supplementary figure 1 select oncogenic target of interest choose an oncogene-driven cell line and a...
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Supplementary Figure 1Select oncogenic target of interest Choose an oncogene-driven
cell line and a cell line to be used as a
counterscreen
OCI-AML3
Ba/F3-NRAS-G12D
Ba/F3
Screen LINCS ChemicalLibrary
(at predeterminedscreening concentration)
384-well plate(Cell Titer Glo)
Validation of LINCS Chemical Library “hits”
Cellular proliferation
assays
96-well plate(Cell Titer Glo)
LINCS Kinase Inhibitor-Focused
Chemical Library
+ AZD6244
Signaling studies
Identificationof LINCS Chemical Library “hits”
Testing in murine models
AZD6244
Choose selective inhibitor of downstream RAS signaling
to test in combination with LINCS Chemical Library
(use at no higher than the IC50)
PI-103 (PIKK inhibitor)GSK2126458 (PI3K inhibitor)
ZSTK474 (PI3K inhibitor)Torin 1 (mTOR inhibitor)
GSK1904529A (IGF-1R inhibitor)
Pe
rce
nta
ge
of
via
ble
ce
lls
PD0325901(MEK inhibitor)
GSK2126458(PI3KInhibitor)
GSK1120212(MEK Inhibitor)
AZD8330(MEKinhibitor)
parental Ba/F3
Ba/F3-NRAS-G12D
OCI-AML3
A
0
20
40
60
80
100
120
0 18.75 37.5 75 150 300
parental Ba/F3 (PI013,PIKK inhibitor)
Ba/F3-NRAS-G12D(PI013, PIKK inhibitor)
parental Ba/F3(GSK2126458, PI3Kinhibitor)
Ba/F3-NRAS-G12D(GSK2126458, PI3Kinhibitor)
parental Ba/F3(ZSTK474, PI3Kinhibitor)
Ba/F3-NRAS-G12D(ZSTK474, PI3Kinhibitor)
[Drug, nM]
Ce
ll N
um
be
r (%
of
Co
ntr
ol)
0
20
40
60
80
100
120
0 18.75 37.5 75 150 300
parental Ba/F3(PD0325901, MEKinhibitor)
Ba/F3-NRAS-G12D(PD0325901, MEKinhibitor)
parental Ba/F3(GSK1120212, MEKinhibitor)
Ba/F3-NRAS-G12D(GSK1120212, MEKinhibitor)
parental Ba/F3(AZD8330, MEK inhibitor)
Ba/F3-NRAS-G12D(AZD8330, MEK inhibitor)
Ce
ll N
um
be
r (%
of
Co
ntr
ol)
[Drug, nM]
B C
0 18.75 37.5 75 150 3000 18.75 37.5 75 150 300
0
20
40
60
80
100
120
0
20
40
60
80
100
120
0
20
40
60
80
100
120
Supplementary Figure 2
Ce
ll N
um
be
r (%
of
co
ntr
ol)
0 2.5 5 10 20 40 80
0 9.375 18.75 37.5 75 150 3000 2.5+
9.3755+18.75
10+37.5
20+75
40+150
80+300
AZD6244ZSTK474
Combination
[Drug, nM]
Ce
ll N
um
be
r (%
of
co
ntr
ol)
0 2.5 5 10 20 40 800 1.25 2.5 5 10 20 400 2.5+
1.255+2.5
10+5
20+10
40+20
80+40
AZD6244
GSK2126458Combination
[Drug, nM]
Ce
ll N
um
be
r (%
of
co
ntr
ol)
0 5 10 20 40 80 160
0 9.375 18.75 37.5 75 150 3000 5+
9.37510+18.75
20+37.5
40+75
80+150
160+300
AZD6244PI-103
Combination
[Drug, nM]
A B C
Ba/F3-NRAS-G12D Ba/F3-NRAS-G12DBa/F3-NRAS-G12D
Ce
ll N
um
be
r (%
of
co
ntr
ol)
0 2.5 5 10 20 40 80
0 9.375 18.75 37.5 75 150 300
0 2.5+9.375
5+18.75
10+37.5
20+75
40+150
80+300
AZD6244
ZSTK474
[Drug, nM]
OCI-AML3
D
Ce
ll N
um
be
r (%
of
co
ntr
ol)
0 5 10 20 40 80 160
0 9.375 18.75 37.5 75 150 300
0 5+9.375
10+18.75
20+37.5
40+75
80+150
160+300
AZD6244
P1013
Combination
[Drug, nM]
OCI-AML3
E
Ce
ll N
um
be
r (%
of
co
ntr
ol)
0 5 10 20 40 80 160
0 1.25 2.5 5 10 20 40
0 5+1.25
10+2.5
20+5
40+10
80+20
160+40
AZD6244
Torin1
Combination
[Drug, nM]
OCI-AML3
F
ZSTK474 (150-300)
AZD6244 (20-40)
Combination(AZD=5-10; ZST=18.75-37.5)
GSK2126458 (10-20)
AZD6244 (20-40)
Combination (AZD=5; GSK=2.5)
PI-103 (37.5-75)
AZD6244 (20-40)
Combination (AZD=5-10;PI=9.375-18.75)
ZSTK474 (150-300)
AZD6244 (20-40)
Combination (AZD=5-10;ZST=18.75-75)
P1013 (40-80)
AZD6244 (20)
Combination (AZD=5-10;PI=9.375-18.75)
Torin 1 (80-160)
AZD6244 (20-40)
Combination(AZD=10;Torin1=2.5)
0
20
40
60
80
100
120
0
20
40
60
80
100
120
0
20
40
60
80
100
120
0
20
40
60
80
100
120
0
20
40
60
80
100
120
0
20
40
60
80
100
120
Supplementary Figure 3
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
Ba/F3-NRAS-G12D(RPMI+10% FBS)
[Drug, nM]
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
Ba/F3-NRAS-G12D(RPMI+10% FBS)
[Drug, nM]
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
Ba/F3-NRAS-G12D(15% WEHI (source of IL-3)
[Drug, nM]
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
Ba/F3-NRAS-G12D(15% WEHI (source of IL-3)
[Drug, nM]
A B
C D
Supplementary Figure 4
Supplementary Figure 5
B CA
D E F
OCI-AML3
Per
cen
tag
e
NB4
Per
cen
tag
e
NOMO-1
Per
cen
tag
e
SKM-1
Per
cen
tag
e
Per
cen
tag
e
HEL
A B
C D E
Supplementary Figure 6 (A-E)
Supplementary Figure 6 (F-I)F
Per
cen
tag
e
NB4
SKM-1
Per
cen
tag
e
NOMO-1
Per
cen
tag
e
H I
G
Per
cen
tag
e
OCI-AML3
0
10
20
30
40
50
60
G0G1
G2M
SP
erc
en
tag
e
G0G1G2MS
0
10
20
30
40
50
60
K
J
Control GSK1904529A AZD6244 GSK1904529A+AZD6244
Control GSK1904529A AZD6244 GSK1904529A+AZD6244
Pe
rce
nta
ge
subG1
0
5
10
15
20
25
30
35
40
45
0
10
20
30
40
50
60
70
%G0G1
%G2M
%S
Control GSK1904529A AZD6244 GSK1904529A+AZD6244
L
Pe
rce
nta
ge
Supplementary Figure 6 (J-L)24 hr
48 hr 48 hr
Control GSK1904529A AZD6244 GSK1904529A+AZD6244
Pe
rce
nta
ge
M 72 hr
Supplementary Figure 6 (M)
Supplementary Figure 6 (N)
N
MOLM14
NB4
OCI-AML3
ControlAZD6244 (300 nM)
GSK1904529A(300 nM)
AZD6244+GSK1904529A
SKM-1 (RPMI+10%FBS)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
SKM-1 (95% HS-5 SCM)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)[Drug, nM]
SKM-1 (95% HS27a SCM)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
NB4-luc+ (RPMI+10%FBS)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
NB4-luc+ (95% HS-5 SCM)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
NB4-luc+ (95% HS27a SCM)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
Supplementary Figure 7 (part 1)
OCI-AML3(RPMI+10%FBS)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
OCI-AML3(95% HS-5 SCM)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)[Drug, nM]
NOMO-1(RPMI+10%FBS)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
NOMO-1(95% HS27a SCM)
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
[Drug, nM]
MOLM14 [PKC412, nM]
Cel
l Nu
mb
er (
% o
f C
on
tro
l)
Supplementary Figure 7 (part 2)
Supplementary Figure 8
No
rmal
ized
gro
wth
Treatment
Supplementary Figure 9
Ce
ll N
um
be
r (%
of
Co
ntr
ol)
GSK1904529A (37.5)ZSTK474 (150)Combination (9.4-18.75)
0
20
40
60
80
100
120
140
0 4.7 9.4 18.75 37.5 75 150 300ZSTK474[Drug, nM]
0 4.7 9.4 18.75 37.5 75 150 300
160
Ba/F3-KRAS-G12D
Ce
ll N
um
be
r (%
of
Co
ntr
ol) Ba/F3-NRAS-G12D
GSK1904529A (>300)ZSTK474 (150-300)Combination (37.5)
0
20
40
60
80
100
120
140
0 4.7 9.4 18.75 37.5 75 150 300ZSTK474[Drug, nM]
0 4.7 9.4 18.75 37.5 75 150 300
160
Ce
ll N
um
be
r (%
of
Co
ntr
ol)
Parental Ba/F3
GSK1904529A (>300)ZSTK474 (>300)Combination (>300)
0
20
40
60
80
100
120
140
0 4.7 9.4 18.75 37.5 75 150 300ZSTK474
0 4.7 9.4 18.75 37.5 75 150 300
160
[Drug, nM]
A
B C
GSK1904529A
GSK1904529A GSK1904529A
Supplementary Figure 10
C D
Phospho-IGF1R/Total IGF1R
2.1 0.7 1.2 1.8 0.89 1.39
A B
OC
I-A
ML
3
NO
MO
-1
NB
4
SK
M-1
HE
L
MO
LM
14
OC
I-A
ML
3
NO
MO
-1
NB
4
SK
M-1
HE
L
MO
LM
14
OC
I-A
ML
3
NO
MO
-1
NB
4
SK
M-1
HE
L
MO
LM
14
Ba/
F3-
KR
AS
-G12
D
Ba/
F3-
NR
AS
-G12
D
Ba/
F3
Supplementary Figure 11
B
A
Supplementary Figure 12
A B
Supplementary Figure 13
Control AZD6244(37.5 nM)
GSK-1904529A(37.5 nM)
Combi-nation
A B
Control NVP-AEW541(300 nM)
GSK-1904529A(300 nM)
AZD6244(300 nM)
AZD6244+
NVP-AEW541
AZD6244+
GSK-1904529A
Supplementary Figure 14
Co
ntr
ol
300
nM
N
VP
AE
W54
1
300
nM
G
SK
1904
529A
300
nM
A
ZD
6244
AZ
D62
44+
NV
PA
EW
541
AZ
D62
44+
G
SK
1904
529A
(Ser 65)
Ab
so
rba
nc
e (
56
0
nm
)A B
E
Ce
ll N
um
be
r (%
of
no
tre
atm
en
t c
on
tro
l)NB4
Supplementary Figure 15NB4
C
Ab
so
rba
nc
e (
56
0
nm
)
HELD HEL
Ce
ll N
um
be
r (%
of
no
tre
atm
en
t c
on
tro
l)
F
GFPControl
IGF-1Rhp1
IGF-1Rhp2
0
GS
K19
0452
9A(1
8.75
nM
)
AZ
D62
44(1
8.75
nM
)
Co
mb
inat
ion
0
GS
K19
0452
9A(3
7.5
nM
)
AZ
D62
44(3
7.5
nM
)
Co
mb
inat
ion
0
GS
K19
0452
9A(7
5 n
M)
AZ
D62
44(7
5 n
M)
Co
mb
inat
ion
GFPControl
IGF-1Rhp1
IGF-1Rhp2
0
GS
K19
0452
9A(1
8.75
nM
)
AZ
D62
44(1
8.75
nM
)
Co
mb
inat
ion
GFP Control
IGF-1R hp1
IGF-1R hp2
GFP Control
IGF-1R hp1
IGF-1R hp2
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
60
70
80
0
100
20
120
40
60
80
0
20
40
60
80
100
120
Supplementary Figure 16
A
B
Gra
ms
NVPAEW541 AZD6244 NVPAEW541+AZD6244
NVPAEW541 AZD6244NVPAEW541+
AZD6244
Supplementary Figure 17
Ce
ll N
um
be
r (%
of
Co
ntr
ol)
NVPAEW541(300 nM)
AZD6244(300 nM)
NVPAEW541 (300 nM)+
AZD6244 (300 nM)
*
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