ATR is required to maintain genome integrity in 53BP1-deficient cells.

<p>H157 cells were transfected with non-targeting (siNT) or 53BP1 siRNA and then treated with 2.5μM ATRi for 24 hours. Cells were then fixed and stained with DAPI to visualize nuclei (A) and γH2AX to identify sites of DNA damage (B). (C) Quantification of the γH2AX intensity of cells shown in B. Box and whiskers plot shows the mean and the range of the samples, * p<0.01, ns (not significant).</p

Loss of 53BP1 is synthetic lethal with ATRi and cisplatin.

<p>(A-F) H157 NSCLC cells were transfected with non-targeting siRNA (siNT) or two siRNAs targeting 53BP1 (number 2 and 3 refer to specific sequences described in the materials and methods). Cells were then treated with ATRi, cisplatin, and ATRi and cisplatin. Cell viability was determined with alamar blue and reported as a percent of the untreated control cells. (A) Sensitivity of 53BP1 knockdown cells to cisplatin. (B and C) Sensitivity of 53BP1 knockdown cells to ATRi and ATRi with 0.5μM cisplatin. Bliss independence synergy between ATRi and cisplatin in control (D) and 53BP1 knockdown cells (E and F). Error bars in all panels are standard deviation (n = 3).</p

ATR inhibition sensitizes cells to cisplatin.

<p>(A and B) U2OS cells were treated with increasing doses of cisplatin or the ATR inhibitor (ATRi) alone or in combination for 72 hours. Cell viability was determined with alamar blue and reported as a percentage of the untreated control cells. Analysis of synergy between cisplatin and ATRi using Bliss Independence (B) and isobologram analysis (C) as described in the materials and methods. (D) U2OS cells were treated with 1μM cisplatin, 1μM ATRi, or both (ATRi + Cis); cells were released into media without drugs after 24, 48, or 72 hours and allowed to form colonies. (E) Cells were treated with 1μM cisplatin or 1μM ATRi for 24hr, ATRi (24hr) followed by cisplatin (24hr) A→C, or cisplatin (24hr) followed by ATRi (24hr) C→A. Error bars in all panels are standard deviation (n = 3).</p

ATR inhibition resensitizes cisplatin-resistant cancer cells to cisplatin.

<p>(A) MDA-MB-468 (468) and MDA-MB-468 cisplatin-resistant (468-CR) cells were treated with increasing doses of cisplatin for 96h prior to measuring cell viability with alamar blue. (B and C) MDA-MB-468 and MDA-MB-468 cisplatin-resistant cells were treated with ATRi alone or ATRi and cisplatin at 0.5μM (B) or 3μM (C) for 96h prior to measuring cell viability with alamar blue. (D and E) Bliss independence synergy between cisplatin and ATRi in MDA-MD-468 (D) and MDA-MB-468 cisplatin-resistant cells (E). Error bars in all panels are standard deviation (n = 3).</p

Loss of homologous recombination is not synthetic lethal with ATRi/cisplatin.

<p>BRCA2-deficient VC8 cells or VC8 cells complemented with a BRCA2 expression vector were treated with ATRi, cisplatin, and ATRi/cisplatin combination. (A) Sensitivity of BRCA2-deficient cells to cisplatin. (B and C) Sensitivity of BRCA2-deficient cells to ATRi alone and ATRi with either 0.05 or 0.1μM cisplatin. Cell viability was measured with alamar blue after 72 hours and reported as a percent of the untreated control cells. Error bars are standard deviation (n = 3).</p

Loss of REV3 is synthetic lethal with ATRi and cisplatin.

<p>(A-F) H157 NSCLC cells were transfected with non-targeting siRNA (siNT) or two siRNAs targeting REV3 (number 2 and 4 refer to specific sequences described in the materials and methods). Cells were then treated with ATRi, cisplatin, and ATRi and cisplatin. Cell viability was determined with alamar blue and reported as a percent of the untreated control cells. (A) Sensitivity of REV3 knockdown cells to cisplatin. (B and C) Sensitivity of REV3 knockdown cells to ATRi and ATRi with 0.1μM cisplatin. Bliss independence synergy between ATRi and cisplatin in control (D) and REV3 knockdown cells (E and F). Error bars in all panels are standard deviation (n = 3).</p

ATR inhibition is synthetic lethal with pharmacologic inhibition of PARP.

<p>(A) U2OS cells were treated with increasing doses of ATR and PARP inhibitors for 96 hours. Cell viability was measured with alamar blue and reported as a percent of the untreated control. Synergy between ATR and PARP inhibition using Bliss Independence (B) and isobologram analysis (C) as described in the materials and methods. (D) Cells were treated with 1μM ATRi, 2nM PARPi, or both (A + P) and cells were released into media without drugs after 72 hours and allowed to form colonies. Error bars in all panels are standard deviation (n = 3).</p

siRNA screen identifies synthetic lethal interactions with cisplatin and ATR inhibitor treatment.

<p>(A) Schematic of the siRNA screen. U2OS cells were transfected with siRNAs and then either left untreated or treated with 1μM ATRi, 0.5μM cisplatin, or ATRi and cisplatin. Cell viability was measured with alamar blue. (B) Viability of the non-targeting (NT) and ATR siRNA controls using the screen conditions. The values represent the mean ± SD of the three independent replicates of the screen. (C-E) The robust z-scores of treated compared with untreated cell viability were determined for each siRNA in the library for ATRi (C), cisplatin (D), and ATRi and cisplatin (E). The red circles represent the 8 unique siRNAs targeting ATR and ATRIP present in the library and the red line indicates a robust z score of -1.3. (F) Summary of the overlap of synthetic lethal relationships with ATRi, cisplatin, or ATRi and cisplatin. (G) Complete list of genes that exhibit a synthetic lethal relationship with ATRi, cisplatin, or ATRi and cisplatin. Genes with 2 or more siRNAs with robust z-scores of less than -1.3 are considered as synthetic lethal. The ATRi single-agent screen was previously published and included for comparison to the other drug treatments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125482#pone.0125482.ref018" target="_blank">18</a>].</p

Clinical parameters of TNBC subtypes.

<p>Clinical parameters of TNBC subtypes.</p

Chemotherapy response and distant relapse-free survival of TNBC treated with neoadjuvant anthracycline and taxane relative to PAM50 or refined TNBCtype-4 subtyping.

<p>Barplots show pCR rates achieved for patients stratified by <b>(A)</b> TNBC, <b>(B)</b> PAM50 or <b>(C)</b> TNBCtype-4. Dotted horizontal line indicates pCR for the individual cohort. Statistical significance determined by Fisher’s exact test. Kaplan-Meier plots display distant relapse-free survival from GSE25066 for <b>(D)</b> TNBC patients; <b>(E)</b> TNBC patients stratified by pCR or RD; <b>(F)</b> TNBC patients stratified by PAM50; and <b>(G)</b> TNBC patients stratified by refined TNBCtype-4.</p