PI3K pathway and its role in cancer biology. One significant caveat to this work is that the characterization of these candidate biomarkers is limited to tumor cell lines. The use of appropriate patient-derived tumor samples, either in vitro or ex vivo, would provide greater context in understanding the biomarkers described in this work as predictors of de novo response to PI3K inhibitors. Furthermore, prior to their use in the identification of patients likely to derive benefit from PI3K inhibitors, the candidate biomarkers of chemotherapeutic response identified here and in other studies need to be subjected to clinical validation using prospective retrospective analyses using patient samples from relevant clinical trials to determine their sensitivity, specificity and reproducibility [42,43]. In conclusion, these data show that GDC-0941 has broad anti- proliferative activity amongst many tumor types represented in the NCI-60 cell panel, suggesting its clinical utility against multiple tumor types, and that a number of tumor cell lines with activated AKT1 are highly sensitive to inhibition of PI3K with GDC-0941. We have also identified several novel genomic and phosphopro- teomic biomarkers of de novo sensitivity to GDC-0941 and validate two of these markers as in vitro regulators of cellular response to inhibition of PI3K signaling. In addition, that in vitro loss of DDN expression sensitizes both de novo GDC-0941-sensitive and - resistant tumor cell lines to PI3K inhibition suggests that the relationship between expression and response may be continuous, with a large dynamic range. However, further work will be required to more completely understand this relationship and to determine the utility of DDN (or OGT) as robust biomarkers. These data also highlight several biomarkers that may serve as pharamacodynamic markers of drug efficacy, or as additional drug targets to increase patient response to PI3K inhibitors. Supporting Information Figure S1 In vitro sensitivity of the NCI-60 tumor cell line collection to GDC-0941 and correlation between in vitro and mutational status of PI3KCA, PTEN, or tumor type. A, GI50 (in mM) of GDC-0941 for 60 cell lines are organized by tumor of origin, as indicated below cell line names. Mean GI50 (0.66 mM) for all cell lines is indicated by dashed line. B, Correlation between GI50 (mM) to GDC-0941 and mutational status of PI3KCA or loss of PTEN protein, amongst all 60 tumor cell lines in the NCI-60 collection. C, Correlation between GI50 (mM) to GDC-0941 and mutational status of PI3KCA or loss of PTEN protein, amongst 16 GDC-0941-resistant and -sensitive tumor cell lines (IGROV1, T-47D, UACC-257, MCF-7, BT-549, HOP-92, HS578T, SK-OV-3, M14, SK-MEL-5, HCC-2998, NCI-H226, MDA-MB-231, NCI-H23, NCI/ADR-RES, OV- CAR-4) analyzed in detail in this study. D, Correlation between GI50 (mM) to GDC-0941 and mutational status of AKT1, PDK1, or MTOR, amongst all 60 tumor cell lines in the NCI-60 collection. E, Correlation between GI50 (mM) to GDC-0941 and mutational status of AKT1, PDK1, or MTOR, amongst 16 GDC- 0941-resistant and -sensitive tumor cell lines (IGROV1, T-47D, UACC-257, MCF-7, BT-549, HOP-92, HS578T, SK-OV-3, M14, SK-MEL-5, HCC-2998, NCI-H226, MDA-MB-231, NCI- H23, NCI/ADR-RES, OVCAR-4) analyzed in detail in this study. F, correlation between GI50 (mM) to GDC-0941 and tumor type of all 60 tumor cell lines in the NCI-60 collection. Tumor types are color coded as follows: Breast (peach), CNS (grey), Colon (green), Leukemia (red), Melanoma (orange), NSCLC (blue), Ovarian (purple), Prostate (indigo), and Renal (taupe). (TIF) Biomarkers of Response to PI3K Inhibitors Figure S2 Gene Set Enrichment Analysis (GSEA) identi- fies biologically-coherent pathways differentially ex- pressed between GDC-0941-resistant and-sensitive tu- mor cell lines. A, Table of KEGG pathways upregulated in GDC-0941-resistant tumor cell lines, with number of genes (‘‘SIZE’’), enrichment score (‘‘ES’’), normalized enrichment score (‘‘NES’’), nominal p-value (‘‘NOM p-val’’), and q-value (‘‘FDR q- val’’) listed. B, Enrichment plot indicating the enrichment score for the KEGG proteasome gene set. C, Heatmap showing relative expression of genes in KEGG proteasome gene set amongst sixteen GDC-0941-resistant and –sensitive tumor cell lines. (TIF) Figure S3 Quantitative analysis of O-GlcNAc levels in OGT siRNA-treated cells, as determined by Western blotting. A, Pixel intensity (greyscale; 0, black-255, white) of total O-GlcNAc levels for MDA-MB-231 tumor cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 mM GDC-0941 for 24 hours. Each treatment condition is indicated below x-axis. Inset is the inverted image of Western blot in Figure 4B used for this analysis. B. % change in pixel intensity of total O-GlcNAc levels for MDA-MB-231 tumor cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 mM GDC-0941 for 24 hours, relative to NS siRNA-treated cells. C, Pixel intensity (greyscale; 0, black-255, white) of total O-GlcNAc levels for OVCAR-4 tumor cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 4 mM GDC- 0941 for 24 hours. Each treatment condition is indicated below x- axis. Inset is the inverted image of Western blot in Figure 4F used for this analysis. D, % change in pixel intensity of total O-GlcNAc levels for OVCAR-4 tumor cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 4 mM GDC-0941 for 24 hours, relative to NS siRNA-treated cells. Data in A and C represent mean 6 SD (n = 2). (TIF) Figure S4 IC50 curves showing dose-response of the MDA-MB-231, OVCAR-4, and MCF7 tumor cell lines treated with GDC-0941. A, IC50 curves for MDA-MB-231 cells treated with GDC-0941 alone (green curve), NS siR- NA+GDC-0941 (red curve), or siRNA targeting OGT+GDC- 0941 (blue curve). B, IC50 curves for OVCAR-4 cells treated with GDC-0941 alone (green curve), NS siRNA+GDC-0941 (red curve), or siRNA targeting OGT+GDC-0941 (blue curve). C, IC50 curves for MCF7 cells treated with GDC-0941 alone (green curve), NS siRNA+GDC-0941 (red curve), or siRNA targeting DDN+GDC-0941 (blue curve). D, IC50 curves for MDA-MB-231 cells treated with GDC-0941 alone (green curve), NS siR- NA+GDC-0941 (red curve), or siRNA targeting DDN+GDC- 0941 (blue curve). Below each curve is the calculated IC50 value under for each condition, as well as the results of statistical comparison of the fitted IC50 values between conditions. (TIF) Figure S5 Loss of OGT expression increases sensitivity of the MDA-MB-231 tumor cell line but not the OVCAR-4 tumor cell line to the dual PI3K/mTOR inhibitor NVP- BEZ235. A, MDA-MB-231 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 10 nM NVP-BEZ235. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with NVP-BEZ235. B, OVCAR-4 cells transfected with OGT-targeting siRNA or non- silencing siRNA, in the presence or absence of 10 nM NVP- BEZ235. Cell viability was assayed at 0, 24, 48, and 72 hours post- transfection and treatment with NVP-BEZ235. All above data PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e46518