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Supplemental Figures and Legends
Rationale for co-targeting IGF-1R and ALK in ALK fusion positive lung cancer
Christine M. Lovly1,20, Nerina T. McDonald1, Heidi Chen2, Sandra Ortiz-Cuaran3, Lukas C. Heukamp4,5, Yingjun Yan1, Alexandra Florin4, Luka Ozretić4, Diana Lim6, Lu Wang6, Zhao Chen7, Xi Chen2, Pengcheng Lu2, Paul K. Paik8, Ronglai Shen9, Hailing Jin1, Reinhard Buettner4, Sascha Ansén10, Sven Perner11, Michael Brockmann12, Marc Bos3,10, Jürgen Wolf10, Masyar Gardizi10, Gavin M. Wright13, Benjamin Solomon14, Prudence A. Russell15, Toni-Maree Rogers16, Yoshiyuki Suehara6, Monica Red-Brewer1, Rudy Tieu17, Elisa de Stanchina17, Qingguo Wang18, Zhongming Zhao18, David H. Johnson19, Leora Horn1, Kwok-Kin Wong7, Roman K. Thomas 3,4, Marc Ladanyi6, William Pao1
Departments of 1Medicine, 2Biostatistics, and 18Biomedical Informatics Vanderbilt University, Nashville, TN, 3Department of Translational Genomics, Center of Integrated Oncology Köln–Bonn, University Hospital Cologne, Cologne, Germany; Departments of 4Pathology and 10Internal Medicine (Department I), Center for Integrated Oncology Köln-Bonn, University Hospital Cologne, Cologne, Germany; 5New Oncology, Cologne, Germany; Departments of 6Pathology, 8Medicine, 9Epidemiology and Biostatistics and 17Anti-tumor Assessment Core Facility, Memorial Sloan Kettering Cancer Center, New York, New York; 7Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; 11Department of Prostate Cancer Research, Institute of Pathology, Center of Integrated Oncology Köln–Bonn, University Hospital of Bonn, Bonn, Germany; 12Department of Pathology, Hospital Merheim, Cologne, Germany; 13University of Melbourne, Department of Surgery, St Vincent's Hospital, Melbourne, Australia; 14Division of Hematology and Medical Oncology, Peter MacCallum Cancer Center, Melbourne, Australia; 15Department of Anatomical Pathology, St Vincent's Hospital, Melbourne, Australia; 16Department of Pathology, Peter MacCallum Cancer Center, Melbourne, Australia; 19Department of Medicine, UT Southwestern School of Medicine, Dallas, TX; 20Corresponding author: Vanderbilt University Medical Center and Vanderbilt-Ingram Cancer Center, 2220 Pierce Avenue, 777 Preston Research Building, Nashville, TN 37232-6307. Phone 615-936-3524; Fax: 615-343-7602; E-mail: christine.lovly@vanderbilt.edu
Nature Medicine: doi:10.1038/nm.3667
SUPPLEMENTAL FIGURE LEGENDS
Supplementary Figure 1: EGFR inhibition does not sensitize ALK+ lung cancer cells to
the effects of ALK inhibitors or IGF-1R inhibitors. (a) H3122 cells were treated with erlotinib
or erlotinib + MAb391. Soft agar assays were performed using hextuplicate biological replicates.
(b) H3122 cells were treated with erlotinib, OSI-906, or the combination. Cell titer blue assays
were performed using hextuplicate biological replicates. (c) STE-1 cells were treated with
crizotinib or crizotinib + MAb391. Soft agar assays were performed using hextuplicate biological
replicates. (d) H3122 cells were treated with crizotinib and OSI-906 as described in Fig. 2b.
Data from this experiment were analyzed as previously described1. The Loewe index provides a
measure of drug interaction. A Loewe index < 1 is indicative of a synergistic drug-drug
interaction. The blue line in the figure represents a Loewe index of 1. The dashed red lines
represent the upper and lower 95% confidence intervals. (e) SUDHL-1 cells were treated with
crizotinib, OSI-906, or the combination. Cell titer blue assays were performed using hextuplicate
biological replicates. (f) Quantification of H3122 xenograft tumor volumes after treatment with
the indicated inhibitors. *P = 0.41 by the Wilcoxon rank sum test. (g) H3122 cells were treated
crizotinib, erlotinib, or the combination. Cell titer blue assays were performed using hextuplicate
biological replicates. (h) H3122 cells were treated crizotinib, lapatinib, or the combination. Cell
titer blue assays were performed using hextuplicate biological replicates. (i) Quantification of
AKT phosphorylation from the western blot shown in Fig. 2f.
Supplementary Figure 2: IGF-1 ligand stimulates phosphorylation of IGF-1R but not ALK.
(a) H3122 cells were serum starved overnight, treated with vehicle or OSI-906, and then
stimulated with IGF-1 for 1min, 5min, or 10min prior to harvest. Lysates were subjected to
immunoblotting with antibodies specific for the indicated proteins. (b) Quantification of AKT
phosphorylation from the western blot shown in Fig. 3b.
Nature Medicine: doi:10.1038/nm.3667
Supplementary Figure 3: IRS-1 knock-down impedes the growth of H2228 ALK+ lung
cancer cells. (a) H2228 cells were transfected with the indicated siRNAs and treated with
500nM crizotinib for 72h.Triplicate biological replicates for each sample were counted on
Coulter Counter. Data are representative of two independent experiments. (b) Western blot
confirming IRS-1 knockdown in the experiment shown in Supplementary Fig. 3a.
Supplementary Figure 4: Characterization of ALK TKI resistant cells. (a) Results from ALK
FISH using the ALK Break Apart FISH probe in H3122 CR cells. (b) H3122 parental and
isogenic H3122 CR cells were treated with 500nM crizotinib. Lysates were subjected to
immunoblotting with the indicated antibodies. (c) Results from ALK FISH using the Vysis ALK
Break Apart FISH probe in H3122 XR cells. (d) Lysates from H3122 parental and isogenic
H3122 XR cells subjected to phospho-RTK arrays according to the manufacturer’s instructions.
(e) H3122 CR cells were treated with increasing amounts of crizotinib, OSI-906, or the
combination crizotinib + OSI-906 at the indicated concentrations for 72h. Cell titer blue assays
were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data
are presented as the percentage of viable cells compared to control. (f) Conditioned
supernatant from H3122 parental, H3122 CR, and H3122 XR cells were subjected to ELISA
assays for IGF-1 according to the manufacturer’s instructions. Data are presented as fold
change in IGF-1 level compared to H3122 parental cells.
Supplementary Figure 5: Efficacy of IGF-1R TKIs in EGFR mutant lung cancer cell lines.
Four different isogenic pairs of EGFR TKI sensitive and EGFR TKI resistant cell lines were used
in these experiments, including PC-9 parental (TKI sensitive cells) and PC-9 ERc1 (Erlotinib
Resistant clone 1) (a), HCC827 parental (TKI sensitive cells) and HCC827 ER (Erlotinib
Resistant) (b), HCC2279 parental (TKI sensitive cells) and HCC2279 ER (Erlotinib Resistant)
Nature Medicine: doi:10.1038/nm.3667
(c), and HCC4006 parental (TKI sensitive cells) and HCC4006 ER (Erlotinib Resistant) (d).
Please see Supplementary Table 1 for a summary of these cell lines. Cell were treated with
increasing amounts of erlotinib alone, OSI-906 alone, or the combination of erlotinib + 1µM OSI-
906 for 72h. Cell Titer Blue Assays were performed as previously described. Each point
represents hextuplicate biological replicates. Data are representative of two independent
experiments. The x-axis indicates the concentration of erlotinib or OSI-906 when these inhibitors
were used as single treatment agents. (e) Lysates from the four different isogenic pairs of EGFR
TKI sensitive (S) and TKI resistant (R) cell lines described above were resolved by SDS-PAGE
and subjected to immunoblotting with the indicated antibodies.
Supplementary Figure 6: Representative pIGF-1R pY1161 immunohistochemistry and
Nanostring data from patients with EGFR mutant lung cancer pre- and post-EGFR TKI
therapy. (a–c) Representative pIGF-1R pY1161 immunohistochemistry showing low (a),
medium (b), and high (c) pIGF-1R staining on lung tumor biopsies. Scalebars are shown and
represent 50 µm. All images viewed correspond to a magnification of 40x. (d–f) RNA was
extracted from 11 different matched pairs of snap-frozen tumor biopsy samples from patients
with EGFR mutant lung cancer pre- and post-EGFR TKI therapy. Panel (d) shows aggregate
data. 6 pairs of matched tumor samples were from patients who underwent biopsy prior to and
2 days post initiation of an EGFR TKI (e). 5 pairs of matched tumor samples were from patients
who underwent biopsy prior to and at the time of acquired resistance to EGFR TKI (f). P values
were determined with the paired T-test.
Supplementary Figure 7: Effects of LDK-378 in ALK+/TKI sensitive and ALK+/TKI
resistant cell lines. (a) STE-1 cells were treated with increasing amounts of crizotinib, LDK-
378, or TAE-684 at the indicated concentrations for 72h. Cell titer blue assays were performed
Nature Medicine: doi:10.1038/nm.3667
to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented
as the percentage of viable cells compared to control (vehicle only treated) cells and are
representative of two independent experiments. (b) H3122 XR cells were treated with increasing
amounts of X-376 or LDK-378 at the indicated concentrations for 72h. Cell titer blue assays
were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data
are presented as the percentage of viable cells compared to control (vehicle only treated) cells
and are representative of two independent experiments. (c) H3122 cells were treated with
crizotinib or LDK-378 for 72 hours prior to harvest. Cells were stained with propidium iodide (PI)
and counted on a FACSCalibur machine. Apoptosis was defined by the percentage of cells
harboring a sub-2N DNA content. This experiment was repeated two times. Representative
results are shown.
Supplementary Figure 8: Characterization of novel STE–1 ALK+ lung cancer cell line. (a)
Results from ALK FISH using the Vysis ALK Break Apart FISH probe in STE-1 cells. (b) cDNA
sequencing of ALK from STE-1 cells reveals the presence of an EML4-ALK E13;A20 (variant 1)
fusion.
Nature Medicine: doi:10.1038/nm.3667
SUPPLEMENTAL TABLE LEGENDS
Supplementary Table 1: Isogenic pairs of EGFR TKI sensitive and EGFR TKI resistant cell
lines. Summary of the isogenic pairs of EGFR TKI sensitive and EGFR TKI resistant cell lines
used in these studies. These cell lines have been previously described2,3.
Supplementary Table 2: Microarray data for crizotinib resistant cell lines. Summary of the
top 20 downregulated genes in the H3122 crizotinib resistant (CR) cells versus isogenic H3122
crizotinib sensitive parental cells as assessed by microarray analysis. Each value shown for a
specific gene represents a unique probe. Changes in IGFBP3 are noted in bold.
Supplementary Table 3: Microarray data for X-376 resistant cell lines. Summary of the top
20 downregulated genes in the H3122 X-376 resistant (XR) cells versus isogenic H3122
crizotinib sensitive parental cells as assessed by microarray analysis. Each value shown for a
specific gene represents a unique probe. Changes in IGFBP3 are noted in bold.
Supplementary Table 4: Summary of the mechanisms of crizotinib resistance evaluated
for in the study cohort. aPCR/Direct sequencing; bWhole exome sequencing; cRNA
sequencing; dWhole genome sequencing.
Supplementary Table 5: Index patient’s clinical genotyping results. Results from SNaPshot
clinical genotyping of the index patient's tumor. Prior to enrollment in the Profile 1007 study of
Nature Medicine: doi:10.1038/nm.3667
crizotinib vs. pemetrexed or docetaxel for patients with advanced ALK fusion positive lung
cancer, the patient’s tumor sample was sent for ALK FISH (as part of the study enrollment) and
also for our institutional SNaPshot lung cancer panel4. ALK FISH testing was positive at the
central laboratory (Esoterix). No other mutations were found based on the SNaPshot results.
Nature Medicine: doi:10.1038/nm.3667
SUPPLEMENTAL REFERENCES
1. Boik, J.C., Newman, R.A. & Boik, R.J. Quantifying synergism/antagonism using
nonlinear mixed-effects modeling: a simulation study. Stat Med 27, 1040-1061 (2008).
2. Chmielecki, J., et al. Optimization of dosing for EGFR-mutant non-small cell lung cancer
with evolutionary cancer modeling. Sci Transl Med 3, 90ra59 (2011).
3. Ohashi, K., et al. Lung cancers with acquired resistance to EGFR inhibitors occasionally
harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1. Proc Natl
Acad Sci U S A 109, E2127-2133 (2012).
4. Su, Z., et al. A platform for rapid detection of multiple oncogenic mutations with
relevance to targeted therapy in non-small-cell lung cancer. J Mol Diagn 13, 74-84
(2011).
Nature Medicine: doi:10.1038/nm.3667
Supplementary Fig. 1 (Lovly et al 2014)
a.
b.
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% g
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0 0.05 0.1 0.5 1 5
0102030405060708090
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% g
row
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Erlotinib (µM)
Erlotinib
OSI-906
Erlotinib + OSI-906
Nature Medicine: doi:10.1038/nm.3667
c.
d.
Loewe Index = 1: Drug combination is additive
Loewe Index > 1: Drug combination is antagonistic
Loewe Index < 1: Drug combination is synergistic
0102030405060708090
100
MAb391 100nM crizotinib 100nM crizotinib +MAb391
% g
row
th re
lativ
e to
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trol
Supplementary Fig. 1 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
f.
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h.
*
*
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1 4 10 14 17 20
Tum
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m3 )
Time (days)
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MAb391
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Erlotinib (µM)
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Crizotinib
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Crizotinib + erlotinib
0 0.05 0.1 0.5 1 5
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Crizotinib
Lapatinib
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0 0.5 1 5 10 50 100
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Crizotinib (μM) OSI-906 (μM)
Supplementary Fig. 1 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
i.
0102030405060708090
100110120130140150160170
Control IGF-1 Crizotinib OSI-906 IGF-1 +crizotinib
IGF-1 +OSI-906
IGF-1 +crizotinib +OSI-906
Pix
el in
tens
ity c
ompa
red
to c
ontro
l
Supplementary Fig. 1 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
1 2 3 4 5 6
– IGF-1 +1’ +5’ +10’ – +10’ – OSI-906 – – – + –
pALK Y1604
pIGF-1R Y1131
Actin
IGF-1R
ALK
a.
b.
0102030405060708090
100110120130140150160170
Control IGF-1 Crizotinib OSI-906 IGF-1 +crizotinib
IGF-1 +OSI-906
IGF-1 +crizotinib +OSI-906
Pix
el in
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ity c
ompa
red
to c
ontro
l
Supplementary Fig. 2 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
IRS-1
Crizotinib
NT siRNA
– – + + – +
IRS-1 siRNA (pool #1)
IRS-1 siRNA (pool #2)
pALK Y1604
Actin
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DMSO
IRS-1 siRNA(pool #2):crizotinib
% c
ell n
umbe
r rel
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e to
con
trol
Supplementary Fig. 3 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
a. b.
c.
H3122 Parental
H3122 XR
IGF-1R d.
Blot: ALK
Crizotinib – + – +
H3122 Parental
H3122 CR
Supplementary Fig. 4 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
e.
f.
0.0
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H3122 parental H3122 CR H3122 XR
Fold
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F-1
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0102030405060708090
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Crizotinib
OSI-906
Crizotinib + OSI-906
Crizotinib (μM)
OSI-906 (μM)
Supplementary Fig. 4 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
a.
c.
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d.
0102030405060708090
100110120
0 10 50 100 500 1,000
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PC-9 parental:erlotinib
PC-9 parental: OSI-906
PC-9 parental: erlotinib + OSI-906 (1μM) PC-9 ER: erlotinib
PC-9 ER: OSI-906
PC-9 ER: erlotinib + OSI-906 (1μM)
0102030405060708090
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Erlotinib (nM)
HCC827 parental:erlotinib
HCC827 parental:OSI-906
HCC827 parental: erlotinib + OSI-906 (1μM) HCC827 ER: erlotinib
HCC827 ER: OSI-906
HCC827 ER: erlotinib + OSI-906 (1μM)
0102030405060708090
100110120
0 10 50 100 500 1,000
% g
row
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lativ
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Erlotinib (nM)
HCC2279 parental:erlotinib
HCC2279 parental:OSI-906
HCC2279 parental: erlotinib + OSI-906 (1μM) HCC2279 ER: erlotinib
HCC2279 ER: OSI-906
HCC2279 ER: erlotinib + OSI-906 (1μM) 0
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HCC4006 parental:erlotinib
HCC4006 parental:OSI-906
HCC4006 parental: erlotinib + OSI-906 (1μM) HCC4006 ER:erlotinib
HCC4006 ER: OSI-906
HCC4006 ER: erlotinib + OSI-906 (1μM)
Supplementary Fig. 5 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
e.
pIGF-1R Y1131
IGF-1R
Actin
IRS-1
EGFR
S R R R R S S S PC-9
HCC- 4006
HCC- 2279
HCC- 827
Supplementary Fig. 5 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
50µM a.
b.
c.
Supplementary Fig. 6 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
Aggregate data (n=11)
Log
(inte
nsity
)
d. e. f. Rebiopsy 2 days post initiation of
EGFR TKI therapy (n=6)
Rebiopsy at the time of acquired resistance to
EGFR TKI therapy (n=5)
Log
(inte
nsity
)
Log
(inte
nsity
)
Pre-EGFR TKI Post-EGFR TKI Pre-EGFR TKI Post-EGFR TKI Pre-EGFR TKI Post-EGFR TKI
P = 0.56 P = 0.51 P = 0.84
Supplementary Fig. 6 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
a. b.
c.
0.0
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Fold
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LDK-378
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Crizotinib
LDK-378
TAE684
Supplementary Fig. 7 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
a.
b.
ALK EML4
Supplementary Fig. 8 (Lovly et al 2014)
Nature Medicine: doi:10.1038/nm.3667
Supplementary Table 1: Results from SNaPshot clinical genotyping from the Index patient's tumor. Prior to enrollment in the Profile 1007 study of crizotinib vs. pemetrexed or docetaxel for patients with advanced ALK fusion positive lung cancer, the patient’s tumor sample was sent for ALK FISH (as part of the study enrollment) and also for our institutional SNaPshot lung cancer panel. ALK FISH testing was positive at the central laboratory (Esoterix), but no other mutations were found based on the SNaPshot results shown above.
Variant Result KRAS c. 34G>A (G12S) Not detected KRAS c. 34G>C (G12R) Not detected KRAS c. 34G>T (G12C) Not detected KRAS c. 35G>A (G12D) Not detected KRAS c. 35G>C (G12A) Not detected KRAS c. 35G>T (G12V) Not detected KRAS c. 37G>T (G13C) Not detected KRAS c. 37G>A (G13S) Not detected KRAS c. 37G>C (G13R) Not detected KRAS c. 38G>A (G13D) Not detected KRAS c. 38G>C (G13A) Not detected KRAS c. 181C>A (Q61K) Not detected KRAS c. 182A>G (Q61R) Not detected KRAS c. 182A>T (Q61L) Not detected KRAS c. 183A>T (Q61H) Not detected KRAS c. 183A>C (Q61H) Not detected BRAF c.1397 G>T (G466V) Not detected BRAF c.1406 G>C (G469A) Not detected BRAF c.1789 C>G (L597V) Not detected BRAF c.1799 T>A (V600E) Not detected NRAS c.182 A>T (Q61L) Not detected NRAS c.181 C>A (Q61K) Not detected NRAS c.182 A>G (Q61R) Not detected PIK3CA c.3140 A>G (H1047R) Not detected PIK3CA c.3140 A>T (H1047L) Not detected PIK3CA c.1624 G>A (E542K) Not detected PIK3CA c.1633 G>A (E545K) Not detected PIK3CA c.1633 G>A (E545Q) Not detected MEK1 c.167 A>C (Q56P) Not detected MEK1 c.171 G>T (K57N) Not detected MEK1 c.199 G>A (D67N) Not detected AKT1 c. 49 G>A (E17K) Not detected PTEN c.697 C>T (R233X) Not detected EGFR c. 2155 G>T (G719C) Not detected EGFR c. 2155 G>A (G719S) Not detected EGFR c. 2156 G>C (G719A) Not detected EGFR c. 2369 C>T (T790M) Not detected EGFR c. 2573 T>G (L858R) Not detected EGFR c. 2582 T>A (L861Q) Not detected EGFR exon 19 deletion Not detected EGFR exon 20 insertion Not detected ERBB2 exon 20 insertion Not detected
Nature Medicine: doi:10.1038/nm.3667
PROBE_ID SYMBOL Log Fold Change (H3122 CR vs. H3122 Parental)
Adj. p value
ILMN_1655595 SERPINE2 -2.951177513 1.08687420563915e-07 ILMN_1788874 SERPINA3 -2.682821686 1.27750672686611e-06 ILMN_1737298 MAT2A -2.333677046 6.21894076548944e-07 ILMN_1685699 PRSS3 -2.293613047 1.12670469894958e-07 ILMN_2396875 IGFBP3 -2.27363455 6.25357513030054e-07 ILMN_1746085 IGFBP3 -2.220717196 1.41234569447561e-05 ILMN_1687978 PHLDA1 -2.205030034 1.6784451887109e-06 ILMN_1748591 ODC1 -2.199597841 1.12670469894958e-07 ILMN_3244117 STMN3 -2.176459125 4.3100094760663e-06 ILMN_1665510 ERRFI1 -2.085008745 4.65866770396597e-07 ILMN_2311166 ITGB5 -2.084209842 1.15443359395256e-07 ILMN_1668374 ITGB5 -2.064248799 2.57943693024856e-08 ILMN_2311537 HMGA1 -2.052228468 3.66553734732614e-07 ILMN_1668411 FHL2 -1.940308946 1.31924833415048e-06 ILMN_2355831 FHL2 -1.894906096 1.12670469894958e-07 ILMN_1744604 CYBA -1.868953167 8.81212340333521e-06 ILMN_2205622 MUC2 -1.853900607 2.60788904180529e-06 ILMN_1656501 DUSP5 -1.816586428 3.10012795310787e-06 ILMN_3258346 LOC100130009 -1.793049641 1.84854551821372e-05 ILMN_1787815 TRIB3 -1.778288075 1.32355473089493e-05
Supplementary Table 2: Summary of the top 20 downregulated genes in the H3122 crizotinib resistant (CR) cells versus isogenic H3122 crizotinib sensitive parental cells as assessed by microarray analysis. Each value shown for a specific gene represents a unique probe. Changes in IGFBP3 are noted in bold.
Nature Medicine: doi:10.1038/nm.3667
PROBE_ID SYMBOL Log Fold Change (H3122 XR vs. H3122 Parental)
adj.P.Val
ILMN_1722489 TFF1 -4.04884302 5.23904145258558e-10 ILMN_1811387 TFF3 -3.93546871 2.58872241664899e-08 ILMN_1774250 PLUNC -3.56948313 7.82194660717942e-08 ILMN_1701603 ALPL -3.35764196 1.70472868421011e-08 ILMN_1744604 CYBA -3.35074577 4.43291985241879e-08 ILMN_2114720 SLPI -3.28339511 2.11244039843639e-08 ILMN_2311537 HMGA1 -3.24325366 3.45511500528556e-09 ILMN_1721818 CLDN10 -3.18038272 1.06732369080335e-09 ILMN_2302757 FCGBP -3.14174831 2.31590355147779e-09 ILMN_1746085 IGFBP3 -3.11316828 3.79570356031621e-07 ILMN_2188862 GDF15 -3.08644 1.63325589861324e-08 ILMN_1656501 DUSP5 -3.0545683 2.5412046779067e-08 ILMN_1774287 CFB -3.03392704 8.23088721115782e-08 ILMN_1687978 PHLDA1 -2.89549654 7.93102825568373e-08 ILMN_1685194 CLDN10 -2.88081299 9.68197089571213e-08 ILMN_1748303 MUC5AC -2.83075488 8.48343484153452e-10 ILMN_1788874 SERPINA3 -2.80410325 2.95669859355518e-07 ILMN_1733904 C20orf114 -2.79660922 2.30621865654443e-08 ILMN_2396875 IGFBP3 -2.7735168 4.66974590243616e-08 ILMN_2321153 MUC4 -2.76025381 4.14618498477288e-07
Supplementary Table 3: Summary of the top 20 downregulated genes in the H3122 X-376 resistant (XR) cells versus isogenic H3122 crizotinib sensitive parental cells as assessed by microarray analysis. Each value shown for a specific gene represents a unique probe. Changes in IGFBP3 are noted in bold.
Nature Medicine: doi:10.1038/nm.3667
Supplementary Table 4: Summary of the isogenic pairs of EGFR TKI sensitive and EGFR TKI resistant cell lines used in these studies. These cell lines have been previously described2,3.
Summary of isogenic pairs of EGFR TKI sensitive and TKI resistant cell lines
Established Cell line
Primary EGFR mutation
Resistant Cell Line
Resistance derived against
Mechanism of acquired resistance
EGFR + IGF-1R TKIs synergistic?
Reference (cited in supplemental referenes)
PC-9 Exon 19 deletion
PC-9 ERc1 Erlotinib EGFR
T790M No 2
HCC827 Exon 19 deletion
HCC827 ER Erlotinib EGFR
T790M No 3
HCC2279 Exon 19 deletion
HCC2279 ER Erlotinib EMT No 3
HCC4006 Exon 19 deletion
HCC4006 ER Erlotinib EMT No 3
Nature Medicine: doi:10.1038/nm.3667
Patient #
Mechanisms of acquired resistance to crizotinib
pIGF-1R IRS-1 ALK mutation ALK
amplification 1 + N/A WTa Negativea 2 + + WTa,b Negativeb 3 + - WTc N/A 4 + N/A G1202Rd Negatived 5 - N/A WTd Negatived
Supplementary Table 5: Summary of the mechanisms of crizotinib resistance evaluated for in the study cohort. aPCR/Direct sequencing; bWhole exome sequencing; cRNA sequencing; dWhole genome sequencing.
Nature Medicine: doi:10.1038/nm.3667
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