Supplementary Figures and Tables legends
Supplementary Figures
Supplementary Figure S1: High-throughput drug sreen flowchart and
results
A: High-throughput drug screen flowchart. Cells were plated at day 1 and
drug was added 24h after plating. After 5 days of drug exposure, cell viability
was assessed by CellTitre-Glo (Promega) luminescence reading. Only
screens fulfilling the pre-established quality criteria (Z prime > 0.2 and
Spearman rank correlation between replicates >0.8) were further considered.
After processing of the data, potentially interesting compounds were selected
based on their ability to show >15% difference in survival fraction between the
ERCC1-proficient
and
ERCC1-deficient populations at

2 different
concentrations. B: Results of the high-throughput drug screen: four PARP1/2
inhibitors are identified as being ERCC1-selective
Supplementary Figure S2: Representative example of the sensitivity to
PARP1/2 inhibitors of two A549 wild-type cell lines from different
suppliers.
Short-term assay evaluating niraparib sensitivity of ERCC1-deficient clones
compared to two wild-type A549 cell lines from different suppliers (Institut
Gustave Roussy and Institute of Cancer Research - originally ATCC);
sensitivity of the two ERCC1 wild-type cell lines to niraparib is similar. Results
obtained with other PARP1/2 inhibitors were similar, with comparable
sensitivity of the two ERCC1 wild-type A549 cell lines to PARP inhibition (data
not shown). Error bars represent the standard deviations from the mean of 6
independent experiments.
Supplementary Figure S3: Correlation between cisplatin sensitivity and
olaparib sensitivity in a panel of 14 non-isogenic NSCLC cell lines.
PARP1/2 inhibitor sensitivity of ERCC1-deficient NSCLC
2
A: Cisplatin and olaparib sensitivity of the panel of 14 non-isogenic NSCLC
cell lines. Cells were exposed to the drug for five days before assessing cell
viability. B: Significant correlation (Pearson r2=0.5409, p<0.05) between
cisplatin SF50 and olaparib SF50 (evaluated in a short-term assay) in a nonisogenic panel of 14 NSCLC cell lines.
Supplementary Figure S4: Selective PARP1/2 inhibitor sensitivity of
ERCC1-deficient cells is not related to overt HR deficiency
A: Representative examples of immunofluorescence staining of RAD51 (488
wavelength, green) and γH2AX (555 wavelength, red) foci in ERCC1-isogenic
cell lines after olaparib exposure for 24h at 10M concentration. B:
Quantification by confocal microscopy of RAD51 foci formation in ERCC1isogenic cell lines after DNA damage by olaparib (24h exposure at 10M
concentration). The percentage of RAD51 foci in untreated cells and A549
cells transfected with BRCA2 siRNA are displayed as negative and positive
controls, respectively.
Supplementary Figure S5: siRNA + olaparib screen flowchart and results
Olaparib was added at a non-toxic (80% survival fraction) concentration to a
siRNA screen of 784 kinases and tumour suppressor genes as well as 127
DNA repair genes 48h after reverse-transfection. The results were obtained
by comparing the ERCC1-proficient vs ERCC1-deficient cells. Hits displayed
followed significance criteria in both ERCC1-deficient clones when compared
to the ERCC1-proficient isogenic cell line. Abbreviations: reps: replicates;
ERCC1-prof.: ERCC1-proficient; ERCC1-def.: ERCC1-deficient.
Supplementary Figure S6: BRCA2-silencing is epistatic with ERCC1deficiency in mediating niraparib sensitivity
Effect of BRCA2 knock-down by siRNA on sensitivity of ERCC1-isogenic cell
lines to niraparib. Cells were reverse-transfected with BRCA2 siRNA and drug
was added 48h after transfection. Cells were exposed to the drug for five
days. Error bars represent the standard deviation from the mean of three
independent experiments.
PARP1/2 inhibitor sensitivity of ERCC1-deficient NSCLC
3
Supplementary Figure S7: Representative images of kinetics of RAD51
and γH2AX foci formation after olaparib treatment
Cells were exposed to 10M olaparib for 24h prior to drug removal. Kinetics of
RAD51 (488 wavelength, green) and γH2AX foci (555 wavelength, red)
formation was then assessed by confocal microscopy at different time points.
Examples of pictures obtained with the A549 ERCC1-proficient cell line and
one ERCC1-deficient clone at olaparib removal, 28h and 76h after olaparib
removal (T0, T28 and T76, respectively).
Supplementary Figure S8: PARP1 silencing effect on olaparib sensitivity
in ERCC1-isogenic cell lines
A: Effect of PARP1 knock-down by siRNA on sensitivity of ERCC1-isogenic
cell lines to olaparib. Cells were reverse-transfected with PARP1 siRNA and
drug was added 48h after transfection. Cells were exposed to the drug for five
days. Error bars represent the standard deviation from the mean of three
independent experiments.
B: Western blot showing PARP1 silencing after siRNA transfection
(corresponding drug curves displayed in Figure 5 and Supplementary Figure
S7)
Supplementary Tables
Supplementary Table S1: EC50 and SF50 values
EC50 and SF50 of each cell line for all experiments displayed in the
manuscript, with experimental error (Standard Error of the Mean, SEM). All
values are displayed in M, unless otherwise notified (BMN673).
Abbreviations: ND = Not Determined
Supplementary Table S2: Complete results of the drug screen in the
ERCC1-isogenic model
PARP1/2 inhibitor sensitivity of ERCC1-deficient NSCLC
4
Survival fractions of all cell lines are displayed for every concentration of each
compound. PARP1/2 inhibitors that came up as “hits” of the screen are
highlighted in bold.
Supplementary Table S3: Cell cycle analysis at different time points
following olaparib removal.
Numbers represent the percentage of cells in each phase of the cell cycle at
different time points following olaparib removal, analysed by FACS.