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1 Supplementary Materials for Pathway-Based Identification of Biomarkers for Targeted Therapeutics: Personalized Oncology with PI3K Pathway Inhibitors Jannik N. Andersen, Sriram Sathyanarayanan, Alessandra Di Bacco, An Chi, Theresa Zhang, Albert H. Chen, Brian Dolinski, Manfred Kraus, Brian Roberts, William Arthur, Rich A. Klinghoffer, Diana Gargano, Lixia Li, Igor Feldman, Bethany Lynch, John Rush, Ronald C. Hendrickson,* Peter Blume-Jensen,* Cloud P. Paweletz *To whom correspondence should be addressed. (R.C.H.); (P.B.-J.) This PDF file includes: Published 4 August 2010, Sci. Transl. Med. 2, 43ra55 (2010) DOI: /scitranslmed Methods Fig. S1. Correlation of basal phospho-erk Thr202/Tyr204 concentrations versus sensitivity to Akt inhibition. Fig. S2. Immunohistochemical stability of phospho-akt Ser474 and phospho- PRAS40 Thr246. Fig. S3. Correlation analysis of phospho-pras40 Thr246 with PI3K-pathway mutation status in breast cancer cell lines. Tables S1 and S2 legend. Table S3. Key information on breast cancer cell line panel. Table S4. Key information on lung cancer cell line panel. Other Supplementary Material for this manuscript includes the following: (available at Table S1. Summary of identified peptides containing the [RXX(s/t)], [PX(s/t)P], and [(F/K)XX(F/Y)(s/t)(F/Y)] motif (Microsoft Excel format). Table S2. Gene ontology summary for 377 identified phosphoproteins (Microsoft Excel format).

2 Supplementary Methods: Reverse Phase Protein Microarray (RPPA) Reverse Phase Protein Microarrays were manufactured as previously described (Paweletz et al., 2001). Briefly, 20 nl of denatured protein lysates (10 mm Tris, 100 mm NaCl, 1 mm EDTA, 20 mm Na 4 P 2 O 7, 1% TX-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 2mM Na 3 VO 4, 1 mm PMSF) were immobilized onto nitrocellulose-coated glass slides (Amersham) using an Auchon 2407 microarrayer in 2-fold dilution curves. Arrays were blocked for 2 hours at room temperature with Casein containing 0.1% Tween-20, followed by blocking of endogenous biotin with avidin (Dako Systems), incubation with primary antibody (1:1000), and finally, incubation with biotinylated secondary antibody (1:5000). Arrays were developed with 3,3 diaminobenzidine terahydrochloride chromogen. Integrated values for each spot were obtained by ImageQuant 3.0. Each assay was performed twice (technical replicas), and on each run two independent measurements for every phosphorylated protein were obtained (biological replicas). The data then was normalized using the double-z score and expression level of a house-keeping gene (GAPDH) followed by averaging across the technical and biological replicas.

3 5 4 Pearson: R = 0.33, P=6e- Spearman: R = 0.36, P=3e- Kendall: R = 0.26, P=2e- 3 2 p-erk AKTi Sensitivity [X/Xo] 1.4 Supplementary Figure 1. Correlation of basal phospho-erk Thr202/Tyr204 concentrations (Y-axis) versus sensitivity to AKT inhibition (X-axis). Lower numbers on the X-axis indicate increasing sensitivity to the AKT inhibitor. Drug sensitivity was determined using Cell Titer Glo (Promega, Madison, WI) at 24, 48 and 72 hours post-inhibitor treatment. Normalized drug sensitivity values [X/X 0 ] were calculated to correct for the differences in basal growth rates (i.e. doubling times) across the cell line panel. All values, including the drug effective concentrations (EC 50 ) at 72 hours post-dose are provided in Table S3.

4 p-akt Ser473 p-pras Thr246 0 hour 0.5 hour 1.0 hour Time to fixation 4.0 hour Supplementary Figure 2. IHC stability of phospho-akt Ser474 and phospho- PRAS40 Thr246. Epitope stability was assessed using GTL-16 tumors fixed at different time points. Consecutive tumor sections were stained with phospho- AKT Ser473 and phospho-pras40 T246. phospho-akt signal is lost as early as 30 minutes, while phosphopras40 is stable and can still be detected in tumors whose fixation was delayed for 1 or more hours.

5 Phospho-Protein Pathway Mutations HCC70 OCUB-F MDA-MB-175-V HCC1395 VP267 T-47D OCUB-M MFM-223 MT-3 BT20 EFM-192C HCC1806 MDA-MB-468 MDA-MB-415 EVSA-T MCF-12A CAL-51 HCC1954 UAAC-893 BT-549 HCC38 HCC1143 HCC1187 MDA-MB-453 KPL-1 MDA-MB-435S ZR-75-1 MDA-MB-361 HMT-3522 S1 HCC1419 EFM-192B CAL-120 MCF-10-2A FR2 BT-483 NM2C5 CAL-148 MCF7 COLO-824 HDQ-P1 HCC2218 SUM1315MO2 CAL85-1 M4A4 HCC1569 AU565 MDA-MB-231 HCC1500 HMT-3522-T4-2 MDA-MB-157 HCC1428 MB 157 MDA-MB-134-V p-akt Ser473 p-pras40 Thr40 PIK3CA mut PTEN mut Supplementary Figure 3. Correlation analysis of phospho-pras40 Thr246 with PI3K-pathway mutation status in breast cancer cell lines. Heatmap of phospho-pras40 Thr246 and phospho-akt Ser473 RPPA values in context of mutations in PTEN and PIK3CA. The phosphorylation level of PRAS40 Thr246 is ranked from high (red) to low (blue) and RPPA values are calculated relative to the mean of all breast cancer cell lines in the experiment. Mutations were retrieved from the Sanger Cancer Cell line Project: (

6 Supplementary Table 1. Summary of identified peptides containing the [RXX(s/t)], [PX(s/t)P], and [(F/K)XX(F/Y)(s/t)(F/Y)] motif (in separate Excel format). Supplementary Table 2. Gene ontology summary for 377 identified phosphoproteins (in separate Excel format).

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8 Supplementary Table 3. Key information on breast cancer cell line panel. RPPA values were normalized using the double-z score and expression level of a house-keeping gene (GAPDH) followed by averaging across the technical and biological replicas. IC50 values were obtained as described in Materials and Methods.

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11 Supplementary Table 4. Key information on lung cancer cell line panel. RPPA values were normalized using the double-z score and expression level of a house-keeping gene (GAPDH) followed by averaging across the technical and biological replicas. The RPPA values are relative to the mean of the 67 breast cancer cell lines in the study.

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