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stm.sciencemag.org/cgi/content/full/11/510/eaaw7999/DC1
Supplementary Materials for
Development of combination therapies to maximize the impact of
KRAS-G12C inhibitors in lung cancer
Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Febe van Maldegem, Edurne Mugarza, Pablo Romero-Clavijo, Eleanor Herbert, Stuart Horswell, Lian-Sheng Li, Matthew R. Janes, David C. Hancock, Julian Downward*
*Corresponding author. Email: [email protected]
Published 18 September 2019, Sci. Transl. Med. 11, eaaw7999 (2019)
DOI: 10.1126/scitranslmed.aaw7999
The PDF file includes:
Materials and Methods Fig. S1. A whole-genome shRNA screen identifies combinatorial drug targets. Fig. S2. Combination of mTOR inhibitors with IGF1R and MEK inhibitors reduces the viability of KRAS-mutant NSCLC cells. Fig. S3. KRAS-mutant cells show increased sensitivity to the combination of mTOR, IGF1R, and MEK inhibitors. Fig. S4. Combination of IGF1R with mTOR inhibitors blocks PI3K/AKT and mTOR pathways. Fig. S5. mTOR inhibition activates the IGF1R pathway in KRAS-mutant cells. Fig. S6. Combination of mTOR, IGF1R, and MEK inhibitors results in regression of KRAS-driven lung tumors. Fig. S7. Drug combinations with a KRAS-G12C inhibitor cause inhibition of viability in KRAS-mutant cells. Fig. S8. Combination of KRAS-G12C inhibitor, IGF1R, and mTOR inhibitors is effective in vivo. Reference (55)
Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/11/510/eaaw7999/DC1)
Data file S1 (Microsoft Excel format). shRNA screen hit lists. Data file S2 (Microsoft Excel format). Primary data.
Materials and Methods
Cell lines and drugs
H23, A549, A427, Calu-1, H520, H2170, EKVX, LUDLU-1, H226, SK-MES-1, and 3LL cells were
obtained from the Francis Crick Institute Cell Services facility; H358, H1792, H2122, SK-LU-1, H441,
H1944, and H1373 were obtained from ATCC; HCC44 cells were obtained from DSMZ; HCC78,
HCC95, and AT2 cells were obtained from Olivier Pardo and Michael Seckl (Imperial College,
London). A549, Calu-1, SK-LU-1, SK-MES-1, HCC78, and HCC95 were maintained in DMEM
supplemented with 10% FBS. The rest of the cell lines were cultured in RPMI with 10% FBS. Cell
lines were tested for mycoplasma and were authenticated by short-tandem repeat (STR) DNA
profiling by the Francis Crick Institute Cell Services facility. RASless MEFs were obtained from the
Frederick National Laboratory and were a kind gift from Rachel Bagni. These cells lack KRAS,
HRAS, and NRAS and have been rescued by expression of endogenous amounts of either wild-type
KRAS or different mutant isoforms. Cells were maintained in DMEM supplemented with 10% FBS.
Trametinib, everolimus, and temsirolimus were obtained from LC Laboratories. Linsitinib was a
generous gift from Astellas. INK-128, BMS-7547807, AZD8055, paclitaxel, GDC0941, gefitinib, and
lapatinib were obtained from Selleck. PP-242 was obtained from Sigma. NVP-AEW541 was
obtained from Cayman. ARS-1620 was obtained under a material agreement with Araxes Pharma,
LLC.
Whole-genome shRNA screen
Whole-genome shRNA was performed using the MISSION Lentiplex Pooled shRNA library from
Sigma (SHPH01). The library contains 82,000 shRNA constructs from the TRC collection, targeting
more than 15,000 genes, and is divided into 10 different pools, which were infected and sequenced
separately.
H23 cells were infected in triplicate with the ready-to-use lentivirus together with 8 g/ml polybrene
at a multiplicity of infection (MOI)<1. 48 hours after the infection, cells were selected in 2 g/ml
puromycin for 48 h, followed by 24 h growth in fresh medium. Then, cells were divided into five
different aliquots. One aliquot was frozen to be used as initial shRNA representation (time=0). The
other four aliquots were seeded in 15 cm dishes, and starting the following day, the cells were
treated with DMSO, 1.5 nM trametinib, 1 M linsitinib, or 1.5 nM trametinib plus 1 M linsitinib for 6
days. Drugs were replaced after three days of treatment. At the end of the treatment, cells were
trypsinized, counted to measure the efficiency of the treatment, and frozen. All the different steps of
the screen including the genomic DNA extraction and the PCR detailed below were done in order to
keep a 400 representation of each shRNA. Each pool contained around 8,200 shRNAs, therefore, a
minimum of 3.3x106 cells were used in each step to ensure that at least 400 cells contained an
individual shRNA.
Genomic DNA was isolated from all the samples using Gentra Puregene Cell kit (Qiagen). shRNA
inserts were retrieved from the genomic DNA by PCR amplification using the following conditions.
(1) 98 C, 30 s; (2) 98 C, 10 s; (3) 60 C, 20 s; (4) 72 C, 1 min; (5) to step 2, 16 or 13 cycles (for PCR1
or PCR2, respectively); 72 C, 5 min. Indexes and adaptors for deep sequencing (Illumina) were
incorporated in the PCR primers. For PCR1, gDNA was amplified using Illuseq_x_PLKO1_f (where x
indicates a different barcode) and P7_pLKO1_r primers. 2.5 l of PCR1 product were used as
templates for PCR2 reaction, together with P5_IlluSeq and P7 primers. The list of primers used is
provided below.
Illuseq_1_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTATCACGCTTGTGGAAAGGACGAAACACCGG
Illuseq_2_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGATGTCTTGTGGAAAGGACGAAACACCGG
Illuseq_3_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCAATCTTGTGGAAAGGACGAAACACCGG
Illuseq_4_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGATCAGCTTGTGGAAAGGACGAAACACCGG
Illuseq_5_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGCTTCTTGTGGAAAGGACGAAACACCGG
Illuseq_6_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCTACCTTGTGGAAAGGACGAAACACCGG
Illuseq_7_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTACTTGTGGAAAGGACGAAACACCGG
Illuseq_8_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATTCCTTGTGGAAAGGACGAAACACCGG
Illuseq_9_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTATTCCTCTTGTGGAAAGGACGAAACACCGG
Illuseq_10_PLKO1_F
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGGCACTTGTGGAAAGGACGAAACACCGG
P7_pLKO1_R
CAAGCAGAAGACGGCATACGAGATTTCTTTCCCCTGCACTGTACCC
P5_IlluSeq
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
P7
CAAGCAGAAGACGGCATACGAGAT
Final PCR product was purified using MiniElute PCR Cleanup (Qiagen) and quantified using
Bioanalyzer. Finally, shRNA representation for each sample was measured by Next Generation
Sequencing (Illumina). The shRNA sequences were extracted from the sequencing reads and
aligned to TRC library. shRNAs not represented by at least 50 reads in each sample were removed
at this stage. Counts were then normalized to the maximum total number of aligned reads across all
samples for that pool. Comparisons were performed by considering the mean log fold change across
triplicates (with all counts incremented by 1 to avoid the possibility of taking the log of zero), with p
value calculated by a paired t-test on these log values. In addition, the following “soft” filters were
applied: t-test p-value must be <= 0.15 and genes must be targeted by at least two distinct shRNAs
with mean ratio of absolute counts between drug treatment and vehicle <= 0.65.
Lentivirus-mediated shRNA knockdown
Lentiviral plasmids (pLKO.1) encoding shRNAs were obtained from Sigma. Lentiviral particles were
generated by transfection of HEK-293 cells with the shRNA-containing vector together with the
packaging vectors pCMV-VSVG and pCMV-8.2 (Addgene). 48 hours after transfection, virus
particles in the supernatant were harvested, filtered, and transduced onto target cells. After 48
hours, transduced cells were selected with puromycin for 48 hours and then plated for viability or
expression experiments.
siRNA transfection
Cells were reverse-transfected with a final concentration of 25 nM siGENOME KRAS siRNA pool or
RISC-free control using DharmaFECT 1 transfection reagent (Dharmacon) in 96-well plates. Drug
treatment was administered 24 hours after the transfection, and cell viability was measured using
Cell Titer Blue (Promega) 72 hours later.
NRAS CRISPR/Cas knockout
To knock out NRAS, 3LL cells were transiently transfected with the pSpCas9(BB)-2A-GFP vector
(PX458, Addgene) expressing a GFP expression cassette and with the gRNA 5’-gRNA-‘3
ACTGGACACAGCTGGACATG, PAM: ATG. GFP-positive cells were sorted using a MoFlo XDP
sorter and single cell cloned manually. Clones were amplified and NRAS protein was quantified by
Western blot.
Western Blotting
Cell lysates were made using Cell Lysis Buffer (Cell Signaling Technology), and protein
concentrations in the samples were quantified using a detergent-compatible modified Lowry reaction
(BioRad DC Protein Assay). The same amount of protein was loaded for each sample, and samples
were subjected to electrophoresis in 4–12% gradient NuPAGE Novex
Bis-Tris gels (Life Technologies) under reducing conditions, and subsequently transferred to
polyvinylidene difluoride membranes (Millipore Immobilon-P). Bound primary antibodies were
incubated with horseradish peroxidase-conjugated secondary antibodies and detected using
chemiluminescence (Luminata HRP substrate, Millipore). Alternatively, membranes were incubated
with secondary conjugates compatible with infrared detection at 700 nm and 800 nm, and
membranes were scanned using the Odyssey Infrared Imaging System (Odyssey, LICOR). Western
blot quantification was done using Image Studio or Image Lab software. Antibodies directed against
phospho-ERK (T202/Y204, 9101), ERK (9107), phospho-AKT (S473 and T308, 9271 and
13038), AKT (2920), phospho-S6 (S235/236, 2211), S6 (2317), phospho-IGF1R (Y1135/1136,
3024), IGF1R (3027), phospho-EGFR (Y1068, 3777), EGFR (4267), phospho-ERBB2
(Y1221/1222, 12708), ERBB2 (4290), phospho-ERBB3 (Y1289, 2842), ERBB3 (12708),
phospho-PRAS40 (T246, 2640), phospho-4EBP1 (T37/46), GRB2 (3972), and cleaved-PARP
(9541) were obtained from Cell Signaling. Antibody directed against NRAS (sc-31) was obtained
from Santa Cruz, and antibodies against vinculin (V4505) and pan-RAS (05-516) were obtained from
Sigma.
RAS-GTP pull-down
After drug treatment, cells were lysed using MBL buffer (5% NP40, 750 mM NaCl, 125 mM Hepes,
50 mM MgCl2, 5 mM EDTA, 10% glycerol) and protein was quantified. To assess RAS-GTP, 500 g
of protein was incubated with RAF-RBD agarose beads (RAS activation assay kit, Merck), rotating at
4 C for 1 hour. Beads were washed 3 times with MBL buffer, resuspended in 1.5x Laemmli buffer,
and boiled for 5 minutes at 95 C.
Cell viability and apoptosis assays
Cells were seeded in 96-well plates, and drugs were added 24 hours later. For 6-day viability
assays, drug was replaced after 3 days of treatment. Starting cell density was optimized to produce
an 80-90% confluent monolayer in mock-treated cells at the conclusion of the experiment. Cell
viability was determined by Cell Titer Blue (Promega). Apoptosis induction was measured using a
caspase-3/7 substrate conjugated to rhodamine 110 (Invitrogen). Alternatively, live-cell proliferation
and apoptosis were measured using Incucyte (Essen Instruments). Cells plated in 96-well format
were imaged every three hours for five days. Proliferation was assessed by bright-field evaluation of
cell confluence, whereas apoptosis was quantified using IncuCyte Caspase-3/7 Green Reagent
(Essen Bioscience). FACS analysis was used as an additional method to quantitate apoptotic and
dead cells. 72 hours after treatment, cells were harvested, resuspended in Annexin V binding buffer,
and stained with FITC Annexin V (BD Biosciences) and DAPI.
For long-term drug treatments, cells were seeded in 24-well plates and treated with drugs for 7 to 17
days. Drugs were replaced every two or three days. Starting cell density was optimized to obtain an
80% confluent monolayer in the DMSO-treated cells after 7 days of treatment (when the first plate
was fixed). At different time points, cells were fixed and stained with a solution containing 2%
ethanol and 0.2% crystal violet.
Synergy scores were calculated using the Chalice Bioinformatic software from Horizon,
http://chalice.horizondiscovery.com/analyzer-server/cwr/. The synergy score is a positive-gated,
inhibition-weighted volume over Loewe additivity. It provides an additional prioritization which favors
combinations at high effect levels and ignores antagonistic portions of the response surface. The
synergy score is always a positive value with no natural scale, which can be used for comparison
between groups.
3D viability assays
1,500 to 3,000 cells were mixed with 2.5% matrigel (Corning) in culture medium and placed in 96-
well ultralow attachment plates (Costar). Spheroid formation was initiated by centrifugation at
1000xg for 5 minutes. Drugs were added 24 hours later and replaced after three days. Cell viability
was determined by CellTiter-Glo 3D Cell Viability Assay (Promega).
Quantitative RT-PCR
RNA was isolated (RNeasy Mini Kit, Qiagen) and reverse transcription was conducted (High
capacity cDNA Reverse Transcription kit, Applied Biosystems) using standard methods. Quantitative
real-time PCR was conducted using gene-specific primers (Quanti-Tect Primer Assays, Qiagen) for
MTOR, RRAGC, TSC2, or GAPDH with Fast SYBR Green Master Mix (Applied Biosystems).
Droplet digital PCR
ddPCR to detect the copy number of wild-type and mutant KRAS was done as previously described
(55). 20x primePCR ddPCR Mutation assays were purchased from Bio-Rad. RNaseP was used as
control.
Phospho-RTK array
Cells were seeded in 6 cm plate format and treated with the indicated drugs for 24 hours. Cell
lysates were prepared using Lysis Buffer 17 and analyzed using Human Phospho-RTK arrays (R&D
Systems, ARY001B) according to the manufacturer’s guidelines.
IGF1 and IGF2 ELISA
Cells were plated in 12-well format. 24 hours later, the cells’ medium was changed. Conditioned
medium was collected 24 hours later, and IGF1 and IGF2 were measured using IGF1 and IGF2
ELISA kits (R&D Systems, DG100 and DG200) according to manufacturer’s guidelines.
Fig. S1. A whole-genome shRNA screen identifies combinatorial drug targets. (A) H23 cells
were treated with 1 M linsitinib (Lins), 1.5 nM trametinib (Tram), or the combination of both. Cell
density was measured every three hours using Incucyte. (B) Cell viability of H23 cells infected with
each individual shRNA pool (the library was divided into 10 pools) during the screen. Infected cells
were treated with 1 M linsitinib, 1.5 nM trametinib, or the combination of both, and cell number was
measured using Countess cell counter. (C) Number of genes that when knocked down increased
sensitivity to the indicated drugs. (D) Dot-plot showing the fold change (log2) in number of reads
between vehicle and treated conditions vs the p-value of the difference between the two treatment
conditions for each shRNA. The plot was generated considering the results from the biological
triplicate of the experiment. Each dot represents one shRNA from the screen. MTOR, RRAGC, and
TSC2 shRNAs with a p-value <0.15 are highlighted. (E) H23 cells were infected with the indicated
shRNAs. After puromycin selection, cells were treated for six days with serial dilutions of trametinib,
and cell viability was measured. Mean ± SD of biological replicates and representative of three
independent experiments. (F) Knockdown efficiencies of H23 cells infected with the indicated
shRNAs. RNA was extracted after puromycin selection, and gene expression was measured by
quantitative PCR. Mean ± SD of 2-4 independent experiments, unpaired Student’s t-test. (G) H358
cells were infected with the indicated shRNAs. After puromycin selection, cells were treated for six
days with serial dilutions of linsitinib, and cell viability was measured. Mean ± SD of biological
replicates and representative of two independent experiments. (H) Knockdown efficiencies of H358
cells infected with the indicated shRNAs. RNA was extracted after puromycin selection, and gene
expression was measured by quantitative PCR. Mean ± SD of two independent experiments,
unpaired Student’s t-test. (I) H23 cells were infected with the indicated shRNAs and after puromycin
selection, cell lysates were probed with the indicated antibodies. Right panel shows the western blot
quantification for S6 phosphorylation. Mean ± SD of three independent experiments, unpaired
Student’s t-test. The numbers in each shRNA indicate the last two numbers in the shRNA name
(data file S1).
Fig. S2
A
B
D
E
H358
100 101 102 1030
25
50
75
100
125
Everolimus (nM)
Via
bilit
y
H358
100 101 102 1030
25
50
75
100
125
Temsirolimus (nM)
Via
bilit
y
H358
100 101 102 1030
25
50
75
100
125
PP-242 (nM)
Via
bilit
y
H358
100 100.5 101 101.5 102 102.50
25
50
75
100
125
INK-128 (nM)
Via
bilit
y
DMSO
Tram
Lins
Lins+Tram
H358
100 100.5 101 101.5 102 102.50
25
50
75
100
125
AZD8055 (nM)
Via
bilit
y
DMSO
Lins
BMS
NVP
H23
100 101 102 1030
1
2
3
4
Everolimus (nM)
Ap
op
tosis
in
du
cti
on
H23
100 100.5 101 101.5 102 102.50
1
2
3
4
AZD8055 (nM)
Ap
op
tosis
in
du
cti
on
H358
100 101 102 1030.0
2.5
5.0
7.5
10.0
Everolimus (nM)
Ap
op
tosis
in
du
cti
on
2.5
H358
100 100.5 101 101.5 102 100.0
2.5
5.0
7.5
10.0
AZD8055 (nM)
Ap
op
tosis
in
du
cti
on
DMSO
Tram
Lins
Lins+Tram
H1792Ever
+Lins
+Tr 5nM
Ever
+Lins
+Tr 20nM
Ever
+GDC
+Tr 5nM
day 7
day 13
day 16
day 10
Veh
HCC95Ever
+Lins
+Tr 5nM
Ever
+Lins
+Tr 20nM
Ever
+GDC
+Tr 5nM
day 7
day 13
day 16
day 10
Veh
KRAS mutant KRAS wild-type
F
Veh
Lins+
Ev
Lins+
Tr
Lins+
Ev+
Tr0
20
40
60
% A
nn
exin
V+
H1792
**
****
C
AZDAZD
+Lins
AZD
+Lins
+Tram AZDAZD
+Lins
AZD
+Lins
+Tram
day 7
day 13
day 16
H23
day 10
AZD8055 20nM AZD8055 100nM
day 7
day 13
day 16
H358
day 10
AZD
AZD
+Lins
AZD
+Lins
+Tram AZD
AZD
+Lins
AZD
+Lins
+Tram
AZD8055 20nM AZD8055 100nM
day 7
day 13
day 16
H1792
day 10
AZDAZD
+Lins
AZD
+Lins
+Tram AZD
AZD
+Lins
AZD
+Lins
+Tram
AZD8055 20nM AZD8055 100nM
Fig. S2. Combination of mTOR inhibitors with IGF1R and MEK inhibitors reduces the viability
of KRAS-mutant NSCLC cells. (A) H358 cells were treated with serial dilutions of the rapalog
temsirolimus or mTOR kinase inhibitors, PP-242 or INK-1258, in the presence or absence of 1 M
linsitinib, 5 nM trametinib, or the combination of both. Cell viability was measured after 6 days. Mean
± SD of biological replicates and representative of two independent experiments. (B) H358 cells
were treated with serial dilutions of everolimus or AZD8055 in combination with a single dose of
IGF1R inhibitor (1 M linsitinib, 150 nM BMS-754807, or 1 M NVP-AEW541). Cell viability was
measured after 6 days. Mean ± SD of biological replicates and representative of two independent
experiments. (C) Percentage of Annexin V-positive cells after 72 h treatment with the indicated
combinations (1 M linsitinib, 5 nM trametinib, and 40 nM everolimus). Mean ± SD, unpaired
Student’s t-test. (D) Apoptosis induction (caspase-3 cleavage) in H23 and H358 cells treated for 48
hours with serial dilutions of everolimus or AZD8055 in presence or absence of 1 M linsitinib, 1 nM
or 5 nM trametinib (1 nM for H23 cells and 5 nM for H358), or the combination of both. Mean ± SD of
biological replicates and representative of two independent experiments. (E) KRAS-mutant cells
were treated with several drug combinations (20 nM or 100 nM AZD8055, 1 M linsitinib, 1 nM or 5
nM trametinib) and stained with crystal violet at various time points. (F) Cells were treated with
several drug combinations (5 nM or 20 nM trametinib, 40 nM everolimus, 1 M linsitinib, or 1 M
GDC-0941) and stained with crystal violet at various time points.
Fig. S3
A
B
C
D
H23 H358 H1792 H2122 Calu-1SK-LU-1 H441 H1944A427A549
0
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Everolimus
312.5
156.2
78.1
39.1
19.5
9.8
4.9
2.4
0
AZD8055
H23 H358 H1792 H2122 Calu-1SK-LU-1 H441 H1944A427A549
0
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64
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821250
625
312
156
78
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0
Ever (nM)+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin
KR
AS
-mu
tan
tK
RA
S-m
uta
nt
MUT WT0.0
0.5
1.0
1.5
2.0
2.5
Syn
erg
y S
co
re
Everolimus + Linsitinib
**
MUT WT0.0
0.5
1.0
1.5
Syn
erg
y S
co
re
AZD8055 + Linsitinib
*
Tram Lin
s
Lins+
Tram
0
25
50
75
100
125
Via
bilit
y
**
***
KRAS-mutant
Tram Lin
s
Lins+
Tram
0
25
50
75
100
125
Via
bilit
y
ns
KRAS wild-type
ns
EKVX LUDLU H226HCC78 HCC95H2170H520
0
39
43
51
54
58
54
55
56
0
33
41
47
46
51
57
55
56
0
37
39
44
39
38
47
49
52
0
69
75
76
77
77
75
72
69
27
75
80
80
80
79
80
80
78
18
81
84
84
83
83
83
85
82
0
65
69
69
69
71
72
72
73
12
79
80
80
81
78
81
80
81
56
85
86
85
85
84
86
85
85
0
0
0
0
0
2
0
0
0
19
46
51
54
52
45
43
43
50
40
63
68
66
59
55
55
60
63
55
52
55
56
53
53
53
58
60
60
59
59
62
59
59
60
69
68
71
71
72
72
68
73
24
27
36
39
39
36
33
36
26
45
56
57
56
57
54
54
55
60
68
75
75
73
73
72
70
72
17
14
14
13
13
11
12
0
33
32
36
36
35
30
23
31
0
42
39
41
41
40
34
28
451250
625
312
156
78
39
19
10
0
KR
AS
wil
d-t
yp
e
Ever (nM)+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin
50 100 0
% Inhibition
AZD (nM)+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin
EKVX LUDLU H226HCC78 HCC95H2170H520
14
29
52
74
87
93
0
2
6
14
29
52
74
87
93
0
2
6
14
29
52
74
87
93
0
21
29
41
54
68
78
88
92
27
51
54
63
69
78
87
92
94
18
41
52
57
69
79
88
94
96
0
3
19
28
43
57
68
79
87
6
24
43
53
65
72
84
89
92
48
62
67
69
75
81
88
91
93
0
0
2
8
9
18
23
33
48
14
39
50
54
58
61
67
75
81
36
66
69
69
73
75
79
84
87
0
29
33
44
53
62
71
75
81
0
34
41
51
60
70
75
79
87
32
55
59
65
71
77
83
88
95
0
8
16
29
39
50
54
64
70
17
40
47
54
59
65
71
74
76
46
65
69
72
75
77
79
80
83
0
10
10
22
31
45
52
56
66
0
30
35
43
52
62
65
69
75
0
24
31
38
52
60
68
68
80312.5
156.2
78.1
39.1
19.5
9.8
4.9
2.4
0
KR
AS
wil
d-t
yp
e
AZD (nM)+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin+Lin
+Tr+Lin
50 100 0
% Inhibition
Fig. S3. KRAS-mutant cells show increased sensitivity to the combination of mTOR, IGF1R,
and MEK inhibitors. (A) Mutational status of 10 KRAS-mutant and 7 KRAS wild-type NSCLC cell
lines. The last two columns show the synergy score for the combination of everolimus or AZD8055
plus linsitinib obtained using Chalice Analyser. (B) Percentage of inhibition of viability of cells treated
with serial dilutions of everolimus (Ever) or AZD8055 (AZD) in the presence of 1 M linsitinib (+Lin)
or 1 M linsitinib plus 5 nM trametinib (+Lin+Tr). (C) Synergy score for everolimus or AZD8055 plus
IGF1R inhibitors obtained using Chalice Analyser. All the data used to obtain the values have been
plotted in panel B. Mean ± SD, unpaired Student’s t-test. (D) Viability of NSCLC cells treated with 5
nM trametinib, 1 M linsitinib, or the combination of both. Mean ± SD, unpaired Student’s t-test.
Fig. S4. Combination of IGF1R with mTOR inhibitors blocks PI3K/AKT and mTOR pathways.
(A) Dose-response and time-course of mTOR inhibitors in KRAS-mutant NSCLC cells. For the dose
response, cells were treated with various doses of everolimus (Ever) or AZD8055 (AZD) for 24
hours. For the time-course, cells were treated with everolimus 40 nM or AZD8055 20 nM and cell
lysates were obtained at various time points. (B) Model of the signaling pathway inhibition produced
by mTOR and IGF1R inhibitors. (C) KRAS-mutant and KRAS wild-type cells were treated for 24 h
with 40 nM everolimus, 20 nM AZD8055, 1 M linsitinib, or the combinations. Cell lysates were
probed with the indicated antibodies. Western blots for AKT phosphorylation are shown in Fig. 3C.
(D) Effect of linsitinib on AKT phosphorylation. Cells were treated with 1 M linsitinib for 24 hours,
and cell lysates were probed with antibodies for phospho-Ser473-AKT and phospho-Thr308-AKT.
For all western blots, see Fig. 3C and panel C of this figure. (E) Correlation between the relative
phosphorylation of AKT and relative viability of cells treated with everolimus plus linsitinib compared
with the values in cells treated with everolimus alone. AKT phosphorylation and viability data of cells
treated with everolimus plus linsitinib were normalized to the values of cells treated with everolimus
alone (set to value 1). This represents the relative decrease of AKT phosphorylation or viability
between the two treatments (Ev+Lins vs Ev). KRAS-mutant cells (MUT) are indicated in black and
wild-type (WT) in gray. (F) Model of the signaling pathway inhibition produced by the combination of
MEK, IGF1R, and mTOR inhibitors.
Fig. S5. mTOR inhibition activates the IGF1R pathway in KRAS-mutant cells. (A) H23 cells
were treated with DMSO, 100 nM everolimus, or 80 nM AZD8055 for 24 h. Cell lysates were
assayed using a phospho-RTK array kit. Phosphorylated RTKs that change their amounts with the
drug treatment are highlighted with boxes. (B) Short exposure of the phospho-RTK array shown in
Fig. 4C. (C) RASless MEFs were treated for 24 hours with either vehicle or 1 M linsitinib. Cell
lysates were probed with the indicated antibodies. (D) RASless MEFs were treated with 40 nM
everolimus, 1 M linsitinib, and/or 5 nM trametinib. Cell viability was measured after 3 days. Mean ±
SD of biological replicates and representative of three independent experiments.
Fig. S5
A B
Vehicle
Everolimus
AZD8055
H23(KRAS-mutant) IGF1R
C
Ctr
l
Lin
s
Ctr
l
Lin
s
Ctr
l
Lin
s
Ctr
l
Lin
s
pIGF1R-Y1135/36
IGF1R
AKT
pERK1/2-202/204
ERK1/2
pAKT-S473
WT G12C G12D G13DKRAS:
RASless MEFs
Veh Ev
Ev+Lin
s
Ev+
Lins+
TrVeh E
v
Ev+Lin
s
Ev+
Lins+
TrVeh E
v
Ev+Lin
s
Ev+
Lins+
TrVeh E
v
Ev+Lin
s
Ev+
Lins+
Tr0
25
50
75
100
125
via
bilit
y
RASless MEFs
WT G12C G12D G13DKRAS:
D
ERBB2
Vehicle
Everolimus
AZD8055
H2170(KRAS wild-type)
Ev+Lins+Tr Pacl+Tr
0
200
400
600
800
-100
Vo
lum
e c
han
ge (
%)
***
KRASLSL-G12D;Trp53flox/flox mice
0 5 10 15 20 2560
70
80
90
100
110
Days%
weig
ht
Vehicle
Lins+Ev+Tr
Fig. S6
Tr
Tr+Lin
s
Tr+L
ins+
Ev
-100
0
100
200
300500
1000
Volu
me c
hange (
%)
**** *
A D
Vehicle Ev Lins+Ev Lins+Ev+Tr
pS6 -S235/236
S6
pAKT-S473
pAKT-T308
pERK1/2
ERK1/2
AKT
IGF1R
pIGF1R-Y1135/6
Vehicle Ev Lins+Ev Lins+Ev+Tr
H
G
Ve
hic
leL
ins
+E
v+
Tra
m
week 0 week 4
Urethane-induced tumors
Veh Ev
Lins+
Ev
Lins+
Ev+
Tr0
1
2
3
4
5
p473-A
KT
/AK
T ** *****
Veh Ev
Lins+
Ev
Lins+
Ev+Tr
0.0
0.5
1.0
1.5
2.0
pS
6/S
6
*** *
*
Veh Ev
Lins+
Ev
Lins+
Ev+Tr
0
1
2
3
4
pE
RK
/ER
K
** **
****
Veh E
v
Lins+
Ev
Lins+
Ev+
Tr0
1
2
3
4
5
6
7
pIG
F1R
/IG
F1R
* ****
B
Urethane-induced tumors
0 5 10 15 20 25 3060
70
80
90
100
110
Days
% w
eig
ht
Vehicle
Lins+Ev+Tr
Lins+
Ev
Lins+
Tram
Ev+Lin
s+Tr
-100
-50
0
50
100
Vo
lum
e c
han
ge (
%)
****** ***
JKRASLSL-G12D;Stk11flox/flox mice
KRASLSL-G12D;Trp53flox/flox mice
-100
-50
0
50
100
Vo
lum
e c
han
ge (
%)
Lins+Ev+Tr
KRASLSL-G12D;Trp53flox/flox mice
week: 2 4 7 9 11
Drug Drug
scan scan
C
KRASLSL-G12D;Trp53flox/flox mice
KRASLSL-G12D;Trp53flox/flox mice
EF
Urethane-induced tumorsI
0 4 7 11
0
250
500
750
-100
1000
1500
Vo
lum
e c
han
ge (
%)
Lins+Ev+Tr Lins+Ev+Trweek:
ns***
**
scan
week: 0 4 7 11
scan
Drug Drug
scan scan
0 4 7 11
0
100
200
300
400
-100
Vo
lum
e c
han
ge (
%)
Lins+Ev+Tr Lins+Ev+Trweek:
*********
scan
week: 0 4 8 12
scan
Drug Drug
scan scan
Fig. S6. Combination of mTOR, IGF1R, and MEK inhibitors results in regression of KRAS-
driven lung tumors. (A) Volume change, between the start and the end of the treatment, of tumors
from Kras LSL-G12D;Trp53Flox/Flox mice treated 4 weeks with trametinib 2 mg/kg as single treatment or in
combination with linsitinib 25 mg/kg and/or everolimus 2.5 mg/kg (4-5 mice per group). Median and
statistics using Mann-Whitney test are shown. (B) Body weight of Kras LSL-G12D;Trp53Flox/Flox mice
treated with vehicle or linsitinib 25 mg/kg, everolimus 2.5 mg/kg, and trametinib 2 mg/kg for the
indicated times. Unless otherwise stated, all experiments with Kras LSL-G12D;Trp53Flox/Flox mice have
been done using the same drug doses. (C) Kras LSL-G12D;Trp53Flox/Flox mice were treated with linsitinib,
everolimus, and/or trametinib. After 4 days, tumors were extracted and protein lysates were probed
with the indicated antibodies. Graphs show the quantification of the western blots. Mean ± SD,
unpaired Student’s t-test. (D) Volume change of tumors from Kras LSL-G12D;Trp53Flox/Flox mice treated 4
weeks with trametinib 2 mg/kg, linsitinib 25 mg/kg, plus everolimus 2.5 mg/kg or trametinib 2 mg/kg
plus paclitaxel 10 mg/kg (4 mice per group). Median and statistics using Mann-Whitney test are
shown. (E) Kras LSL-G12D;Trp53Flox/Flox mice were treated with linsitinib, everolimus, and trametinib (4
mice per group). Tumor volume change after 4 weeks of treatment is shown, followed by
discontinuation of the treatment for 3 weeks and a second 4-week cycle of treatment. Median,
Mann-Whitney test. (F) Waterfall representation of the response of each tumor after the second
cycle of 4 weeks of treatment. (G) Body weight of mice with urethane-induced lung tumors treated
with either vehicle or linsitinib 17 mg/kg, everolimus 1.7 mg/kg, and trametinib 1.3 mg/kg for the
indicated times. (H) Representative micro-computed tomography 3D reconstruction of the lungs from
urethane-treated mice before and after 4-week treatment. Each color represents an individual tumor.
(I) Mice with urethane-induced lung tumors treated with linsitinib 17 mg/kg, everolimus 1.7 mg/kg,
and trametinib 1.3 mg/kg. Tumor volume change after 4 weeks of treatment is shown, followed by
discontinuation of the treatment for 4 weeks and a second cycle of treatment of 4 weeks. Median,
Mann-Whitney test. (J) Volume change of tumors from Kras LSL-G12D;Stk11Flox/Flox mice treated with 25
mg/kg linsitinib, 2.5 mg/kg everolimus, and/or 2 mg/kg trametinib (4 mice per group). Median and
statistics using Mann-Whitney test are shown.
Fig. S7
A
D
Vehicle
Ev
Ev+Lins
Ev+Lins+Tram
0 24 48 72 96 120 1440
25
50
75
100
Time elapsed (hours)
% c
on
flu
en
cy
H23
0 24 48 72 96 120 1440
25
50
75
100
Time elapsed (hours)
% c
on
flu
en
cy
H358
E
H
0 24 48 72 96 120 1440
25
50
75
100
Time elapsed (hours)
% c
on
flu
en
cy
Vehicle
ARS
ARS+Lins
ARS+Ev
ARS+Lins+Ev
H1792
2D ( non KRAS-G12C cells)
100 101 102 103 1040
25
50
75
100
125
ARS1620 (nM)
Via
bilit
y
3D ( non KRAS-G12C cells)
100 101 102 103 1040
25
50
75
100
125
ARS1620 (nM)
Via
bilit
y
A549
HCC95
(KRAS-G12S)
(KRAS-wild-type)
ARS1620 + Everolimus + Linsitinib
IGF1R
IRS1/2
PI3K
AKT
mTORC1
TSC1TSC2
Rheb
S6K
ShcSOS
Grb2
RTK
MEK1/2
ERK1/2
RAF
KRASG12C
T308
S473
mTORC2
IGF1/2
p85
S6
p110
Everolimus
Linsitinib
ARS-1620
BC
2D (KRAS-G12C cells)
100 101 102 103 1040
25
50
75
100
125
ARS1620 (nM)
Via
bilit
y
3D (KRAS-G12C cells)
100 101 102 103 1040
25
50
75
100
125
ARS1620 (nM)
Via
bilit
y
H23
H1792
H2030
HCC44
Calu-1
H358
H1373
L
G
day 5
day 10
day 13
day 8
Veh EvEv
+Lins
Ev
+Lins
+Tram
A549 (KRAS-G12S mutant)
ARS
+Lins
ARS
+Ev
ARS
+Lins
+EvARS
day 7
day 13
day 17
day 10
Veh EvEv
+Lins
Ev
+Lins
+Tram
H226 (KRAS wild-type)
ARS
+Lins
ARS
+Ev
ARS
+Lins
+EvARS
day 7
day 13
day 17
day 10
Veh EvEv
+Lins
Ev
+Lins
+Tram
AT2 (KRAS wild-type)
ARS
+Lins
ARS
+Ev
ARS
+Lins
+EvARS
pS6-S235/236
S6
pAKT-T308
pERK1/2-202/204
ERK1/2
AKT
pAKT-S473
C
A549
AR
S
AR
S+
Lin
s
AR
S+
Ev
Lin
s+
Ev
Lin
s
Ev
Tr+
Lin
s+
Ev
AR
S+
Lin
s+
Ev
0
25
50
75
100
125
Via
bilit
y
RISC-f
KRAS siRNA
ARS-1620
Lins + Ev
+ +
H358(KRAS-G12C)
A549(KRAS-G12S)
+ + + - -
+ +- - - -
+ + - -- -
+ + +- - -
+ + + - -
+ +- - - -
+ + - -- -
+ + +- - -
F
H23 (KRAS-G12C mutant)
VehLins
+Ev
ARS
+Lins
+EvARSARS
+GDCGDC
day 7
day 13
day 17
day 10
ARS
+GefGef
ARS
+LapLap
H358 (KRAS-G12C mutant)
VehLins
+Ev
ARS
+Lins
+EvARSARS
+GDCGDC
day 7
day 13
day 17
day 10
ARS
+GefGef
ARS
+LapLap
HCC95 (KRAS wild-type)
VehLins
+Ev
ARS
+Lins
+EvARSARS
+GDCGDC
day 7
day 13
day 17
day 10
ARS
+GefGef
ARS
+LapLap
H1792 (KRAS-G12C mutant)
VehLins
+Ev
ARS
+Lins
+EvARSARS
+GDCGDC
day 7
day 13
day 17
day 10
ARS
+GefGef
ARS
+LapLap
pS6-S235/236
S6
pAKT-T308
pERK1/2-202/204
ERK1/2
cleaved-PARP
AKT
pAKT-S473
C
H1792
AR
S
AR
S+
Lin
s
AR
S+
Ev
Lin
s+
Ev
Lin
s
Ev
Tr+
Lin
s+
Ev
AR
S+
Lin
s+
EvI J K
day 7
day 13
day 17
day 10
Veh EvEv
+Lins
Ev
+Lins
+Tram
H1792 (KRAS-G12C mutant)
ARS
+Lins
ARS
+Ev
ARS
+Lins
+EvARS
Ctrl
Lins+
EvARS
ARS+L
ins+
Ev0
20
40
60
80
% A
nn
exin
V+
H358
**
******
Ctrl
Lins+
EvARS
ARS+L
ins+
Ev0
10
20
30
40
% A
nn
exin
V+
H23
***
H23 H358 H17920.0
0.5
1.0
1.5
2.0
2.5
Rela
tiv
e c
op
y n
um
ber
(KR
AS
/ R
NaseP
)
KRAS-G12C
KRAS WT
Fig. S7. Drug combinations with a KRAS-G12C inhibitor cause inhibition of viability in KRAS-
mutant cells. (A) KRAS-mutant cells were treated with everolimus as single treatment or in
combination with the indicated drugs. Cell confluence was monitored over time using Incucyte. (B)
Percentage of Annexin V-positive cells after 72 h treatment with the indicated combinations. Mean ±
SD, unpaired Student’s t-test. (C) Model of the signaling pathway inhibition produced by the
combination of KRAS-G12C, IGF1R, and mTOR inhibitors. (D) NSCLC cells and non-transformed
lung cells (AT2) were treated with several drug combinations and stained with crystal violet at
various time points. (E) A549 cells were treated for 24 h with several drug combinations, and cell
lysates were probed with the indicated antibodies. (F) Cell viability of H358 and A549 cells
transfected with either control (RISC-f) or KRAS siRNA and treated 24 h later with 1 M ARS-1620
or 1 M linsitinib plus 40 nM everolimus. Cell viability was measured 72 hours after drug treatment.
(G) Cells were treated with several drug combinations and stained with crystal violet at various time
points. GDC-0941 (GDC), gefitinib (Gef), and lapatinib (Lap) were used at 1 M. (H) H1792 cells
were treated with the KRAS-G12C inhibitor ARS-1620 (1 M) as single treatment or in combination
with the indicated drugs. Cell confluence was monitored over time using Incucyte. (I) H1792 cells
were treated with several drug combinations and stained with crystal violet at various time points. (J)
H1792 cells were treated for 24 h with several drug combinations, and cell lysates were probed with
the indicated antibodies. (K) ddPCR was used to detect copy number. Wild-type KRAS and G12C
mutant KRAS were amplified from genomic DNA using ddPCR. Data were normalized to RNaseP,
used as reference control. (L) NSCLC cells were grown in either 2D-adherent monolayers or 3D-
spheroid suspension and treated with serial dilutions of ARS-1620. Cell viability was measured after
5 days. Mean ± SD of three independent experiments. When not otherwise indicated, treatments
have been done using 1 M linsitinib (Lins), 40 nM everolimus (Ev), 1 M ARS-1620 (ARS), and/or 5
nM trametinib.
Fig. S8. Combination of KRAS-G12C inhibitor, IGF1R, and mTOR inhibitors is effective in
vivo. (A) Sanger sequencing of Kras and Nras genes of parental 3LL cells. Mutated amino acid,
compared with wild-type sequence, is indicated in red. (B-C) Body weight of C57BL/6 mice with 3LL
NRAS-63 tumors treated with the indicated drug combinations. (B) Data at each indicated time
point are shown as meanSEM (5-7 mice per group). (C) Percentage of mouse weight at day 14 of
treatment compared with the weight at the start of the treatment. Mean ± SD, unpaired Student’s t-
test.
Fig. S8
AKras Nras
Vehic
le
ARS+L
ins+
Ev
Tr+L
ins+
Ev80
90
100
110
120
% w
eig
ht
Vehicle
ARS+Lins+Ev
Tr+Lins+Ev
*
0 5 10 1580
90
100
110
120
Days
% w
eig
ht
Vehicle
ARS+Lins+Ev
Tr+Lins+Ev
B C
C12A11 G13 V14G10
G T G G T T G G A G C T G G T G G C G T A G G C A A G A
G12A11 G13 V14G10
3LL
WT
H61G60A59 E62 E63
G A C A C A G C T G G A C A A G A G G A G T A C A G T G
Q61G60 E62 E63A59
3LL
WT
