si appendix experimental procedures cell culture conditions · 1 si appendix experimental...

1

SI Appendix Experimental Procedures

Cell culture conditions

Human NSCLC H3122, H460, H522, H1299 and A549 cells, human breast cancer SKBR3 and

BT474 cells were cultured in RPMI1640 supplemented with 10% FBS (RPMI growth medium).

Mice myeloma Ba/F3 cells were cultured in DMEM supplemented with 10% FBS with or

without 0.5ng/mL IL-3 (Invitrogen).

Generation of H3122 CR cells

H3122 cells were seeded at ~70% confluence in 15-cm dishes in RPMI 1640 with 10% FBS.

Crizotinib was added at a starting concentration of 30 nM, and cells were maintained in fresh

drug-containing medium changed every ~72 hours. Cells were passaged once they reached

confluence. After every two passages at a given concentration of drug, the concentration of

crizotinib was increased in half-log intervals until a final concentration of 1 uM was achieved.

The resulting pool of resistant cells (designated H3122 CR) were maintained in RPMI with 10%

FBS containing 1 uM crizotinib. From the H3122 CR pool, we derived 11 clones from single

cells by limiting dilution.

2

Survival assays

For 72-h drug treatments, 3000 cells were plated in replicates of six into 96-well plates.

Following drug treatments, cells were incubated with CellTiter-Glo assay reagent (Promega) for

10 min and luminescence was measured using a Centro LB 960 microplate luminometer

(Berthold Technologies).

Fluorescence in situ hybridization

Two-color fluorescence in situ hybridization (FISH) was done on 3:1 methanol/acetic acid–fixed

cell lines using the LSI ALK Dual Color, Break Apart Rearrangement Probe (Abbott-Vysis)

following the manufacturer’s protocols. Images were captured with an Olympus BX61

fluorescent microscope equipped with a charge-coupled device camera, and analysis was done

with Cytovision software (Applied Imaging).

Immunoblotting

Cells were resuspended in lysis buffer (20 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 10%

glycerol, 1 mM EDTA, 1 mM EGTA, and protease and phosphatase inhibitors), incubated on ice

for 10 min and centrifuged for 5 min (15,000 rpm). Protein concentration determination and

immunoblotting were performed as previously described (36). The phospho-ERK (T202/Y204),

3

ERK, phospho-AKT (S473 and T308), total Akt, phospho-ALK (Y1604), and ALK antibodies,

were obtained from Cell Signaling Technology. The actin antibody was purchased from Sigma.

Apoptosis assay

Cells were collected and stained with AnnexinV and 5ug/ml PI for 10 minutes. Cells were then

assayed using a FACS Diva (BD Bioscience) flow cytometer, and the data analyzed using Flow

Jo software (Treestar).

Retroviral infection

cDNAs encoding EML4-ALK variant1 or EML4-ALK variant1 L1196M were cloned into 1520

retroviral expression vectors, and virus was produced as previously described (37). After

retroviral infection, Ba/F3 cells were selected in puromycin (0.5ug/mL) for 2 weeks. IL-3 was

withdrawn from the culture medium for 2 wks before experiments.

RNA preparation and quantitative real-time PCR

Quantitative RT-PCR was performed essentially as described (17). Total RNA from cell lines

was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA). RNA (1 µg) was reverse

transcribed using Transcriptor High Fidelity cDNA Synthesis Kit (Roche), according to the

4

manufacturer’s instructions. mRNA was quantified with CYBR Green using a PCR LightCycler

480 (Roche Diagnostics) and normalized by the amount of LINE1 mRNA. Primer sequences are

provided in Supplementary Table S3.

Isolation of gDNA preparation and L1196M mutation specific PCR

Genomic DNA was isolated from cell pellets with a DNeasy kit (QIAGEN) according to the

manufacturer’s protocol. Exon 23 of ALK was PCR-amplified from genomic DNA using Pfu

Ultra II (Agilent Technologies, Santa Clara, CA) and sequenced bidirectionally by Sanger

dideoxynucleotide sequencing with the primers described in the supplementary methods.

ALK-Exon23 or L1196M mutation-specific qPCR was performed by a LightCycler 480 (Roche

Diagnostics) with CYBR Green Master Mix (Roche). Primer sequences are provided in

Supplementary Table S3.

siRNA transfection

ALK siRNA and Silencer negative control #1 siRNA were obtained from Ambion (Austin, Tx).

H3122, H3122 CR and A549 cells were plated in 6-well plates and reverse transfected with 20

nM siRNA and HiPerFect Reagent (QIAGEN) according to the manufacturer’s protocol.

Transfected cells were cultured at 37C for 72 hours before analysis.

5

Xenograft Study

For H3122 CR xenografts, cells (5 x 106) were injected s.c. in the left flank of 6- to 8-week-old

male athymic nude mice. The mice were maintained in laminar air-flow units under aseptic

conditions and the care and treatment of experimental animals were in accordance with

institutional guidelines. Mice were monitored on a daily basis for adverse effects, signs of tumor

development and treatment toxicity. Tumors were measured twice a week with calipers and

tumor volume in mm3 was calculated according to the formula: volume x width x length x 0.52.

Before starting treatment, mice (n = 5 per group) were randomized to: a) control group, b)

crizotinib only (100mg/kg/day), c) AP26113 only (50 mg/kg/day), or d) NVP-TAE684 only (25

mg/kg/day) and treated daily for 17 days.

Statistical analysis

All data except for xenograft experiments are shown as mean ± SD. Statistical analysis was

performed using two-tailed Student’s t test. Significance was established for p values < 0.05.

siRNAs and primers for sequence and PCR

siRNAsiALK 5'- CCGCUUUGCCGAUAGAAUA -3'

Primers(cloning)EML4-ALK-F 5'- CACCATGGACGGTTTCGCCGGCAGTC -3'EML4-ALK-R 5'- TCAGGGCCCAGGCTGGTTCATGC -3'(sequencing)EML4-ALK-seq1F 5'- TCCAGAAAGCAAGAATGCTACTCC -3'EML4-ALK-seq1R 5'- GTCAACATCGGAAGGAATGAACATGG -3'EML4-ALK-seq2F 5'- TGGAGTTTCACCCAACAGATGC -3'EML4-ALK-seq2R 5'- AGCTTGCTCAGCTTGTACTCAGG -3'EML4 ALK 3F 5' TTGCCTGTGGCTGTCAGTATTTG 3'EML4-ALK-seq3F 5'- TTGCCTGTGGCTGTCAGTATTTG -3'EML4-ALK-seq3R 5'- GGTGACAAACTCCAGAACTTCC -3'EML4-ALK-seq4F 5'- ACCGCTTTGCCGATAGAATATGG -3'(mutagenesis)EML4-ALK-L1196M-F 5'- GCCCCGGTTCATCCTGATGGAGCTCATGGCGGG -3'EML4-ALK-L1196M-R 5'- CCCGCCATGAGCTCCATCAGGATGAACCGGGGC -3'( RT PCR)(qRT-PCR)ALK-Cterm-F 5'- AAATGGAACTCCTGTGGAGCCTG -3'ALK-Cterm-R 5'- ATCTTCTGTCCATTCTCTTCCAGCCAGTC -3'GAPDH-Exon8-F 5'- GCTCTCCAGAACATCATCCCTGCCTCTAC -3'GAPDH-Exon8-R 5'- GAGTGGGTGTCGCTGTTGAAGTCAGAG -3'LINE-1-F 5'- AAAGCCGCTCAACTACATGG -3'LINE-1-R 5'- TGCTTTGAATGCGTCCCAGAG -3'LINE-1-R 5 - TGCTTTGAATGCGTCCCAGAG -3(L1196M mutation specfic PCR)ALK-L1196M-specific-F 5'- ATCCCTGCCCCGGTTCATCCTGA -3'ALK-L1196M-specific-R 5'- CTGCCCACTCTTGCTCCTTCCATC -3'

rol)

100

A B

rol)

100

Katayama et al, SI Appendix Figure S1m

ber (

% o

f con

tr

50

100

mbe

r (%

of c

ontr

50

100

0 10 100 1000

cell

num

00 1 10 100

concentration of TAE684 (nM)

cell

num

01000

concentration of crizotinib(nM)

C

rol)

100

Don

trol

)

100

mbe

r (%

of c

ontr

50

100

num

ber (

% o

f co

50

0 1 10 100concentration of AP26113 (nM)

cell

num

01000 0 1 10 100

concentration of 17AAG (nM)

cell

n

01000

H3122H3122 CR

A549H1299 SI Appendix Figure S1. Cell survival assay of H3122, H3122 CR and the A549SKBR3BT474H522H460

inidcated cancer cell lines following treatment with ALK inhibitors or 17AAG.(A-D) H3122, H3122 CR, A549, H460, H522, H1299, SKBR3 and BT474 cells were treated with the indicated doses of crizotinib, NVP-TAE684, AP26113, or 17AAG for 72 hr. After the incubation, the cell survival was assayed by Cell-Titer-Glo.

A B

6M)

Katayama et al, SI Appendix Figure S2

A

mbe

ron

trol

) 100

pare

nt

EML4

-ALK

(WT)

EML4

-ALK

(L11

96

0 10 100 1000

cell

nu(%

of c

o

0

50

pALK(pY1604)

ALK

p E E

Ba/F3 cells

*0 10 100 1000concentration of crizotinib (nM)

Ba/F3Ba/F3_EML4-ALK (WT)Ba/F3_EML4-ALK (L1196M)

ACTB

*

SI Appendix Figure S2. Exogenous expression of EML4-ALK L1196M transforms the Ba/F3 cells and confers resistance to crizotinib.(A) Ba/F3 cells were transformed by expression of the wild type or mutant (L1196M) EML4-ALK. Parental (cultured with IL-3) or transformed Ba/F3 cells (cultured without IL-3) were treated by the i di t d d f i ti ib f 72 h Aft th i b ti th ll i l d b C llindicated doses of crizotinib for 72 hr. After the incubation, the cell survival was assayed by Cell-Titer-Glo. (B) Ba/F3 parental and transformed cells were lysed and examined the expression of EML4-ALK by western blotting.

H3122 H3122 CR0 6


on 00 n

M

00 n

M

00 n

M

000

nM

crizotinib (6hr) crizotinib(6hr)H3122 H3122 CR0.6

on 0 nM

00 n

M

00 n

M

000

nM

0 nM

00 n

M

pALK

ALK

no 10 30 60 10 no 30 30 60 1030 10pAKT (pS473)

AKT

pERK

ERK

ActinActin

SI Appendix Figure S3. H3122 CR0.6 cells require higher doses of crizotinib to inhibit ALK and downstream signaling.H3122 parental and intermediately resistant (CR0.6) cells were treated the indicated concentration of crizotinib for 6 hr. After the treatment, the cells were lysed and examined the phosphorylation of ALK AKT and ERK by western blottingexamined the phosphorylation of ALK, AKT and ERK by western blotting.


1000

cts E1)

L1196M mutation specific qPCR

106 8

949.2 p<0.01

10

100

tive

PCR

pro

duc

6M s

peci

fic/L

INE

1 21.8

5.4

18.8

106.8

0.1

1

122 4%1% H3122 CR0.4% 20%0.1%0% 100%

Rel

at(L

119 1.2

0.7 0.7

H31 H3122 CR0.696%99%99.6% 80%99.9%100% 0%

SI Appendix Figure S4. Allele-specific PCR is highly sensitive for detection of the L1196M mutation.Allele-specific quantitative PCR reactions were performed to measure the L1196M mutation. PCRs were performed in tripricate in 25uL reactions containing 30ng genomic DNA purified from H3122 H3122 CR0 6 H3122 CR In addition H3122genomic DNA purified from H3122, H3122 CR0.6, H3122 CR. In addition, H3122 CR and H3122 CR0.6 were mixed in the indicated ratios to determine the limit of the sensitivity of the assay.

4s

A


2

3

4

ve P

CR

pro

duct

sA

LK/L

INE-

1)

H31

22

lone

1

lone

2

22 C

R

lone

3

lone

6

lone

4

lone

5

orm

al

CR

0.6

lone

7

one1

0

lone

9

lone

8

one1

10

1

Rel

ativ (A

H

CR

cl

CR

cl

H31

2

CR

cl

CR

cl

CR

cl

CR

cl

Fem

ale

no

H31

22 C

CR

cl

CR

clo

CR

cl

CR

cl

CR

clo

)

6B

PC

R p

rodu

cts

spec

ific/

GA

PDH

)

4

2

3122

one1

one2

2 C

R

one3

one6

one4

one5

orm

al

CR

0.6

one7

ne10

one9

one8

ne11

Rel

ativ

e(L

1196

M s

0

2

H

CR

clo

CR

clo

H31

22

CR

clo

CR

clo

CR

clo

CR

clo

Fem

ale

no

H31

22 C

CR

clo

CR

clo

CR

cl o

CR

clo

CR

clo

SI Appendix Figure S5. All clones from H3122 CR harbor both the ALK amplification and L1196M mutation.Quantitative PCRs measuring Exon23 of ALK (A) or specific for the L1196M mutation (B) were performed in triplicate containing 30ng genomic DNA purified from H3122, H3122 CR0.6, H3122 CR and clones derived from H3122 CR cells. Please note that the parental H3122 cells have ~ 4 copies of wt ALK and ~1 copy of EML4-ALK (Fig. 2A). Thus, the specific amplification of EML4-ALK in the CR0.6 and CR cells does not have a proportional effect on total ALK.


H3122

control 1000 nM300 nM100 nM30 nMA

9.6% 6.2% 3.3% 19 2% 63 5%

H3122 CR

Crizotinib

9.6% 6.2% 3.3% 19.2% 63.5%

9.6% 7.0% 7.4% 9.3% 9.5%

H3122 CR

H3122

AP26113

3 nM 300 nM100 nM30 nM10 nM

PI 9.1% 7.7% 60.2% 62.9% 49.8%

H3122 CR

H3122

3 nM 300 nM100 nM30 nM10 nM

8.0% 9.8% 11.4% 44.4% 62.0%

7.6% 5.8% 60.6% 55.7% 72.6%

H3122 CR

TAE684

AnnexinV-APC

7.6% 9.5% 27.8% 61.0% 74.1%

AP26113 TAE684Crizotinib

cont

rol

100n

M30

nM

300n

M10

00nM

cont

rol

100n

M30

nM

300n

M

10nM

3nM

cont

rol

100n

M30

nM

300n

M

10nM

3nM

10nM

cleaved PARP

B

cleaved-PARP

Actin

cleaved-PARP

Actin

H3122

H3122 CR

SI Appendix Figure S6. TAE684 and AP26113, but not crizotinib, induce apoptosis in H3122 CR cells. (A) H3122 parental and resistant (CR) cells were treated with the indicated concentrations of crizotinib, TAE684, or AP26113. After 72 hr, cells were stained with Alexa-633 labeled Annexin-V and PI, and analyzed by flow cytometry. The percent of cell undergoing apoptosis is shown in red. (B) Cells were treated as in (A). Lysates were immunoblottedwith an anti-PARP antibody to detect PARP cleavage.

AB


B

mbe

rnt

rol)

100

150

mbe

ron

trol

) 100

10 100 1000ce

ll nu

m(%

of c

on

0

50

0 1 10 100

cell

num

(% o

f co

0

50

0 10 100 1000concentration of AP26113 (nM)

0 1 10 100concentration of TAE684 (nM)

Ba/F3 (with IL-3)Ba/F3_EML4-ALK (WT)Ba/F3_EML4-ALK (L1196M)

Ba/F3 (with IL-3)Ba/F3_EML4-ALK (WT)Ba/F3_EML4-ALK (L1196M)

C

ol) 100

mbe

r (%

of c

ontr

o

50

0 10 100 1000Concentration of 17AAG (nM)

cell

num

0SI Appendix Figure S7. TAE684, 17AAG and AP26113 effectively inhibit both wild type and L1196m EML4-ALK. (A-C)

l / ll d h f dBa/F3 (with IL-3)Ba/F3_EML4-ALK (WT)Ba/F3_EML4-ALK (L1196M)

Parental Ba/F3 cells and those transformed with EML4-ALK (WT), EML4-ALK L1196M were treated by the indicated dose of NVP-TAE684 (A), AP26113 (B), and 17-AAG(C) for 72 hr. After the incubation, cell survival was assayed by Cell-Titer-Glo.


100

LK)

o-A

LK in

tens

ityK

/tota

l EM

L4-A

L

50

Rel

ativ

e ph

osph

oos

pho-

EML4

-ALK

10 100 10000

concentration (nM)

R(p

ho

H3122H3122 CR

H3122H3122 CR

H3122

AP26113

crizotinib

concentration (nM)

H3122H3122 CR NVP-TAE684

SI Appendix Figure S8. The effect of ALK inhibitors on EML4-ALK phosphorylation in H3122 and H3122 CR cells.The intensity of phospho-ALK and total ALK in Fig.1C, 4C, 4E were quantified and plotted.Y-axis indicate relative value of phospho-EML4-ALK/total EML4-ALK relative to untreated controls.

ATumor Volume (H3122 CR)


ControlCrizotinib (100mg/kg)AP26113 (50mg/kg)TAE684 (25mg/kg)

3

4

5

Volume

( )

1

2

3

Relative Tum

or V

0

0 5 10 15 20

Day

28

32

(g)

Body WeightB

20

24

0 5 10 15 20

Weight

0 5 10 15 20Day

SI Appendix Figure S9. The structurally different ALK inhibitors NVP-TAE684 or AP26113 are effective treatment strategy against in EML4-ALK L1196M mutant harboring crizotinib resistant H3122 CR cells in vivo. (A, B) H3122 CR xenografts were treated with the indicated drug regimens ( , ) g g g(as described in SI Methods), and relative tumor volumes (A) (S.E.M.) and body weight (B) were plotted over time.

SI Appendix Table S1. In vitro Kinase assay data of AP26113

A

BKm for ATP Crizotinib AP26113 Crizotinib AP26113

ALK (wild type) 30.7 uM 0.69 0.09 competitivecompetitive,tight‐binding

ALK (L1196M) 27.2 uM 8.2 0.08 competitivecompetitive,tight‐binding

Ki (nM) MOI with ATP

SI Appendix Table S1; A, Kinase Selectivity Profile of AP26113. AP26113 was profiled against >250 kinases by Reaction Biology Corporation (Malvern, PA) using the Kinase Hotspot assay, which utilizes 10 µM [33P]-ATP, recombinant kinase domain, peptide substrate, and a single inhibitor concentration of 1 µM. For those kinases exhibiting ≥ 80% inhibition in this assay, full 10-pt curves were obtained to establish an IC50 value (http://www.reactionbiology.com/pages/kinase.htm). Using the same experimental system, IC-50

g g

of crizotinib for ALK was 3.6 nM.B, Km of ALK wildtype and L1196M, and Ki of ALK inhibitors. Crizotinib and AP26113 were profiled against ALK (wild type or L1196M) by Reaction Biology Corporation (Malvern, PA) using HotSpot Kinase Ki Determination studies, which utilize 25 µM [33P]-ATP, 2nM recombinant ALK kinase domain (wild type and L1196M), 0.2 mg/ml poly [Glu, Tyr] 4:1 substrate, and multiple inhibitor concentrations (http://www.reactionbiology.com/pages/kinase.htm).

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