Download Table S3 Compounds in the isogenic cell line high throughput drug

Miller et al Supplementary Material
Supplementary Data
Figure S1 ARID1A mutations in a panel of OCCC tumour cell line models
with protein expression. (A) Schematic illustrating the domain structure of
the ARID1A protein. ARID DNA binding domain shown in blue and the
DUF3518 domain in red. Amino acid positions of truncating mutations in the
panel of OCCC cell lines is shown. Black = frameshift, red = nonsense, blue =
missense. (B) Western blot analysis of total cell lysates from OCCC cell lines
and from the colorectal HCT116 ARID1A isogenic cell line (See Figure S5).
Full length ARID1A protein expression is absent in OVAS, OVISE, SMOV2,
OVTOKO, OVAMANA, KOC7C, TOV21G and HCH1 but is present in the
three ARID1A wild type cell lines; ES2, KK and RMG-1. One cell line,
OVSAYO harbored a non-synonymous ARID1A mutation. No detectable
ARID1A protein was detected for OVSAYO, although ARID1A protein
expression has been reported in this cell line (18). OVSAYO was excluded
from subsequent analysis.
Figure S2 Un-cropped western blots from main and supplementary
figures
Uncropped western blots from main and corresponding figures with
corresponding labels. For Figures 2E, 2H, 3F, S6B, S7F and S9E protein
bands were visualised using enhanced chemiluminescence (ECL, GE
Healthcare, UK) and Kodak BioMAX XAR film (Kodak). For figures 5D, S7D
and S7E images were taken using the Odyssey Infrared Imaging system from
Licor ®.
1
Miller et al Supplementary Material
Figure S3. (A) Heatmap showing selected AUC data from the high-throughput
screen (only compounds with a candidate ARID1A selective effect are shown).
Compounds are ranked according to difference in median AUC between
ARID1A mutant and wild-type cohorts. ARID1A_mut and ARID1A_WT depict
the median effects in mutant and wild type cohorts, respectively. Dasatinib is
highlighted by an arrow. (B) Heatmap showing SF50 data from the highthroughput screen, as per (A). Data is only shown for drugs in which the SF50
was achieved in >4 cell line models.
Figure S4 Inhibitors of the PI3K/mTOR signalling pathway and ARID1A
selectivity. Cell inhibition dose response curves are shown. (A) Dose
response curves for (A) the panel of OCCC cell lines models, (B) the median
of the ARID1A wild-type (n=3) and ARID1A mutant (n=8) OCCC cohorts from
(A). Cells were plated in 96 well plates and continuously exposed to drug at a
range of concentrations for 5 days.
Figure S5 Dasatinib is a synthetic lethal drug in ARID1A mutant OCCC
tumour cell line models. (A and B) Pooled (A) and individual (B) dasatinib
surviving fraction (SF) short term, dose-response curves in ARID1A mutant
and wild-type cell lines from Figure 2A. (C and D) Pooled (C) and individual
(D) Clonogenic (15 day exposure) dasatinib dose response curves in
individual ARID1A mutant and wild-type OCCC cell lines from Figure 2C. (E)
Dasatinib dose response survival data from OCCC ARID1A wild type cell line
KK transfected with multiple siRNA species targeting ARID1A or control
siRNA. 48 hours following transfection cells were plated into 96 well plates
2
Miller et al Supplementary Material
3
and exposed to dasatinib for five days. Error bars represent SEM from six
replica experiments. For each ARID1A siRNA dasatinib survival curve vs.
control siRNA two-way ANOVA p<0.001. (F) Dasatinib dose response survival
data from breast tumour cell lines DU4475 (ARID1A wild type) and CAL51
(ARID1A null) transfected with siRNA. For DU4475 siARID1A vs. control
siRNA two-way ANOVA p<0.001. For CAL51 p= ns. (G) Dasatinib dose
response survival data from HCT116 ARID1A
WT/WT
cells transfected with
siRNA. siARID1A vs. control siRNA two-way ANOVA p<0.001. Experiments in
F and G were performed as per (E).
Figure S6 Dasatinib causes synthetic lethality in ARID1A mutant
isogenic HCT116 colorectal tumour cell line model. (A) Schematic
illustrating the targeting of a p.Q456* ARID1A mutation in the isogenic cell line
in the human human HCT116 colorectal cancer cell line. Top. Schematic of
the wild type ARID1A gene around exons 2 and 3, indicating the intro/exon
structure and the position of the p.Q456 codon in exon 3. Middle. Structure of
an AAV targeting vector encompassing an p.Q456* ARID1A mutation 3’ to by
a loxP flanked selectable marker gene.
The modified allele following
recombination is shown at the bottom. (B) Western blot analysis of ARID1A
expression in HCT116 isogenic cell lines models demonstrating loss of
ARID1A expression in ARID1A Q456*/Q456* clone with retained expression in the
ARID1AWT/WT parental clone and the ARID1AWT/Q456* heterozygous clone. The
ARID1A wild-type OCCC cell line, ES2 is included as a positive control.
ACTIN is included as a loading control. (C) Heatmap illustrating surviving
fraction (SF) data from the high-throughput screen in HCT116 ARID1A
Miller et al Supplementary Material
isogenic models. Compounds are ranked according to difference in SF
between ARID1AWT/WT and ARID1AQ456*/Q456* models. R1, R2, R3 = replica
screens 1, 2 and 3 respectively. (D) Dasatinib dose response survival data
(five-day dasatinib exposure) from subsequent validation experiments in
HCT116 ARID1AWT/WT and ARID1AQ456*/Q456* isogenic models. Error bars
represent SEM from six replicas per cell line. 2-way ANOVA p<0.001,
ARID1A Q456*/Q456* cells when compared to the parental ARID1AWT/WT line.
Figure S7 Genetic screens for dasatinib targets. (A) Bar chart plots
demonstrating NPI values for each of the siRNA oligos in the ARID1A mutant
and wild-type cohorts of OCCC cell lines. (B) Waterfall plots of NPI values for
each OCCC cell line model transfected with YES1 siRNAs. Data used to
generate the median values shown in Figure 3E. (C) Bar chart plot of YES1
siRNA NPI values in subsequent validation experiments in the ARID1A
HCT116 isogenic model. Median NPI values from six replica experiments are
shown and error bars represent SEM. (D) Western blot showing reduction in
YES1 activation treatment in three OCCC cell lines with dasatinib but not with
saracatinib, a SRC inhibitor, which is not ARID1A selective (data not shown).
(E) Western blot showing reduction in YES1 activation (pYES1) with dasatinib
treatment but not total YES1 levels in the ARID1A isogenic cell line model. (F)
Western blot analysis of YES1 expression in the panel of OCCC cell lines.
Figure S8 Apoptosis assay in four OCCC cell line models. Two ARID1A
wild-type cell lines (ES2 and KK) in black and two ARID1A mutant cell lines
(OVISE and TOV2G) in red. Caspase 3/7 activity in response to dasatinib at
4
Miller et al Supplementary Material
increasing concentrations is plotted. Cells were treated as in Figure 4A and
caspase 3/7 activity assessed using the ApoTox Glo triplex assay (Promega)
corrected for cellular viability.
Figure S9 Dasatinib sensitivity in ARID1A mutant OCCC is dependent
upon G1/S checkpoint effectors (A) Dasatinib dose response curves for the
ARID1A mutant OCCC cell line OVSAYO following transfection with the
CDKN1A siRNA SMARTpool. Silencing of CDKN1A expression results in
dasatinib resistance. Each point represents the mean and SEM of six
replicates. CDKN1A siRNA dasatinib survival curve vs. control siRNA two-way
ANOVA p<0.001. (B) Dasatinib dose response curves for the ARID1A mutant
OCCC cell lines OVISE, TOV21G and HCH1 and the ARID1AQ456*/Q456*
isogenic clone following transfection with RB1 siRNA pool. Silencing of RB1 in
ARID1A mutant cell lines results in dasatinib resistance. For each cell line
RB1 pool siRNA dasatinib survival curve vs. control siRNA two-way ANOVA
p<0.001. (C) Dasatinib dose response curves for the ARID1A wild-type OCCC
cell line KK and the ARID1AWT/WT isogenic clone following transfection with
RB1 siRNA pool. Silencing of RB1 in ARID1A wild-type cell lines does not
alter dasatinib sensitivity. For each cell line, RB1 pool siRNA dasatinib
survival curve vs. control siRNA two-way ANOVA p=ns (D) Dasatinib dose
response curves for the ARID1A mutant OCCC cell line OVISE and the
ARID1AQ456*/Q456* isogenic clone following transfection with four individual RB1
siRNA oligos. Each point represents the mean and SEM of six replicates. For
each RB1 siRNA dasatinib survival curve vs. control siRNA two-way ANOVA
p<0.001. (E) Western blot demonstrating the on-target nature of the RB1
5
Miller et al Supplementary Material
siRNA. TOV21G cell line was transfected with RB1 siRNA and whole cell
lysates collected 48 hours later. Lysates were probed with the RB1 antibody
and a fluorescent-dye labeled secondary antibody used.
Figure S10 Establishing the optimal method of dasatinib delivery. Both
Intraperitoneal (IP) and oral dasatinib treatment at a range of concentrations
cause target inhibition (reduction in phosphorylated-SRC). Animals were
treated with varying doses of dasatinib by either oral gavage or IP injection for
72 hours. Tumour and normal tissue was then collected and lysates made.
The ARID1A mutant cell line, TOV21G grown in vitro and treated with either
DMSO or 10 μM dasatinib for 24 hours, is included as a control. There is a
profound reduction in SRC activation in both the in vivo tumour (90%) and in
the OCCC cell line, TOV21G (80%) reduction.
Table S1. Compounds used in the high-throughput drug screen. This
table lists the supplier and catalogue number of each compound included in
the drug screen.
Table S2. Compounds used in the high throughput drug screen. The
screen includes 68 compounds that are either already used in routine clinical
practice for the management of cancer or in early phase clinical trials.
Compounds were present in the screen at a range of concentrations (1 nM,
10 nM, 100 nM and 1 μM).
6
Miller et al Supplementary Material
Table S3 Compounds in the isogenic cell line high throughput drug
screen. This table lists the additional compounds included in the drug screen
performed on the isogenic cell line. This screen includes additional
compounds to that described in Supplementary Table S2 and each compound
is present at six different concentrations (0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM,
100 nM, 500 nM and 1 μM).
Table S4 siRNA library gene list. This table lists the genes included in the
siRNA library alongside the gene accession number and the product code
(Dharmacon).
Table S5. ARID1A mutation status in OCCC panel. The ARID1A status of
the OCCC cell lines was confirmed by deep exon sequencing. Whole exome
sequencing was performed on the panel of OCCC cell lines (see
Supplementary materials and methods). Mutations were defined as
homozygous if the observed allele frequency > 0.75.
Table S6. Pearson’s correlation and Z prime values for OCCC panel
demonstrating robust reproducibility and dynamic range. Pearson’s
correlation values and Z prime for the three replicate plates (R1-3) for each
OCCC model. Pearson’s correlation co-efficient > 0.75 was considered
acceptable. One replicate plate of HCH1 (R1), OVAS (R1) and OVMANA (R3)
failed to produce satisfactory values and were excluded from further analysis.
The dynamic range of each screen was assessed by calculation of Z’ values
for each plate in the screen using the cell viability estimate in DMSO exposed
7
Miller et al Supplementary Material
cells as the negative control and the cell viability estimate in cells exposed to
5µM puromycin as the positive (cell inhibition) control. Each plate delivered a
Z’ > 0.5 with the exception of one replicate of each of OVAS and HCH1 which
also failed to achieve satisfactory Pearson’s correlation values and were
exclude from further analysis.
Table S7 Results from the high throughput drug screen in the nonisogenic OCCC cell line panel. This table lists the median surviving fraction
values from the three replicate screens for each cell line at every
drug/concentration pairing tested. Each cell line is annotated with ARID1A
mutation status and the median values for the ARID1A wild-type and mutant
cohorts are listed with the difference in median values.
Table S8 Pearson’s correlation and Z prime values for isogenic cell lines
demonstrating robust reproducibility and dynamic range. Pearson’s
correlation values and Z prime for the three replicate plates (R1-3) for each
ARID1A isogenic model. Pearson’s correlation co-efficient > 0.75 was
considered acceptable. The dynamic range of each screen was assessed by
calculation of Z’ values for each plate in the screen using the cell viability
estimate in DMSO exposed cells as the negative control and the cell viability
estimate in cells exposed to 5µM puromycin as the positive (cell inhibition)
control. Each plate delivered a Z’ > 0.75.
Tables S9 and S10. MS Stats output from dasatinib bead proteomics:
These tables list the results of the complete set of pairwise MS stats
8
Miller et al Supplementary Material
comparisons of purified kinases from pooled biological replicates of ARID1A
wild-type and mutant OCCC lines (S7), and then those kinases represented
by more than 1 peptide with an adjusted p value < 0.05 from the comparison
of the combined ARID1A wild-type and mutant lines.
Table S11. Results from the dasatinib target screen. This table lists the
median NPI values for the ARID1A wild-type (n=3) and mutant (n=7) OCCC
cohorts for each dasatinib target siRNA oligo and SMARTpool examined,
alongside the difference in the median NPI between the ARID1A mutant and
ARID1A wild-type cohorts.
Table S12. Results from the siRNA kinase and tumour suppressor
dasatinib resistance screen. This table lists each siRNA and the Z score
data for the dasatinib and DMSO arms of the screen with the DE Z score for
each of the screens (dasatinib SF50 and SF16). The median DE Z score for
each siRNA is calculated.
9
Miller et al Supplementary Material
Supplementary Materials and Methods
High throughput drug screen analysis
Luminescence data was log2 transformed and centred on a per plate basis
according to the plate median value. Using this data we assessed the
reproducibility of replica screens by the calculation of r2 values and the
dynamic range of each screen by the calculation of Z prime values (1). Z
prime values were calculated based on a positive (puromycin) and negative
(DMSO) control exposed samples in each plate. A Z prime >0.3 and r2 >0.75
were used to define acceptable screen data (2, 3). Surviving fractions were
calculated relative to DMSO treated wells and this data was used to generate
AUC (area under the curve) and SF50 data.
Cell-based siRNA assays
Cell lines were transfected with individual and SMARTpool siRNAs
(Dharmacon) targeting the gene of interest and non-targeting control siRNA
(Dharmacon or Qiagen) or siPLK1 (Dharmacon) using RNAiMax (Invitrogen)
or Dharmafect 4 (Dharmacon).
To screen dasatinib target siRNAs, each of the OCCC tumour cell lines was
transfected with either a SMARTpool of four siRNAs for each gene or
individual siRNA species (four per gene). After siRNA transfection, cells were
cultured for a subsequent five days at which point cell viability was estimated
as before. To estimate the extent by which each siRNA caused tumour cell
inhibition, and to take account of varying transfection efficiency effects, we
10
Miller et al Supplementary Material
calculated Normalised Percent Inhibition (NPI) values for each siRNA. These
were calculated by scaling Cell Titre Glo luminescence readings for each of
the siRNA transfections according to luminescence signals from cells
transfected with either a non-targeting control siRNA or a siRNA designed to
target PLK1. In total, each OCCC cell line was transfected with each siRNA
four times.
1.
Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter
for Use in Evaluation and Validation of High Throughput Screening Assays. J
Biomol Screen. 1999;4:67-73.
2.
Iorns E, Turner NC, Elliott R, Syed N, Garrone O, Gasco M, et al.
Identification of CDK10 as an important determinant of resistance to
endocrine therapy for breast cancer. Cancer Cell. 2008;13:91-104.
3.
Turner NC, Lord CJ, Iorns E, Brough R, Swift S, Elliott R, et al. A
synthetic lethal siRNA screen identifying genes mediating sensitivity to a
PARP inhibitor. EMBO J. 2008;27:1368-77.
11