amongst a panel of cell lines representing a highly diverse range of tumor types, to aid in the development of more generalizable predictors of drug response. To this end, we carried out a drug sensitivity screen using the NCI-60 panel of tumor cells to profile their cellular response to GDC-0941. The NCI-60 cancer cell line collection is a highly characterized panel of 60 diverse human cancer cell lines, representing multiple tumor types (breast, colon, NSCLC, renal, ovarian, prostate, melanoma, leukemia and CNS) which has been screened for drug sensitivity to .100,000 compounds [9]. Multiple genomic profiling experiments using the NCI-60 collection have been published, reporting expression profiles for mRNAs and miRNAs [10–15], proteomic [13,16], as well as SNP and CNV analyses [17–19]. We stratified the cell lines into groups either sensitive or resistant to GDC-0941, and then correlated the phenotype of these cell lines with basal levels (in the absence of GDC-0941) of mRNA expression and the phosphorylation state of multiple signaling nodes to identify predictive biomarkers of de novo drug sensitivity. Prominent amongst genes significantly up-regulated in the GDC- 0941 resistant cell lines were the genes O-linked N-acetylgluco- samine transferase (OGT) and dendrin (DDN). Validation experiments using siRNA-mediated loss of expression of either OGT or DDN resulted in increased in vitro sensitivity to GDC- 0941 in multiple cell lines, and concomitant alterations in effectors of both the PI3K and MAPK signaling cascades. These results demonstrate that OGT and DDN are novel in vitro regulators of cellular response to inhibition of PI3K signaling and highlight their potential roles as additional predictive biomarkers for de novo resistance to GDC-0941 and other PI3K inhibitors. We also found that GDC-0941 has broad anti-proliferative activity amongst the nine tumor types represented in the NCI-60 cell panel, suggesting its clinical utility against multiple tumor types. Materials and Methods NCI-60 cell lines and drug screening The NCI-60 tumor cell line collection [9] was obtained directly from the National Cancer Institute’s Developmental Therapeutics program (NCI DTP) and maintained in RPMI 1640 media (Invitrogen) containing 10% Fetal Bovine Serum (ATCC). Upon receipt, the identities of all cell lines in the NCI-60 collection were independently authenticated via STR profiling performed by the ATCC Cell Authentication Testing Service using the PowerPlexH 18D System (Promega). All experiments conducted in this study were performed using the authenticated NCI-60 tumor cell line collection obtained directly from the NCI DTP [9]. GDC-0941 and NVP-BEZ235 were obtained from Selleck and dissolved in DMSO prior to cell treatments. PUGNAc was obtained from Sigma and dissolved in DMSO prior to cell treatments. In vitro drug sensitivity testing of the NCI-60 tumor cell line collection against GDC-0941 was performed, with two biological replicates, by the National Cancer Institute’s Developmental Therapeutics Program as previously described [20], on June 14, 2010 and August 9, 2010. GI50 was defined as the drug concentration resulting in a 50% reduction in net protein increase (as measured by SRB staining) as compared to control cells. Briefly, following treatment with a 5-point, 10-fold serial dilution with GDC-0941 for 48 hours, cells were fixed in TCA and a 0.4% (w/v) sulforhodamine B (SRB) solution in 1% acetic acid is added. Cells are washed with 1% acetic acid, air dried, bound stain solubilized with 10 mM trizma base, and absorbance measured at 515 nm [20]. Following standardization, we defined cell lines with a standardized GI50 (z-score) $0.8 as being resistant and cell lines with a z-score #20.8 as being sensitive to GDC-0941. Biomarkers of Response to PI3K Inhibitors Microarray data, identification of differentially expressed genes, and statistical analysis Normalized log2 mRNA expression data for the non-drug treated NCI-60 tumor cell line collection [11] were downloaded from CellMiner database (http://discover.nci.nih.gov/cellminer) and imported into GenePattern 3.3 for all downstream analyses (http://www.broad.mit.edu/cancer/software/genepattern). Miss- ing values were estimated with a weighted K-nearest neighbors algorithm (KNNimpute), and two-way average linkage hierarchi- cal cluster analysis was performed using a UPGMA algorithm, with the data being displayed relative to the median expression for each gene. To identify genes differentially expressed between GDC-0941 resistant- and sensitive-tumor cell lines, we utilized the Comparative Marker Selection Module [21] of GenePattern 3.3 employing a nonparametric t-test with a P value cutoff of 0.01. All P values were corrected for multiple testing with the Bonferroni correction. The Gene Set Enrichment Analysis (GSEA) [22] module of GenePattern 3.3 was used to determine the extent to which expression profiles were enriched for a priori defined sets of genes from biologically coherent pathways. To correct for multiple hypotheses testing, the false discovery rate (FDR) threshold was set at ,0.25. Correlations between drug sensitivity and gene expression, levels of phosphorylation, mutational status, tumor type, or tissue of origin were estimated by Spearman’s rank correlation method, and differences between groups were calculated with Student’s t test using Prism 5.0 (GraphPad). All tests of significance were two- sided, and P values ,0.05 were considered significant. siRNA knockdown and cell viability assays ON-TARGETplus siRNAs (Thermo Scientific), containing pools of 4 siRNAs per gene, were utilized for siRNA knockdown experiments as described previously [23]. Briefly, 70 ml of cells (1.06105 cells/ml) were plated in black, clear-bottomed 96-well plates in antibiotic-free RPMI 1640 medium and allowed to adhere overnight. Cells were then transfected with siRNA using DharmaFECT transfection reagent (Thermo Scientific) at a final concentration of 25 nM. Following a 4 hour incubation, 10 ml per well of GDC-0941 or NVP-BEZ235 were then added for a total assay volume of 100 ml. Assays were performed in triplicate, with ON-TARGETplus Non-Targeting siRNA (Thermo Scientific) as a negative control, with biological replicates. For experiments with downstream protein analyses, cells were transfected with siRNAs as above. 60 hours post-transfection, cells were treated with 300 nM, 1 mM, or 4 mM GDC-0941 for 24 hours prior to harvesting. For experiments to determine the combined effects of siRNA knockdown and PI3K inhibition on cell proliferation, cell viability was measured at 24 hour intervals using the CellTiter 96H AQueous One Solution Cell Proliferation Assay (Promega) with the differences between cell numbers at 72 hours calculated using Student’s t test using Prism 5.0 (GraphPad). For determination of IC50 values for GDC-0941, and the effect of siRNA knockdown of OGT or DDN on sensitivity to GDC- 0941, cells were transfected as above. Four hours post transfection, 10 ml per well of an 11-point, 2-fold serial dilution of GDC-0941 (10 mM maximum concentration) was then added, for a total assay volume of 100 ml. Assays were performed in triplicate, with ON- TARGETplus Non-Targeting siRNA (Thermo Scientific) as a negative control, with biological replicates. Cell viability was measured 72 h later using the CellTiter 96H AQueous One Solution Cell Proliferation Assay (Promega), and IC50 values calculated by fitting a four-parameter logistic curve of dose- response data using Prism 5.0 (GraphPad). Comparison of the fitted log IC50 values for GDC-0941 in drug treated cell lines PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e46518