Supplemental Figures and Legends Rationale for co …...Supplemental Figures and Legends Rationale for co-targeting IGF-1R and ALK in ALK fusion positive lung cancer Christine M. Lovly1,20,

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: [email protected]

Nature Medicine: doi:10.1038/nm.3667

mailto:[email protected]

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.


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)


(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


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.


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


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.


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).


Supplementary Fig. 1 (Lovly et al 2014)

a.

b.

0

10

20

30

40

50

60

70

80

90

100

MAB391 100nMerlotinib

500nMerlotinib

1,000nMerlotinib

100nMerlotinib +MAb391

500nMerlotinib +MAb391

1,000nMerlotinib +MAB391

% g

row

th re

lativ

e to

con

trol

0 0.05 0.1 0.5 1 5

0102030405060708090

100110120

0 0.05 0.1 0.5 1 5OSI-906 (µM)

% g

row

th re

lativ

e to

con

trol

Erlotinib (µM)

Erlotinib

OSI-906

Erlotinib + OSI-906


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

con

trol



f.

g.

e.

h.

*

*

0

100

200

300

400

500

600

700

1 4 10 14 17 20

Tum

or v

olum

e (m

m3 )

Time (days)

Vehicle

Crizotinib

MAb391

Crizotinib + MAb391

0 0.1 0.5 1 5 10

0

10

20

30

40

50

60

70

80

90

100

110

120

0 0.01 0.05 0.1 0.5 1

Erlotinib (µM)

% g

row

th re

lativ

e to

con

trol

Crizotinib (µM)

Crizotinib

Erlotinib

Crizotinib + erlotinib

0 0.05 0.1 0.5 1 5

0

10

20

30

40

50

60

70

80

90

100

110

120

0 0.05 0.1 0.5 1 5

Lapatinib (µM)

% g

row

th re

lativ

e to

con

trol

Crizotinib (µM)

Crizotinib

Lapatinib

Crizotinib + lapatinib

0 0.5 1 5 10 50 100

0

20

40

60

80

100

120

140

160

0 0.5 1 5 10 50 100

% g

row

th re

lativ

e to

con

trol

Crizotinib

OSI-906

Crizotinib + OSI-906

Crizotinib (μM) OSI-906 (μM)



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



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

tens

ity c

ompa

red

to c

ontro

l



IRS-1

Crizotinib

NT siRNA

– – + + – +

IRS-1 siRNA (pool #1)

IRS-1 siRNA (pool #2)

pALK Y1604

Actin

a.

b.

0

10

20

30

40

50

60

70

80

90

100

110

NT siRNA:DMSO

NT siRNA:Crizotinib

IRS-1 siRNA(pool #1):

DMSO

IRS-1 siRNA(pool #1):crizotinib

IRS-1 siRNA(pool #2):

DMSO

IRS-1 siRNA(pool #2):crizotinib

% c

ell n

umbe

r rel

ativ

e to

con

trol



a. b.

c.

H3122 Parental

H3122 XR

IGF-1R d.

Blot: ALK

Crizotinib – + – +

H3122 Parental

H3122 CR



e.

f.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

H3122 parental H3122 CR H3122 XR

Fold

cha

nge

in IG

F-1

0 0.1 0.5 1 5 10

0102030405060708090

100110120130

0 0.01 0.05 0.1 0.5 1

% g

row

th re

lativ

e to

con

trol

Crizotinib

OSI-906

Crizotinib + OSI-906

Crizotinib (μM)

OSI-906 (μM)



a.

c.

b.

d.

0102030405060708090

100110120

0 10 50 100 500 1,000

% g

row

th re

lativ

e to

con

trol

Erlotinib (nM)

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

100110120

0 10 50 100 500 1,000

% g

row

th re

lativ

e to

con

trol

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

th re

lativ

e to

con

trol

Erlotinib (nM)



HCC2279 parental: erlotinib + OSI-906 (1μM) HCC2279 ER: erlotinib

HCC2279 ER: OSI-906

HCC2279 ER: erlotinib + OSI-906 (1μM) 0

10

20

30

40

50

60

70

80

90

100

110

120

0 10 50 100 500 1,000

% g

row

th re

lativ

e to

con

trol

Erlotinib (nM)



HCC4006 parental: erlotinib + OSI-906 (1μM) HCC4006 ER:erlotinib

HCC4006 ER: OSI-906

HCC4006 ER: erlotinib + OSI-906 (1μM)



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



50µM a.

b.

c.



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



a. b.

c.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Crizotinib LDK-378

Fold

cha

nge

in s

ub-2

n D

NA

co

nten

t com

pare

d to

con

trol

0

10

20

30

40

50

60

70

80

90

100

110

0 50 100 500 1000 5,000

% g

row

th re

lativ

e to

con

trol

Drug Concentration (nM)

X-376

LDK-378

0

10

20

30

40

50

60

70

80

90

100

110

120

0 5 10 50 100 500 1,000

% g

row

th re

lativ

e to

con

trol

Drug concentration(nM)

Crizotinib

LDK-378

TAE684



a.

b.

ALK EML4



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


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.


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.


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 ER Erlotinib EMT No 3


HCC4006 ER Erlotinib EMT No 3


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.


Documents

Supplemental Figures and Legends Rationale for co …...Supplemental Figures and Legends Rationale for co-targeting IGF-1R and ALK in ALK fusion positive lung cancer Christine M. Lovly1,20,