si appendix experimental procedures cell culture conditions · 1 si appendix experimental...
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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.
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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.
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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
Katayama et al, SI Appendix Figure S3
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.
Katayama et al, SI Appendix Figure S4
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
Katayama et al, SI Appendix Figure S5
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.
Katayama et al, SI Appendix Figure S6
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
Katayama et al, SI Appendix Figure S7
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.
Katayama et al, SI Appendix Figure S8
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)
Katayama et al, SI Appendix Figure S9
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).