A
110
(% o f D a y 1 )
R e la tiv e b o d y w e ig h t
120
C y c lo d e x t r in ip 1 q w
100
o n a le s p ib 5 5 m g /k g ip 1 q w
90
E r lo t in ib 1 2 . 5 m g / k g p o q d
E r lo t in ib + o n a le s p ib
80
70
0
50
100
150
200
250
300
350
400
450
D ay
B
120
(% o f D a y 1 )
R e la t iv e b o d y w e ig h t
130
110
C o n tro l
O n a le s p ib 5 5 m g /k g ip 1 q w
100
C r iz o t in ib 5 0 m g / k g p o q d
C r iz o t in ib + o n a le s p ib
90
80
0
3
2
0
1
0
1
0
1
1
0
0
0
0
1
9
8
0
0
0
7
6
5
4
0
3
2
0
0
1
0
70
D ay
C
(% o f D a y 1 )
R e la t iv e b o d y w e ig h t
110
100
C r iz o tin ib 5 0 m g /k g p o q d
C r iz o tin ib + o n a le s p ib 5 5 m g /k g ip q w
90
80
70
0
2
4
6
8
10
12
Day
Figure S1: Effects of onalespib combinations on body weight of mice bearing tumor xenografts
Relative body weight change of mice bearing HCC827 (A), H2228 (B) or H2228-CR6 (C) tumour xenografts treated with 55 mg/kg onalespib in
combination with 12.5 mg/kg erlotinib (A) or 50 mg/kg crizotinib (B, C).
1400
3
T u m o u r v o lu m e (m m )
1
1200
2
1000
3
4
800
5
6
600
7
400
200
0
1
29
57
85
113
141
169
197
225
253
281
D ay
Figure S2: Generation of crizotinib-resistant models
H2228 tumours were grown subcutaneously in BALB/c SCID mice and continuously treated with crizotinib (50 mg/kg, p.o) from the day after cell
implantation until they acquired resistance to treatment.
A
H 2 2 2 8 -C S
3 M
40
1 M
0 .3  M
30
0 .1  M
0 .0 3  M
0 .0 1  M
20
0 .0 0 3  M
DMSO
10
3 M
50
100
IC50 Proliferation (µM)
1 M
0 .3  M
30
0 .1  M
Crizotinib
Onalespib
H2228-CS
1.3
0.043
H2228-CR2
6.2
0.03
H2228-CR4
2.9
0.04
H2228-CR6
2.8
0.058
0 .0 3  M
20
0 .0 1  M
DMSO
10
0
0
B
10 M
40
0
0
150
50
100
150
T im e (h )
T im e (h )
H 2 2 2 8 -C R 2
H 2 2 2 8 -C R 2
c r iz o tin ib
150
O n a le s p ib
100
10 M
3 M
1 M
0 .3  M
100
0 .1  M
0 .0 3  M
0 .0 1  M
50
0 .0 0 3  M
DMSO
M e a n p e rc e n t c o n flu e n c e
M e a n p e rc e n t c o n flu e n c e
o n a le s p ib
50
10 M
M e a n p e rc e n t c o n flu e n c e
M e a n p e rc e n t c o n flu e n c e
H 2 2 2 8 -C S
c r iz o tin ib
50
0
10 M
3 M
80
1 M
0 .3  M
60
0 .1  M
0 .0 3  M
40
0 .0 1  M
0 .0 0 3  M
DMSO
20
0
0
50
100
150
0
50
100
150
T im e (h )
T im e (h )
H 2 2 2 8 -C R 4
c riz o tin ib
10 M
M e a n p e rc e n t c o n flu e n c e
100
3 M
80
1 M
0 .3  M
60
0 .1  M
0 .0 3  M
40
0 .0 1  M
20
0 .0 0 3  M
O n a le s p ib
80
M e a n p e rc e n t c o n flu e n c e
H 2 2 2 8 -C R 4
0
50
100
3 M
1 M
60
0 .3  M
0 .1  M
40
0 .0 3  M
0 .0 1  M
0 .0 0 3  M
20
DMSO
0
DMSO
0
10 M
0
150
50
100
150
T im e (h )
T im e (h )
H 2 2 2 8 -C R 6
H 2 2 2 8 -C R 6
c r iz o tin ib
10 M
3 M
1 M
60
0 .3  M
0 .1  M
40
0 .0 3  M
0 .0 1  M
0 .0 0 3  M
20
DMSO
o n a le s p ib
80
M e a n p e rc e n t c o n flu e n c e
M e a n p e rc e n t c o n flu e n c e
80
10 M
3 M
1 M
60
0 .3  M
0 .1  M
40
0 .0 3  M
0 .0 1  M
0 .0 0 3  M
20
DMSO
0
0
0
50
100
T im e (h )
150
0
50
100
T im e (h )
150
Figure S3: The crizotinib-resistant cell lines are sensitive to
onalespib
A - H2228 cell lines generated from the crizotinib-resistant
tumours presented in Figure S2 (CR2, CR4, CR6 originated
from tumours 2, 4, 6 respectively) or from a crizotinibsensitive tumour (CS) were treated in vitro with
concentrations of crizotinib or onalespib ranging from 0 to
10 µM for 7 days. Proliferation was measured in real-time
using cell live microscopy and expressed as the mean
percent confluency. B – Relative proliferation for each
concentration of crizotinib and onalespib was calculated
from the area under the curve (AUC) and IC50s determined.
Figure S4: Exome-sequencing for crizotinib-resistant H2228-CR1, -CR5 and -CR7 cell lines
Circos plot displaying non-synonymous somatic mutations and copy number variations in H2228-CR1, -CR5 and –CR7 samples (compared to H2228-CS)
that were analysed after exome sequencing. Mutations are displayed as coloured radial lines. The inner ring displays copy number data for each sample.
A
pALK IC50 (µM)
H2228-CS
H2228-CR6
Onalespib
0.29
0.55
Crizotinib
0.20
1.70
H 2 2 2 8 -C R 6
100
50
H 2 2 2 8 -C S
p A L K ( % c o n tr o l)
p A L K ( % C o n tr o l)
H 2 2 2 8 -C S
H 2 2 2 8 -C R 6
100
50
0
0
-4
-3
-2
-1
0
1
-4
2
17AAG (nM) 0
ALK
ALK
ERK
ERK
pERK
pERK
AKT
AKT
pAKT
pAKT
S6
S6
pS6
pS6
HSP70
β-actin
HSP70
H2228-CR6
(ALKamp, ALKC1156Y)
0
30
100
300
1000
0
H2228-CS
(ALK+)
30
100
300
1000
1000
300
100
30
Onalespib (nM) 0
1000
H2228-CR6
(ALKamp, ALKC1156Y)
H2228-CS
(ALK+)
30
100
300
B
-3
-2
-1
0
1
2
L o g 1 0 [ o n a le s p ib ] µ M
L o g 1 0 [ C r iz o t in ib ] µ M
C
Proliferation IC50 (µM)
H2228-CS
H2228-CR6
Onalespib
0.043
0.058
17AAG
0.047
0.043
Ganetespib
0.028
0.023
β-actin
Figure S5: Further characterisation of the H2228-CR6 cell line
Inhibition of phospho-ALK was measured for H2228-CS and –CR6 lines treated with crizotinib or onalespib for 6h and IC50 values determined. PhosphoALK levels were measured using the PathScan® Phospho-ALK (Tyr1604) Chemiluminescent Sandwich ELISA. Representative curves are shown (A). H2228CS and –CR6 lines were treated with onalespib or 17-AAG at the indicated concentrations for 24h. The effect on protein levels associated with the ALK
signalling pathway was measured by western blot (B). H2228-CS and –CR6 lines were treated in vitro with onalespib, 17-AAG or ganetespib and
proliferation was measured in real-time using cell live microscopy. The area under the growth curve (AUC) was calculated for each concentration and
used to calculate IC50 values (C).