Splitomicin and Glipizide treatment reduce the incidence of microtubule growth and density and increase the amount of acetylated tubulin.

<p>(A) Live-cell images of HeLa^EB3-TdTomato cells treated with 40 <b>μ</b>M glipizide or 20 <b>μ</b>M splitomicin, as indicated. HeLa^EB3-TdTomato expressing cells were treated with the indicated drugs for 1 h and filmed using time-lapse microscopy once every 10 seconds for a period of 5 minutes. Scale bar as indicated. (B) Graph shows drug treatment induced reduction in EB3 comet velocity. Instantaneous velocity of EB3 comets between consecutive time-frames were considered using movies acquired as described in (A). (C) Graph shows drug treatment induced reduction in EB3 comet density. Comet density plotted as percentage of EB3-comets observed per square μm area in interphase cells. Values are normalised using unperturbed negative control conditions. N = number of cells. (D) Graph showing increased incidence of cold stable and acetylated tubulin bearing microtubules following drug treatments. Cells were treated with two different concentrations of glipizide (20 μM; 1x and 40 μM; 2x) or splitomicin (10μM; 1x and 20μM; 2x) and cold treated as described in S2A prior to immunostaining with antibody against acetylated tubulin. * indicates statistically significant difference assessed using binomial confidence interval.</p

Validation of the predictive ability of the refined signatures.

<p>(A) Validation of the signatures through the signature reversion paradigm. The basal expression profiles of the cell lines in the GDSC panel are queried against the refined multiple signatures and connection scores are computed. Cell lines negatively connected to the signatures are predicted to be more sensitive to paclitaxel and its analogue, for which drug response data is available in the GDSC database. (B) Scatter plots showing that the cancer cell lines negatively connected to the Paclitaxel/PIs inconsistent signature and the microtubule stabilising signature (simultaneously) tend to be more sensitive to docetaxel, vinorelbine and paclitaxel. Purple points refer to cell lines significantly negatively connected to the two signatures for which drug response data is available. Gray points refer to the cell lines in the rest of the panel. Coordinates on the y-axis indicate the log IC<i>50</i> of the drug under consideration. P-values of an unpaired two sample t-test quantifying the extent of difference in drug response across the two groups of cell lines are reported in the insets together with the difference between the two log IC<i>50</i> means (ΔM).</p

Paclitaxel initial network.

<p>(A) The initial neighbourhood of paclitaxel in the drug similarity network (DN). Each node represents a drug whose consensual transcriptional response is significantly similar to that elicited by paclitaxel. Edge thickness is proportional to the distance between the connected drugs and different colours indicate different drug communities. In the inset some of the mode of action enriched in the communities are reported. (B) List of drugs contained in the paclitaxel neighbourhood. First column contains drug distance scores between paclitaxel and the drug under consideration. The second column contains the percentile in which the distance falls when sorting all the possible pair-wise distances between the 1,309 drugs in the cMap dataset. Third column contains the name of the drug while the fourth column contains the identifiers of the network community containing the drug under consideration. Fifth column contains the occurrence of that community when considering the first <i>n</i> neighbours (where <i>n</i> is the position in the list). Sixth and seventh columns contains, respectively community enrichment <i>p-values</i> (i.e. the probabilities of observing a given number of drug from a given community, in the first <i>n</i> neighbours given the total number of drugs belonging to that community in the whole DN) and the same p-values after correction for multiple hypothesis testing.</p

Iterative network guided connectivity mapping pipeline.

<p>(A) A drug similarity network (DN) is assembled from the drug response signatures contained in the connectivity-map database; the DN is queried by using the transcriptional signature of a seed compound, composed by up- and down-regulated genes, indicated in red and blue respectively, following treatment with the seed compound. (B) The resulting neighbourhood is analysed. By using a supervised approach the drugs and drug communities connected to the seed compound are investigated for enriched modes of action (MoAs); to dilute effects on drug similarity due to commonalities in secondary MoAs, refined signatures are computed, taking into account inconsistent effects of drug treatment on transcription (i.e. genes up-regulated by the seed treatment and down-regulated when treating with the classes of compounds characterised by the common secondary MoA and vice versa, indicated in orange and cyan respectively). This subdivides the original seed signature into a refined consistent signature and a refined inconsistent signature; the drug network is queried again with the computed multiple refined signatures and resulting connection scores are combined yielding a new seed neighbourhood; (C) after a number of refinement iterations the final neighbourhood of the seed compound is considered as output of the procedure; compounds in the final network, predicted to share the principal MoA of the seed compound, are finally selected for experimental validation.</p

Paclitaxel refined networks.

<p>(A and B) First iteration of the network guided connectivity-mapping: (A) Expression percentile heat-map for the genes in the paclitaxel optimal-signature, along the prototype ranked lists (PRLs) of paclitaxel and some of its neighbouring drugs: 3 proteasome inhibitors (PIs) and 2 benzimidazoles (included for comparison). Rows corresponding to genes whose rank positions are inconsistent between the PRLs of paclitaxel and PIs (Paclitaxel/PIs inconsistent signature) are highlighted in yellow while those corresponding to genes with consistent rank positions are highlighted in cyan. (B) Network of drugs connected to paclitaxel when querying the connectivity-map (cMap) with the multiple signatures highlighted in the heat-map. Each node represents a drug whose consensual transcriptional response is significantly similar to the computed multiple signatures simultaneously, edge thickness is proportional to the normalised connectivity scores (NCS) between the connected drugs. Recovered microtubule perturbing drugs are highlighted in pink. (C and D) Second iteration of the network guided connectivity-mapping: (C) Expression percentile heat-map for the genes in the microtubule stabilising signature, along PRLs of paclitaxel and the 4 recovered benzimidazoles. (D) Network of drugs connected to paclitaxel when querying the cMap with the multiple signatures in the two heat-maps. The graphic notation is the same of (B).</p

Paclitaxel final network.

<p>Paclitaxel final network.</p

EWSCs are sensitive to PARP1 trapping.

<p><b>(A)</b> Relative viability of mock-transfected and PARP1 siRNA-transfected ES8 cells treated with vehicle or olaparib. Asterisks indicate <i>student’s paired t-test P</i> value **P<0.01, ns = not significant. <b>(B)</b> PARP1 expression in cells transfected with a scrambled control or a titration of PARP1_1 siRNA and their relative viability following treatment with vehicle or olaparib. <b>(C)</b> IC50 values of parental ES8 and PARPi-resistant OLAR5 cells to five different PARPi and the fold difference between them. <b>(D)</b> Western blot of PARP1 expression in ES8 and OLAR5 cells. Viability values are the mean of technical triplicates and representative of 3 independent experiments.</p

EWSCs are sensitive to PARP inhibition and S-phase DNA-damaging agents.

<p><b>(A)</b> and <b>(C)</b> Scatter plots of IC₅₀ (μM) values on a log scale comparing drug sensitivity of <i>EWS-FLI1</i>-positive and wild-type (WT) <i>EWS-FLI1</i>-negative cell lines to (A) four PARPi and (C) camptothecin and cisplatin. The sample size (n) is indicated and each circle represents the IC₅₀ of one cell line. The red bar is the geometric mean and the drug name is depicted above each plot along with the significance of the association as determined by an unpaired two-sample t-test. <b>(B)</b> Long term viability assays in EWSCs were performed in the presence of vehicle (-) or increasing concentrations of four PARPi as indicated. Non-EWSC lines (U-2-OS and HeLaSF) are included for comparison. These data are representative of 3 independent experiments.</p

Temozolomide enhances PARP inhibitor sensitivity in multiple tumour types.

<p>List of cell lines screened against a combination of olaparib and temozolomide. Whether enhancement of PARP inhibitor sensitivity with temozolomide is observed (✔) or not (✖) is indicated.</p

DNA DSB repair by HR is functional in EWSCs.

<p><b>(A)</b> Western blot of ES8 cells treated with olaparib for the times indicated. Markers are grouped as part of ATM or ATR signaling. Tubulin served as a loading control. (<b>B)</b> Western blot of ES8 cells treated with camptothecin and harvested at various time points following drug washout. GAPDH served as a loading control. <b>(C)</b> Percentage of EdU-positive and EdU-negative ES8 cells with >5 nuclear RAD51 foci following 6-hour treatment with vehicle or olaparib (ola). <b>(D)</b> Olaparib log GI₅₀ (μM) of cell lines mock-transfected or transfected with CtIP or BRCA1 siRNA as indicated.</p