Supplementary Tables 1 - 6, Figures 1 - 4 from Drug Repurposing for Gastrointestinal Stromal Tumor

PDF file - 1279K, Table S1: List of FDA-approved drug library; Table S2: Screen parameters; Table S3: Curve classification criteria; Table S4: Screening of FDA-approved drugs on Hs 919.T. cell line hits summary; Table S5: Drug structures of candidate hits; Table S6: Known pharmacological data of auranofin (Prometheus Laboratories Inc. (San Diego, CA, USA); Figure S1: Drug screen assay development; Figure S2: TrxR1, TrxR2, TrxR3 transcript expression analysis from a GIST microarray dataset GS31802; Figure S3: In vitro effects of auranofin treatment on TrxR/Trx activity; Figure S4: GIST-T1, GIST T1-10R, and GIST 882 cells were treated with or without auranofin at the concentration indicated in the figure.</p

Drug combination using dasatinib and CX-4945.

<p><b>A.</b> The Chou-Talalay method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref078" target="_blank">78</a>] was used to perform drug combination studies of dasatinib and CX-4945. The points represent the average viability ± standard error of mean following 72 h of drug treatment at the indicated concentrations of CX-4945 (•) and CX-4945 + dasatinib (◆; constant molar ratio of 20:1 of CX-4945:dasatinib) for the various EOC cell lines as a percentage of vehicle treated cells. The curve-fit lines were generated using non-linear regression analysis in GraphPad Prism. Data for the other molar ratios that were evaluated are presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s002" target="_blank">S2 Fig</a>. B.</b> The dose response data were used to calculate the Combination Index (CI) values for each cell line at the various molar ratios using CalcuSyn software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref079" target="_blank">79</a>]. CI values less than 1 suggest that the drugs are working synergistically. Shown is the average calculated CI value ± standard error of the mean.</p

Gene expression in clinical samples.

<p>Agilent gene expression data from TCGA on 518 serous cystadenocarcinomas and 8 fallopian tube samples derived from healthy individuals were queried for 29 dasatinib sensitizing genes. The six Agilent probes that showed ≥ 1.5-fold increase in the average gene expression of the respective genes in the tumor samples (gray boxes) relative to the controls (white boxes) are shown. The whiskers of each box plot represent the expression values at the 5^th and the 95^th percentiles. The p-values were calculated using an unpaired two-tailed t-test using GraphPad Prism. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s007" target="_blank">S4 Table</a></b> lists the average expression values of the Agilent probes across the tumor and normal samples for all 29 genes.</p

Correlation of gene expression to dasatinib sensitivity.

<p><b>A.</b> The basal level of gene expression of 29 dasatinib-sensitizing genes in seven EOC cell lines was measured by using quantitative PCR performed with a 96☓96 dynamic array on the Fluidigm BioMark microfluidic platform. Shown is a representative heat map of the dynamic array. Delta C_t values were calculated for each gene in each cell line (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#sec010" target="_blank">Materials and Methods</a> for details). <b>B.</b> Data on the dose response to dasatinib for seven EOC cell lines were generated and cell viability at 1 μM dasatinib was calculated for each cell line as a percentage of vehicle treated cells using GraphPad Prism. Shown is the average ± standard error of mean for each data point. <b>C.</b> Delta C_t and dasatinib sensitivity data (i.e. viability at 1 μM drug concentration) were subjected to Spearman Correlation analysis using GraphPad Prism. The magnitude of correlation (Spearman r value) is shown for the four genes which showed a statistically significant correlation (p < 0.05). Each point represents an EOC cell line with the color matching the code shown in panel 2B. The line through the data points is for illustrative purposes only. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s006" target="_blank">S3 Table</a></b> lists the r and p-values for the other genes evaluated but which did not show significance.</p

Quantification of cell cycle and apoptosis assays.

<p>Cell cycle and apoptosis data were quantified for the indicated fold-changes relative to vehicle treated cells and are presented as bar graphs showing the average fold-change ± standard error of mean. In all three assays, single (das, 0.5 μM; CX-4945, 10 μM) and combination drug treatments (das, 0.5 μM; CX4945, 10 μM) were for 72 h. P-values were calculated using a t-test comparing the combination treatment group to each single agent treatment group. The dashed line indicates the theoretical value if the drugs act additively calculated using the Bliss independence model (Bliss additivity value = FC_Das + (FC_CX-4945 * (100—FC_Das))/100 where FC is fold-change [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref051" target="_blank">51</a>]. Observed values larger than the Bliss additivity value indicate synergy. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#sec010" target="_blank">Materials and Methods</a> for additional assay details.</p

Overview of the design and work flow of experiments.

<p><b>A</b>-<b>F.</b> A general schematic of the experimental workflow of the primary and secondary siRNA screening and subsequent validation and refinement experiments performed to identify the second-site sensitizers for dasatinib. Details for each set of experiments are provided in the subsequent Figures and Supplementary Figures and Tables throughout the Results section.</p

Secondary

<p><b>screens on a panel of EOC and HIO cell lines. A.</b> Eight EOC cell lines were reverse transfected with siRNAs targeting 300 genes identified from the primary screening of the A1847 cell line using transfection parameters optimized for each cell line (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047086#pone.0047086.s009" target="_blank">Table S3</a></b>). Each circle represents an averaged viability score from technical replicates following silencing of a particular gene. The grey dotted line represents the cut off value for the viability score (0.85) to select hits. <b>B.</b> Three HIO cell lines were transfected using parameters optimized for each (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047086#pone.0047086.s009" target="_blank">Table S3</a></b>).</p

Validation of hits via siRNA deconvolution screening, qRT-PCR and Western blotting.

<p><b>A.</b> The seven hits selected for further studies were validated by performing deconvolution screens where individual siRNAs which were initially a part of a pool of four siRNAs in the secondary screens were evaluated for their effects on cell viability. For each hit being validated, the average normalized viability scores (±SD) following gene silencing using each of the four species of siRNAs evaluated in the eight EOC cell lines are shown. Hits were considered on-target if viability scores of less than 0.85 were observed for all eight EOC cell lines for at least two independent siRNA species targeting a gene. The bar graphs represent the eight EOC cell lines in the following order: A1847, A2780, C30, CP70, OVCAR5, OVCAR8, SKOV3, and UPN275. <b>B.</b> The two most effective siRNAs targeting each gene were pooled (12.5 nM each siRNA species) and the effect on cell viability was quantified. The bars represent the eight EOC cells lines as described in Panel A. <b>C.</b> qRT-PCR was performed on all of the eight EOC cell lines following gene silencing for 72 h using a pool of the two most effective siRNAs identified from the deconvolution studies from panel A for the four hits that were determined to be on target. The asterisk represents <i>PTN</i> mRNA levels which are below the level of detection following siRNA treatment (i.e. complete knockdown of mRNA). <b>D.</b> Western blot analysis was performed following gene silencing for either 72 h (<i>HSPA5</i>, <i>NDC80</i>, and <i>PTN</i>) or 120 h (<i>NUF2</i>) to determine the level of knockdown at the protein level in A1847 cells. Immuno blots were quantified using AlphaView software, version 3.3 (Cell Biosciences).</p

List of hits validated following deconvolution of siRNA pools.

<p>List of hits validated following deconvolution of siRNA pools.</p

Effect of gene silencing on cell survival and cell cycle progression.

<p>A1847 and A2780 cells were transfected with <i>HSPA5</i>, <i>NDC80</i>, <i>NUF2</i>, <i>PTN</i> or <i>GL2</i> siRNAs. Seventy-two hours post-transfection, cells were harvested and processed for analysis of apoptosis or cell cycle progression. <b>A.</b> & <b>C.</b> The fraction of apoptotic cells was measured by annexin V staining followed by enumeration by using a Guava flow cytometer (Millipore). The fold-change in apoptotic cells is shown (mean ± SD, n = 3). <b>B</b> & <b>D.</b> The fraction of cells in each phase of the cell cycle was measured by propidium iodide staining followed by enumeration using the Guava instrument. The fold-change for each cell cycle phase is shown (mean ± SD, n = 3).</p