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Supplementary Materials for
Systematic identification of signaling pathways with potential to confer
anticancer drug resistance
Colin A. Martz, Kathleen A. Ottina, Katherine R. Singleton, Jeff S. Jasper,
Suzanne E. Wardell, Ashley Peraza-Penton, Grace R. Anderson, Peter S. Winter,
Tim Wang, Holly M. Alley, Lawrence N. Kwong, Zachary A. Cooper, Michael Tetzlaff,
Pei-Ling Chen, Jeffrey C. Rathmell, Keith T. Flaherty, Jennifer A. Wargo,
Donald P. McDonnell, David M. Sabatini,* Kris C. Wood*
*Corresponding author. E-mail: [email protected] (D.M.S.); [email protected] (K.C.W.)
Published 23 December 2014, Sci. Signal. 7, ra121 (2014)
DOI: 10.1126/scisignal.aaa1877
This PDF file includes:
Fig. S1. Schematic of the screen.
Fig. S2. Stable transfection of constructs.
Fig. S3. Results of a screen in UACC-62 melanoma cells (BRAFV600E) for pathways
conferring resistance to MEK1/2 inhibitor AZD6244.
Fig. S4. Meta-analysis of screening results across 13 targeted therapies.
Fig. S5. Resistance to etoposide mediated by stable expression of non-cleavable
caspases in MCF-7 breast cancer cells.
Fig. S6. Immunoblotting of BRAFV600 melanoma cells expressing pathway-activating
constructs and treated with MAPK pathway inhibitors.
Fig. S7. Resistance to targeted and cytotoxic drugs in breast cancer cells treated with
a soluble Notch agonist.
Fig. S8. Differentiation markers and signaling in MCF-7 breast cancer cells
expressing Notch1.
Fig. S9. Characterization of dedifferentiation-associated phenotypes in breast cancer
cells with activated Notch1.
Fig. S10. Analysis of Notch1 pathway members and EMT markers in a mouse model
of tamoxifen-resistant breast cancer (TamR).
Fig. S11. Analysis of Notch1 pathway members and EMT markers in human breast
cancer patients.
Fig. S12. Estrogen receptor–driven resistance to MAPK inhibitors.
Fig. S13. Notch1 target gene expression in melanoma cells with activated Notch1.
Fig. S14. Western blot analysis of differentiation markers and signaling in BRAFmutant melanoma cells expressing Notch1.
Fig. S15. Characterization of dedifferentiation-associated phenotypes in melanoma
cells with activated Notch1.
Fig. S16. Notch1 hairpin validation.
Fig. S17. Characterization of evolved MAPK inhibitor–resistant, BRAF-mutant
melanoma cell lines.
Fig. S18. Analysis of patient tumors in cohort 1.
Fig. S19. Analysis of patient tumors in cohort 2.
Fig. S20. Resistance pathway inhibitors sensitize intrinsically resistant melanoma
cells to VX-11E independently of inhibitor effects on cell viability.
Fig. S21. Vector maps for vectors used in this study.
Legends for tables S1 to S7
Legend for date file S1
Other Supplementary Material for this manuscript includes the following:
(available at www.sciencesignaling.org/cgi/content/full/7/357/ra121/DC1)
Table S1 (Microsoft Excel format). List of pathway-activating constructs and
controls used in this study.
Table S2 (Microsoft Excel format). List of all drugs, drug concentrations, and cell
lines screened.
Table S3 (Microsoft Excel format). Results of primary screens.
Table S4 (Microsoft Excel format). Characterization of cell lines and clonal derivates
with evolved resistance to MAPK inhibitors.
Table S5 (Microsoft Excel format). Metastatic melanoma patient characteristics
(cohorts 1 and 2).
Table S6 (Microsoft Excel format). List of attB1/B2 primers used to barcode and
amplify constructs by PCR.
Table S7 (Microsoft Excel format). Sequences of additional primers used in this
study.
Data file S1 (Microsoft Word format). Nucleotide sequences.
Figure S1. Schematic of the screen. Schematic depicting design, construction, validation, and
screening with the library of pathway-activating constructs used in this study.
Figure S2. Stable transfection of constructs. Relative abundances of all screened constructs in
(A) BT-20 cells and (B) MDA-MB-453 cells immediately after infection (t=0) and after 4 weeks
of culture (t=f), indicating that all constructs are present and stably persist in pooled cell
populations. (continued next page)
Figure S2. (continued) (C) Relative abundances of all screened constructs in T47D cells
immediately after infection (t=0) and after 4 weeks of culture (t=f), indicating that all constructs
are present and stably persist in pooled cell populations.
150 nM AZD6244
MAPK
14
750 nM AZD6244
12
1.5 µ M AZD6244
10
PI3K
NF-κB Notch1
8
6
ER
4
p53 (DN)
AR-V7
TGFBR1
ERa (Y537S)
Lats2 DN
YAP2 (5SA)
Mkk7-JNK2
JNK2
BCL-XL
BCL-2
Cas-8 (C360A)
Cas-3 (C163A)
RalA (G23V)
H-Ras (E37G)
Rlf-CAAX
MKK6(EE)
p38
Notch1 ICD
Notch3 ICD
MEK5 (DD)
myr-MEK5
IKKa (EE)
FLAG-IKKb (EE)
GSK3b (K85A)
b-catenin (S33Y)
b-catenin (3A)
JAK2 (V617F)
Stat3 (CC)
SmoM2
Gli2 trunc
myr-PI3K
myr-Akt
Rheb (Q64L)
0
Hras (G12V)
Kras (G12V)
MEK1 (DD)
2
HcRed
Luciferase
MEK1
Fold enrichment (drug/control)
16
Figure S3. Results of a screen in UACC-62 melanoma cells (BRAFV600E) for pathways
conferring resistance to MEK1/2 inhibitor AZD-6244. The fold enrichment for each
construct, which represents the fractional representation of that construct in the presence of drug
normalized to the same quantity in the absence of drug, is shown. Shaded region represents no
enrichment. Hit pathways are indicated by arrows.
B
% of screens
30
25
20
15
10
5
Ral
TGF-β
p53
AR
JNK
Hippo
ERK5
Apoptosis
p38
Wnt
NF-κB
Hedgehog
ER
JAK-STAT
Notch
0
PI3K-mTOR
100
90
80
70
60
50
40
30
20
10
0
Ras-MAPK
% of screens
A
1
2
3
4
5
6
7
# of
pathwaysscoring
scoring
# of
pathways
pathways scoring
Viability
(normalized to vehicle control)
Figure S4. Meta-analysis of screening results across 13 targeted therapies. (A) Percentage
of screens in which at least one construct for each indicated pathway scored. (B) Percentage of
screens in which the indicated number of resistance pathways were identified for a given drug
and cell line. Resistance pathways were considered hits when at least one construct activating
that pathway yielded an Enrichment Score of >1.5 and scored in at least 2 of 3 drug
concentrations screened.
1.5
MCF-7 cells
*
1.25
1
*
*
*
0.75
0.5
0.25
Caspase 8 (C360A)
Caspase 3 (C163A)
10 µM
Luciferase
100 µM
0
[Etoposide]
Figure S5. Resistance to etoposide mediated by stable expression of non-cleavable caspases
in MCF-7 breast cancer cells. Cells expressing indicated constructs were incubated for four
days in the presence of the indicated concentrations of etoposide, then viability was measured
using the Cell Titer Glo assay (Methods). Data are means ± S.D. from three experiments.
*p < 0.05.
Figure S6. Immunoblotting of BRAFV600 melanoma cells expressing pathway-activating
constructs and treated with MAPK pathway inhibitors. A375 melanoma cells were treated
with a RAF inhibitor (RAFi, P), MEK inhibitor (MEKi, A), or ERK inhibitor (ERKi, V). Blots
are representative of two experiments.
100
0 uM DSL
1 uM DSL
10
GI50 (μM)
10 uM DSL
***
***
*** ***
1
***
**
0.1
0.01
0.001
0.0001
Cell line:
Drug:
MCF7
MCF7
MCF7
Fulvestrant
BEZ-235
Doxorubicin
Figure S7. Resistance to targeted and cytotoxic drugs in breast cancer cells treated with a
soluble Notch agonist. GI50 values for MCF-7 cells treated with the indicated drugs and
incubated with either vehicle or the indicated concentrations of a Notch1-activating ligand, the
DSL peptide. Data are means ± S.D. from three experiments. ** p < 0.05; *** p < 0.01.
A
B
Luc
N-cadherin
Vimentin
Slug
N1ICD
-
+
Luc
+
-
BEZ-235
N1ICD
P-AKT (Thr308 )
T-AKT
AKT
Figure S8. Differentiation markers and signaling in MCF7 breast cancer cells expressing
Notch1. (A) Western blotting for mesenchymal differentiation markers in cells expressing
luciferase (Luc) or Notch1 ICD (N1ICD). (B) Western blotting for phosphorylated and total AKT
in cells expressing indicated cDNAs and treated with vehicle (-) or PI3K–mTOR inhibitor BEZ235 (+). Blots are representative of three experiments.
B
T47D
**
8
6
4
2
N
ot
ch
on
tr
o
C
Apoptosis
*
2.5
2.0
1.5
1.0
0.5
1u
yc
m
m
ap
a
M
R
R
M
R
M
3u
T47D
Luciferase
in
in
yc
SO
in
M
D
m
ap
a
ap
a
R
M
1u
yc
yc
m
M
SO
in
0.0
D
120
hrs
C
Fold change in
% Annexin V+ / 7AAD- cells
48
hrs
Sl
ug
0
l
0
hrs
**
10
3u
T47D
Slug
1
T47D
N1 ICD
Sphere Forming Efficiency (%)
T47D
Luciferase
ap
a
A
T47D
Notch1 ICD
Treatment
Figure S9. Characterization of dedifferentiation-associated phenotypes in breast cancer
cells with activated Notch1. Migration (A), mammosphere formation (B), and apoptosis
resistance (C) in T47D cells stably expressing Notch1 ICD, the EMT transcription factor Slug, or
luciferase. Images in (A) are representative of three experiments and data in (B and C) are means
± S.D. from three experiments. *p < 0.1, **p < 0.01.
Figure S10. Analysis of Notch1 pathway members and EMT markers in a mouse model of
tamoxifen-resistant breast cancer (TamR). (A) Expression of Notch1 pathway genes in TamR
versus parental MCF-7 tumors by qRT-PCR. (continued next page)
Figure S10. (continued) (B) qRT-PCR analysis of the Notch1 pathway in TamR cells. Pathway
map was modified using PathVisio.
Figure S11. Analysis of Notch1 pathway members and EMT markers in human breast
cancer patients. (A) Metaplot showing the Log2 DMFS hazard ratios for Notch1 pathway
members in tumors from patients with luminal B breast cancer (Figure 3, E and F). Indicators are
colored according to the expression of the Notch1 high group described in Figure 3E. (B)
Correlation plot of Notch1, Notch1 pathway, Notch1 target genes, and dedifferentiation markers
in luminal B breast tumors. (continued next page)
Figure S11. (continued) (C) Gene Set Enrichment Analysis (GSEA) results based on the
signature of Notch1 coexpressed genes in luminal B breast tumors. The top 20, as ranked by the
Nominal Enrichment Score (NES), are plotted as a bar graph and colored according to gene-set
size.
UACC-62 cells
3
2
Parental +
10 nM Estradiol
ERα (Y537S)
O/E
0
Luciferase O/E
1
Parental
VX-11E GI50 (normalized)
4
Figure S12. Estrogen receptor–driven resistance to MAPK inhibitors. VX-11E GI50 values
for BRAF-mutant UACC-62 melanoma cells expressing luciferase, ERα Y537S, or treated with
estradiol. O/E, overexpression. Data are means ± S.D. from three experiments.
1.00#
0.50#
0.00#
***
Hes2
HES2%
12.00#
Fold change
1.50#
14.00#
10.00#
8.00#
6.00#
4.00#
2.00#
(transcript)
Fold%
Change%
(Transcript)%
**
Fold change
Hes1
HES1%
(transcript)
Fold%
Change%
(Transcript)%
0.00#
Notch1#ICD#
Notch1
ICD
6.00#
***
4.00#
3.00#
2.00#
1.00#
0.00#
Hes4
HES4%
***
40.00#
30.00#
20.00#
10.00#
Notch1#ICD#
Notch1
ICD
Hey1
HEY1%
5.00#
50.00#
0.00#
Luciferase#
Luciferase
Fold change
Fold change
(transcript)
Fold%
Change%(Transcript)%
Luciferase#
Luciferase
(transcript)
Fold%
Change%
(Transcript)%
Fold change
(transcript)
Fold%
Change%
(Transcript)%
2.00#
6.00#
Luciferase#
Luciferase
Hey2
HEY2%
Notch1#ICD#
Notch1
ICD
***
5.00#
4.00#
3.00#
2.00#
1.00#
0.00#
Luciferase#
Luciferase
Notch1#ICD#
Notch1
ICD
Luciferase#
Luciferase
Notch1#ICD#
Notch1
ICD
Figure S13. Notch1 target gene expression in melanoma cells with activated Notch1.
Expression of canonical Notch1 target genes in UACC-62 melanoma cells expressing Notch1
ICD or luciferase measured by qRT-PCR. Data are means ± S.D. from three experiments.
** p< 0.05; *** p < 0.01.
A
Luc N1ICD
TYR
TRP-2
B
-
-
+
+
PLX4720
N1ICD Luc N1ICD Luc
P-ERK
NGFR
T-ERK
Slug
β-actin
Figure S14. Western blot analysis of differentiation markers and signaling in BRAFmutant melanoma cells expressing Notch1. (A) Expression of melanocyte differentiation
markers in UACC-62 cells expressing luciferase (Luc) or Notch1 ICD. (B) ERK phosphorylation
in cells expressing the indicated cDNAs and treated with PLX4720 (RAF inhibitor; Colo679
cells). Blots are representative of two experiments in each.
UACC62
Slug
B
UACC62
0 hrs
**
30
20
10
0
C
N
ot
on
tr
ch
ol
12 hrs
***
40
Sl
ug
UACC62
N1 ICD
1
UACC-62
Luciferase
Sphere Forming Efficiency (%)
A
Apoptosis
*
4
3
2
1
1u
M
VX
-1
1E
3u
M
VX
-1
1E
D
M
SO
1u
M
VX
-1
1E
3u
M
VX
-1
1E
0
D
M
SO
36 hrs
C
Fold change in
% Annexin V+ / PI- cells
24 hrs
5
48 hrs
UACC-62
Luciferase
UACC-62
N1 ICD
Treatment
Figure S15. Characterization of dedifferentiation-associated phenotypes in melanoma cells
with activated Notch1. (A) Migration, (B) melanosphere formation, and (C) apoptosis
resistance in UACC-62 cells stably expressing Notch1 ICD, Slug, or luciferase. Images in (A)
are representative of three experiments, and data in (B and C) are means ± S.D. from three
experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure S16. Notch1 hairpin validation. Notch1 knockdown by the indicated shRNAs in
melanoma cell lines assessed by immunoblotting. Image is representative of two experiments.
Figure S17. Characterization of evolved MAPK inhibitor–resistant, BRAF-mutant
melanoma cell lines. (A) Apoptosis induction by VX-11E in Notch1-dependent, and PLX- or
AZD-resistant derivatives (+, 1 μM; ++, 3 μM). Data are means ± SD from three experiments. *
p < 0.05, ** p < 0.01. (B) Western blot of phosphorylated (P) and total (T) ERK in parental cell
lines and Notch1-dependent, MAPK inhibitor (MAPKi)-resistant derivatives. S, cells derived
using the slow or step-wise method; F, cells derived using the fast or constant-dose method
(Methods). Blots are representative of two experiments. (C) Notch1-dependent and independent
clonal derivatives of cell lines in Fig. 4C. (D) Representative clonogenic assays that are used to
calculate S Score and dependence. S Score is defined in table S4; S Scores significantly less
than 1.0 indicate Notch1-dependent resistance.
C
4.0
HES1
Fold change
(Relapse/Pre-Rx)
Fold change
(Relapse/Pre-Rx)
A
2.0
0.0
3a
3b
8a
HES4
Candidate resistance genes sequenced
BRAF
MEK1
MEK2
NRAS
AKT1
4.0
2.0
0.0
12
3a
3b
8a
12
Pretreatment
Relapse
1.0
0.0
Fold change
(Relapse/Pre-Rx)
3a
4.0
DLL1
3.0
2.0
1.0
0.0
3a
3b
Fold change
(Relapse/Pre-Rx)
JAG1
2.0
8a
12
3b
8a
2.0
3.0
JAG2
1.0
3a
DLL4
1.5
1.0
0.5
0.0
3b
8a
3b
12
8a
12
15.0
Fold-change
(Relapse/Pre-Rx)
Patient Mechanism Notch1 HES2 HES5
9.2
10.3
10 IGF-1R O/E 2.5
0.0
12
3a
D
2.0
Fold change
(Relapse/Pre-Rx)
3.0
Fold change
(Relapse/Pre-Rx)
Fold change
(Relapse/Pre-Rx)
B
DLK1
10.0
5.0
0.0
3a
3b
8a
12
Figure S18. Analysis of patient tumors in cohort 1. Expression of Notch1-driven reporter
genes (A) and genes encoding Notch1 ligands (B) in relapsed patient tumors (coded 3a, 3b, 8a,
or 12) from cohort 1 with evidence of Notch1 activation (normalized to pretreatment expression).
(C) Candidate resistance genes queried from tumor cDNA in all tumors from cohort 1. (D)
Patient 10 from cohort 1 harbored evidence of coincident Notch1 activation and a second
putative resistance mechanism, IGF-1R overexpression (O/E) (34). Rx, treatment.
B
Fold change
(Transcript)
A
100.0
HEY2
10.0
**
**
1.0
0.1
3
24
Candidate resistance genes assayed
AKT1
AKT2
AKT3
BRAF
CTNNB1
KRAS
MAP2K1
MAP2K2
NF1
NRAS
PIK3CA
PIK3R1
PTEN
RAC1
RB1
TP53
Pretreatment
Relapse
Figure S19. Analysis of patient tumors in cohort 2. (A) Expression of HEY2 in relapsed
patient tumors (coded 3 and 24) from cohort 2 with evidence of Notch1 activation (normalized to
pretreatment transcript abundance). Data are means ± SD from three technical replicate
measurements; **p<0.05. (B) Candidate resistance genes queried from tumor cDNA in all
tumors from cohort 2.
100
90
1.2
80
1
70
60
0.8
50
0.6
40
30
0.4
20
0.2
Hs294T cells
BEZ-235 +
shNotch1
BEZ-235
BMS345541
IKKi IV
BEZ-235 +
shNotch1
BEZ-235
IMD0354
Bay 11-7085
Inhibitor(s):
10
IKKi IV
0
Cell Viability
(+inhibitors / -inhibitors)
VX-11E GI50
(+inhibitors / -inhibitors)
1.4
0
WM1745 cells
Figure S20. Resistance pathway inhibitors sensitize intrinsically resistant melanoma cells to
VX-11E independently of inhibitor effects on cell viability. Blue bars (left axis): VX-11E
GI50 values in the presence of pathway inhibitors normalized to GI50 in the absence of
inhibitors. Sensitizing drug interactions are indicated by GI50 ratios less than 1.0. GI50 ratios
represent the ratio of mean GI50 values in the “+” and “-“ inhibitor conditions, each calculated
from three experiments. Red boxes (right axis): Cell viability in the presence of pathway
inhibitors normalized to viability in the absence of inhibitors. Data are means ± SD from three
experiments.
Figure S21. Vector maps for vectors used in this study. The Gateway-compatible donor
vector pDONR223 and destination (expression) vector pcw107 are shown (C-terminal V5 tag not
shown).
Table S1. List of pathway-activating constructs and controls used in this study. Thirty-six
unique pathway-activating constructs, three ORF controls. Constructs containing a C-terminal
V5 tag are indicated with a “+”, whereas those lacking the tag are indicated with a “-”. In some
cases, both the V5-tagged and untagged versions of a construct were produced (indicated with
“+/-” designation). Table S1 is provided as an Excel file.
Table S2. List of all drugs, drug concentrations, and cell lines screened. Totals: 13 drugs,
12 cell lines, 110 screens. Table S2 is provided as an Excel file.
Table S3. Results of primary screens. Fold enrichment (drug/vehicle treatment) for each
pathway activating construct in each primary screen. Primary screens (columns) are annotated as
Cell line_Drug_Drug dose. Table S3 is provided as an Excel file.
Table S4. Characterization of cell lines and clonal derivates with evolved resistance to
MAPK inhibitors. Six BRAF-mutant melanoma cell lines were evolved to resistance to
PLX4720 (PLX), AZD6244 (AZD), or VX-11E (VX) by continuous selection using the slow (S)
or fast (F) methods (refer to Methods). Pathway reactivation was assessed by immunoblotting for
phospho-ERK and total-ERK in each cell line in the presence of treatment with the indicated
MAPK inhibitor. Evolved resistance indicates the GI50 value for the indicated drug in the
indicated cell line normalized to the same value in the parental cell line from which it arose.
Notch1-dependent sensitization indicates the GI50 value for the indicated drug in the indicated
cell line with concurrent Notch1 knockdown normalized to the GI50 of the same drug in the
parental cell line. In the Summary column, Complete indicates complete resensitization of
resistant derivatives via Notch1 knockdown (to GI50 levels at or below that of the parental cell
lines); Partial indicates resensitization to intermediate levels between the parental and resistant
derivative lines. S Score reflects the ratio of the area of cell growth in Notch1-knockdown wells
normalized to GFP-knockdown wells in the presence of treatment with the indicated drug
normalized to the same quantity in the absence of drug treatment (clonogenic growth assay; refer
to Methods). S Score of less than 1.0 indicates sensitization by Notch1-knockdown. White
boxes under the columns "Notch1-dependent Sensitization" and "S Score" indicate cell lines or
clones with evidence of Notch1-dependent resistance while pink boxes indicate cell lines or
clones whose resistance is Notch1-independent. Table S4 is provided as an Excel file.
Table S5. Metastatic melanoma patient characteristics (cohorts 1 and 2). For each patient,
the patient sample identifier, BRAF mutation, drug treatment (Rx), RECIST (Response
Evaluation Criteria In Solid Tumors) response category, response (percentage change in size of
target lesions), progression-free survival interval, putative resistance mechanism, and putative
Notch1 activation status are provided (34). Table S5 is provided as an Excel file.
Table S6. List of attB1/B2 primers used to barcode and amplify constructs by PCR.
Gateway recombination sequences are indicated in red text, construct barcodes in black text,
spacer sequences in green text, and construct-specific 5' and 3' complementary sequences in blue
text. F and R designations in primer names signify "forward" and "reverse". Table S6 is provided
as an Excel file.
Table S7. Sequences of additional primers used in this study. For primers, blue text indicates
P5 and P7 sequences, red text indicates complementary sequences, and green text indicates index
barcode sequences (one example is shown). For reads, black text indicates the 4 nucleotide
construct barcode sequence, green text indicates the 17 nucleotide spacer sequence, and red text
indicates the 6 nucleotide index barcode sequence. Table S7 is provided as an Excel file.
Data File S1: Nucleotide sequences. Full nucleotide sequences for all pathway-activating and
control ORFs used in this study. Red = Tag sequence; Black = Coding sequence. The data file is
provided as an Excel file.