C AKT2 R371H G1112A AKT2 S302G A904G AKT3 E17K G49A AKT3 G171R G511A ALK 1174I T3520A ALK A877S G2G29T ALK D1091N G3271A ALK F1174L C3522A ALK F1245C T3734G ALK F1245V T3733G ALK I1171N T3512A ALK I1250T T3749C ALK L560F G1680C ALK M1166R T3497G ALK R1275QL G3824AT BRAF D594GV A1781GT BRAF D594E T1782A BRAF G464EVA G1391ATC BRAF G464R G1390C BRAF G466EVA G1397ATC BRAF G466R G1396C BRAF G469R G1405CA BRAF G469EVA G1406ATC BRAF K601E A1801G BRAF K601N A1803T BRAF L597VL C1789GT BRAF L597R T1790G


BRAF S605CG A1813TG BRAF V600K G1798AC, T1799A BRAF V600D T1799A G1800AT BRAF V600EKD T1799ACG CDK4 R24C C70T CDK4 R24H G71A CTNNB1 D32AGT A95CGV CTNNB1 D32HNY G94CAT CTNNB1 G34EVA G101ATC CTNNB1 S33APT T97GCA CTNNB1 S37APT T109GCA CTNNB1 S37CFY C110GTA CTNNB1 S45APT T133GCA CTNNB1 S45CFY C134GTA CTNNB1 T41APS A121GCS TRIM62 R187W R187W_C559T EGFR A750 V3 deletion EGFR A750 V3 deletion EGFR A750 V3 deletion EGFR E746 V3 deletion EGFR E746 V3 deletion EGFR E746 V3 deletion EGFR G719VD G2155TA EGFR K860I A2579T EGFR L747 V3 deletion EGFR L747 V3 deletion EGFR L747 V3 deletion EGFR L858R T2573G EGFR L861QR T2582AG EGFR P753S C2257T EGFR S720P T2158C EGFR T790M C2369T EGFR T854I C2561T EGFR Y813C A2438G Era G400V G400V FBWX7 R465C C1393T FBWX7 R465H G1394A FBWX7 R479QL G1436AT


FBWX7 R479G C1435G FBWX7 R505HLP G1514ATC FBWX7 R505C C1513T FBWX7 S582L C1745T FBXW7 H460R A1379G FGFR1 S125L C374T FGFR2 K659E A1975C FGFR2 W290C G870C FGFR2 Y375C A1124G FGFR3 A391E C1172A FGFR3 G370C G1108T FGFR3 G697C G2089T FGFR3 K650EQ A1948GC FGFR3 K650MT A1949TC FGFR3 R248C C742T FGFR3 S131L C392T FGFR3 S249C C746G FGFR3 S371C A1111T FGFR3 Y373C A1118G FLT3 D835HNY G2503CAT FLT3 D835EE T2505AG FLT3 D835V A2504 FRAP M135T T404C FRAP N2343K C7029AG GNAQ Q209KEX C625AGT GNAQ Q209H C627T IDH1 R132C C394T IDH1 R132HL G295AT IDH2 R172GW A514GT JAK2 V617F G1849T KIT A829P G2485C KIT D816HNY G2446CAT KIT D816GVA A2447GTC KIT D820GAV A2459GAT KIT D820NHY G2458ACT KIT D820E T2460AG KIT K558N G1674CT KIT K642E A1924G


KIT L576P T1727C KIT M541L A1621C KIT N556D A1696G KIT N822YHD A2464TCG KIT N822KNK T2466GCA KIT R634W C1900T KIT T670I C2009T KIT V559DAG T1676ACG KIT V560DAG T1679ACG KIT V654A T1961C KIT V825A T2474C KIT Y553N T1657A KIT Y823HND T2466CAG KRAS A146PT G436CA KRAS G10R G28A KRAS G12 G34N KRAS G12 G35N KRAS G13 G37N KRAS G13 G38N KRAS Q61 C181N KRAS Q61 A182N KRAS Q61 A182N MC1R D294H G880C MC1R D84E C252A MC1R G89R G265C MC1R R142H G425A MC1R R151C C451T MC1R R160W C478T MC1R R163Q G488A MC1R T155I C464T MC1R T95M C284T MC1R V60L G178T MC1R V92M G274A MC1R Y152Stp C456A MEK1 D67N G119A MEK2 D71N G211A MET H1112RL A3335GT MET H1112Y C3334T


MET H1124D C3370G MET M1268T T3803C MET N375S A1124G MET N848S A2843G MET R988C C2962T MET T1010I C3029T MET Y1248HD T3742CG MET Y1248C A3743G MET Y1253D T3757G NRAS G12 G34N NRAS G12 G35N NRAS G13 G37N NRAS G13 G38N NRAS Q61 A182N NRAS Q61 A183N NRAS Q61 C181N PDGFRA D842V A2525T PDGFRA D842E G2524A PDGFRA E996K G2986A PDGFRA N659K C1977A PDGFRA N659Y A1975T PDGFRA V561D T1682A PDGFRA V824L G2470C PDPK1 D527E C1581G PDPK1 T354M C1061T PHLPP2 L1016S T3047C PIK3CA A1046V C3137T PIK3CA C420R T1258C PIK3CA E110K G328A PIK3CA E418K G1252A PIK3CA E453K G1357A PIK3CA E542K G1624AC PIK3CA E542VG A1625GT PIK3CA E545K G1633AC PIK3CA E545AGV A1634GT PIK3CA E545D G1635CT PIK3CA F909L G2727G PIK3CA G1049R G3145C


PIK3CA H1047RL A3140GT PIK3CA H1047Y C3139T PIK3CA H701P A2102C PIK3CA K111N G333C PIK3CA M1043I G3129ATC PIK3CA M1043V A3127G PIK3CA N345K T1035A PIK3CA P539R C1616G PIK3CA Q060K C178A PIK3CA Q546EK C1636GA PIK3CA Q546LPR A1637TGC PIK3CA R088Q G263A PIK3CA S405F C1214C PIK3CA T1025SA A3073TG PIK3CA Y1021HN T3061CA PIK3CA Y1021C A3062G PIK3R1 D560Y G1678T PIK3R1 G376R G1126CA PIK3R1 N564K C1693AG PRKAG1 R70Q G209A PRKAG2 N488I A1463T PRKAG2 R531Q G1592A PTEN C124S G371C PTEN C124S T370A PTEN G129E G386A PTEN G129R G385AC RAF1 Q335H G1005C RAF1 S259A T775G RAF1 Y340D T1018G RET C634FSY G1901TCA RET C634RSG T1900CAG RET M918T T2753C RICTOR M675I G2025A RICTOR S159F C476T TNK2 E346K G1036A TNK2 R99Q G296A


Table S3. Copy number variation for genes associated with dasatinib targeting and/or NSCLC

Gene symbol UniGene ID

UniGene Homo

Sapiens Gene name Chromosome

aCGH analysis region

Copy number status

Number of copies

NRAS 701661 486502 Neuroblastoma RAS viral (v-ras) oncogene

homologue

1 1p13.2 No change 2

LCK 685786 470627 Lymphocyte-specific protein tyrosine kinase


EPHA2* 158085 171596 EPH receptor A2 1 1p36 No change 2

EPHA8 176755 283613 EPH receptor A8 1 1p36.12 No change 2

EPHB2* 914076 523329 EPH receptor B2 1 1p36.1-p35 No change 2

FGR* 1293058 533683 Fibroblast growth factor receptor 2

1 1p36.2-p36.1

No change 2

DDR2* 2062844 593833 Discoidin domain receptor tyrosine kinase

2

1 1q23.3 No change 2

ARG (ABL2)* 155654 159472 V-abl Abelson murine leukemia viral oncogene

homologue 2

1 1q25.2 Gain >2

EPHA4* 199139 371218 EPH receptor A4 2 2q36.1 No change 2

EPHA3* 146616 123642 EPH receptor A3 3 3p11.2 Gain >2

EPHB1* 144742 116092 EPH receptor B1 3 3q21-q23 No change 2

EPHB3* 131320 2913 EPH receptor B3 3 3q21-qter No change 2


PIK3CA 140872 85701 Phosphoinositide 3-kinase, catalytic, alpha

polypeptide


ACK (TNK2)* 909260 518513 Tyrosine kinase, non-receptor, 2

3 3q29 No change 2

GAK* 198183 369607 Cyclin G-associated kinase

4 4p16 No change 2

FGFR3 130997 1420 Fibroblast growth factor receptor 3


KIT* 694913 479754 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologue

4 4q11-q12 No change 2

PDGFRA* 139697 74615 Platelet-derived growth factor receptor, alpha

polypeptide

4 4q12 No change 2

EPHA5 2723810 654492 EPH receptor A5 4 4q13.1 No change 2

DDR1* 2138882 631988 Discoidin domain receptor tyrosine kinase

1

6 6p21.3 Gain >2

FYN* 208483 390567 FYN oncogene related to SRC, FGR, YES

6 6q21 No change 2

FRK 141245 89426 Fyn-related kinase 6 6q21-q22.3 No change 2

EGFR* 703452 488293 Epidermal growth factor receptor

7 7p12 Gain >2

EPHB4 232045 437008 EPH receptor B4 7 7q22 No change 2

EPHA1* 141318 89839 EPH receptor A1 7 7q34 No change 2

BRAF* 1436871 550061 v-raf murine sarcoma viral oncogene homologue B1

7 7q34 No change 2


FGFR1 172147 264887 Fibroblast growth factor receptor 1

8 8p12 No change 2

BLK 152206 146591 B lymphoid tyrosine kinase

8 8p23-p22 Loss <2

LYN* 706926 491767 V-yes-1 Yamaguchi sarcoma viral-related oncogene homologue

8 8q13 No change 2

CDKN2A 903346 512599 Cyclin-dependent kinase inhibitor 2A (melanoma, p16,

inhibits CDK4)

9 9p21 Loss <2

JAK2 2725531 656213 Janus kinase 2 9 9p24 No change 2

ABL1 227738 431048 c-abl oncogene 1, non-receptor tyrosine kinase


RET 193761 350321 Ret proto-oncogene 10 10q11.2 No change 2

PTEN 715625 500466 Phosphatase and tensin homologue


HRAS 136481 37003 v-Ha-ras Harvey rat sarcoma viral oncogene

homologue


KRAS* 720192 505033 V-Ki-ras2 Kirsten rat sarcoma viral oncogene

homologue


CDK4 141747 95577 Cyclin-dependent kinase 4

12 12q14 No change 2

FLT3 722749 507590 Fms-related tyrosine kinase 3

13 13q12 No change 2

TP53 2723799 654481 Tumor protein p53 17 17p13.1 No change 2


ERBB2 241389 446352 V-erb-b2 erythroblastic leukemia viral oncogene

homologue 2, neuro/glioblastoma-derived oncogene

homologue

17 17q21.1 Gain >2

YES1 161156 194148 v-yes-1 Yamaguchi sarcoma viral oncogene

homologue

18 18p11.31-p11.21

No change 2

STK11 905752 515005 Serine/threonine kinase 11


AKT2 2138429 631535 V-akt murine thymoma viral oncogene homologue 2

19 19q13.1-q13.2

Gain >2

HCK* 2724528 655210 Hemopoietic cell kinase 20 20q11-q12 Gain >2

SRC* 161422 195659 v-src sarcoma (Schmidt-Ruppin A-2) viral

oncogene homologue (avian)

20 20q12-q13 No change 2

BRK (PTK6)* 138032 51133 PTK6 protein tyrosine kinase 6


* Dasatinib target-associated gene


Table S4. Mutational statuses of patients with NSCLC treated with dasatinib EGFR KRAS BRAF Response

Patient no. Histology Status Exon Alteration Status Alteration Status Alteration Activity RECIST PET

1 AC wt Mutant GGT12TGT wt PD NE 2 NSCLC wt wt wt SD SD

3 AC wt wt wt SD PR

4, PX AC wt Silent GGT12GGG (synonymous)

Mutant Y472C Reduced PR PR

5 AC wt wt wt NE NE

6 AC wt wt wt NE NE

7 AC wt Mutant CAA61CTA wt SD PR

8 AC Mutant 19 Deletion 746E-750A

wt wt SD SD

9 AC wt wt wt PD SD

13 SCC wt wt wt PD PD

14 SCC wt wt wt PD SD

16 AC wt wt wt PD NE

17 AC Mutant 19 Deletion 747L-752S

wt wt PD PD

18 AC Mutant 21 CGC836CGT (nonsense)

wt wt PD NE

20 SCC wt wt Mutant G469E Intermediate

PD SD

21 NSCLC wt wt wt PD NE

22 AC wt wt Mutant V600E Increased SD PD

23 AC wt Mutant GGT12CGT wt PD PD

34 AC Mutant 19 Deletion 747L-752S and GAA746GTT(E746V)

wt wt SD SD

RECIST, Response Evaluation Criteria in Solid Tumors; AC,adenocarcinoma; PD, progressive disease; NE, not evalauted; SD, stable disease; PR, partial response; SCC, squamous cell carcinoma.

supplementary materials for - science translational …...2012/05/25 · sen and peng, supplemental...

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