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Supplementary Information Material and Methods MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Supplementary Information Material and methods Chemicals The EGFR inhibitor NVP-AEE788 (Novartis), the Jak inhibitor I (Merck Calbiochem, #420099) and anisomycin (Alomone labs, # A-520) were prepared as 50 mM stock solutions in 100% DMSO. Doxorubicin (Adriablastin, Pfizer), EGF (Sigma Ref: E9644), PDGF (Sigma, Ref: P4306) and IL-4 (Sigma, Ref: I-4269) stock solutions were prepared as recommended by the manufacturer. For in vivo administration: Temodal (20 mg Temozolomide capsules, Essex Chemie AG, Luzern) was dissolved in 4 mL KZI/glucose (20/80, vol/vol); Taxotere was bought as 40 mg/mL solution (Sanofi Aventis, France), and prepared in KZI/glucose. Antibodies The primary antibodies used were as follows: anti-S473P-Akt (#9271), anti-T308P-Akt (#9276,), anti-S9P-GSK3β (#9336), anti-T389P-p70S6K (#9205), anti-YP/TP-Erk1/2 (#9101), anti-YP/TP-p38 (#9215), anti-YP/TP-JNK1/2 (#9101), anti-Y751P-PDGFR (#3161), anti- p21Cip1/Waf1 (#2946), anti-p27Kip1 (#2552) and anti-Ser15-p53 (#9284) antibodies were from Cell Signaling Technologies; anti-Akt (#05-591), anti-T32P-FKHRL1 (#06-952) and anti- PDGFR (#06-495) antibodies were from Upstate; anti-IGF-1R (#SC-713) and anti-EGFR (#SC-03) antibodies were from Santa Cruz; anti-GSK3α/β (#44610), anti-Y641P-Stat6 (#611566), anti-S1981P-ATM (#200-301), anti-T2609 DNA-PKcs (#GTX24194) and anti- 1 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Y1316P-IGF-1R were from Bio-Source International, Becton-Dickinson, Rockland, GenTex and internal production, respectively. The 4G10 antibody was from Millipore (#05-321MG). Cell lines The MCF7 (ATCC, #HTB-22), HT-29 (ATCC, #HTB-38), PC3M (MD Anderson Cancer Center, Houston, Texas, USA), and NCI-N87 (ATCC, #CRL-5822) cell lines were cultivated as described (3,4) and were authenticated by SNP analysis. CSF1R cell based assays A clone of HEK293 cells stably transfected with human CSF1R was used in a capture immunosorbant assay to measure autophosphorylation changes. One day before the assay 15000 cells/well were seeded into Poly-D-Lysine 96 well plates (Greiner Bio-One Cat. 655940). Serial dilutions in DMSO of test compounds were added in medium to cells for 90 min and then stimulated with CSF1R ligand MCSF (R&D Systems Cat. 216-MC) for 5-10 min. After removal of supernatant, cells were lyzed and lyzates transferred to a blocked MSD (MesoScale Discovery (MSD), Gaithersburg, MD, USA) plate that was pre-spotted with an antibody to CSF1R (SC-692, Santa Cruz Biotechnology) and incubated at 4oC overnight with shaking. The plates were washed and phosphorylation of captured CSF1R was detected with 10 nM SULFO-tag PY20, anti-phosphotyrosine detection antibody (MSD Cat. 32AP) added in 1% BSA/TBS/0.02% Tween-20. After shaking for one hour at room temperature, the plates were washed again and read on a Sector 6000 instrument (MSD) in 1.5 X Read T solution (MSD Cat. R93T). FGFR2 ELISA assay 2 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor The full description of the ELISA methodology has been recently described in Guagnano et al (2). In vivo angiogenic assays The chamber implant assay has been described previously (1). Tumor vascular permeability was assessed in rats bearing orthotopic BN472 tumors of approximately 2500 mm3 in size. Animals first received the Evans blue dye (0.5%, 5 ml/kg) via the femoral vein. Thirty minutes later, the high molecular weight vascular compartment marker FITC-dextran dye (Sigma, 100 mg/kg in PBS) was injected via the vena cava with the animals under isoflurane anaesthesia. Tumors were then excised 2 min later and snap frozen and sections prepared for the visualization and quantification of fluorescence as described in legend of Sup Figure 7B. 3 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Supplementary Table 1 Activity of NVP-BKM120 in the Invitrogen kinase panel Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) ABL1 3 EPHB2 0 p38 alpha 4 PRKX 0 ABL1 E255K -4 EPHB3 -1 ERK1 7 PTK2 (FAK) 4 ABL1 G250E -4 EPHB4 -4 MAPKAPK2 -4 PTK6 (Brk) -10 ABL1 T315I 3 ErbB2 -4 MAPKAPK3 1 RET -4 ABL1 Y253F 1 EebB4 10 MAPKAPK5 -1 ROCK1 -2 ABL2 (Arg) -3 FER 1 HYL 0 ROCK2 3 GRK2 1 FES (FPS) 0 cMER 3 ROS1 27 GRK3 2 FGFR1 4 cMet 5 RSK1 5 Akt1 2 FGFR2 2 MET M1250T 1 RSK3 4 Akt2 0 FGFR3 2 MINK1 4 RSK2 2 Akt3 3 FGFR4 5 MST4 -3 MSK2 6 ALK 1 FGR 15 MUSK 5 MSK1 8 Aurora B 11 VEGFR1 2 skMLCK 8 p70S6K 8 BLK -3 FLT3 18 NEK1 8 SGK1 25 BMX 3 FLT3 D835Y 11 NEK2 7 SGK2 10 BTK 3 VEGFR3 2 TRKA 9 SGK3 20 CaMKI delta 12 PTK5 2 TRKB 9 SRC 10 CaMKII alpha 2 FYN 9 TRKC 4 SRC N1 7 CaMKII beta 9 GRK4 2 PAK3 -1 SRC N2 -1 CaMKII delta 1 GRK5 1 PAK4 -2 SRMS 13 CDK1/cyclin B 9 GRK6 4 PAK6 0 SRPK2 7 CDK2/cyclin A 2 GRK7 0 PASK -1 TSSK2 -6 CDK5/p35 -1 GSK3 alpha 5 PDGFRA 5 TSSK1 -4 CHK1 3 GSK3 beta 7 PDGFRA D842V 1 MST3 7 CHK2 17 HCK 10 PDGFRA T674I 11 YSK1 6 CLK1 32 IGF1R 1 PDGFRB 5 MST2 5 CLK2 15 IKK beta -2 PDK1 4 MST1 14 FMS (CSF1R) 85 INSR 6 PHKG1 11 Aurora A 34 CSK 5 INSRR (IRR) 3 PHKG2 3 SYK 1 CK1 alpha 1 -1 IRAK4 12 PIM1 6 TAOK2 6 CK1 delta 4 ITK 3 PIM2 3 TBK1 7 CK1 epsilon 11 JAK2 18 PKN1 (PRK1) 9 TEK 5 CK1 gamma 1 1 JAK3 7 PLK1 1 RSE 3 CK1 gamma 2 0 VEGFR2 3 PLK2 -2 YES1 12 CK1 gamma 3 -3 KIT 39 PLK3 1 CK2 alpha 1 1 KIT T670I 13 PKA 0 CK2 alpha 2 -2 LCK 7 PKC alpha 4 DAPK3 1 LYN A 6 PKC beta I 30 DYRK3 11 LYN B 3 PKC beta II 8 DYRK4 3 MEK1 4 PKC delta 4 EGFR 3 MEK2 3 PKC epsilon 2 EGFR L858R 12 MKK6 6 PKC gamma 5 EGFR L861Q 6 MLK1 2 PKC eta 4 EPHA1 -2 GCK 6 PKC iota 9 EPHA2 8 HGK 1 PKD3 8 EPHA3 -2 KHS1 10 PKC theta 5 EPHA4 9 ERK2 6 PKC zeta 2 EPHA5 0 p38 beta 4 PKC mu 5 EPHA8 3 p38 gamma 3 PKD2 2 EPHB1 3 p38 delta 2 PKG2 10 The data set is shown in full. No entries have been removed. 4 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Supplementary Table 2 Activity of NVP-BKM120 in the Ambit kinase panel Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) AAK1 0 CDC2L2 19 EGFR(S752-I759del) 0 ABL1 0 CDK11 0 EPHA1 2 ABL1(E255K) 0 CDK2 30 EPHA2 97.4 ABL1(F317I) 41 CDK3 0 EPHA3 0 ABL1(F317L) 0 CDK5 0 EPHA4 2 ABL1(H396P) 0 CDK7 0 EPHA5 0 ABL1(M351T) 0 CDK8 0 EPHA6 3 ABL1(Q252H) 0 CDK9 12 EPHA7 0 ABL1(T315I) 2 CDKL2 0 EPHA8 0 ABL1(Y253F) 0 CDKL3 0 EPHB1 0 ABL2 0 CDKL5 50 EPHB2 0 ACVR1 0 CHEK1 1 EPHB3 4 ACVR1B 3 CHEK2 0 EPHB4 4 ACVR2A 5 CIT 0 EPHB6 3 ACVR2B 0 CLK1 58 ERBB2 24 ACVRL1 38 CLK2 0 ERBB3 22 ADCK3 4 CLK3 29 ERBB4 7 ADCK4 0 CLK4 70 ERK1 1 AKT1 0 CSF1R 0 ERK2 0 AKT2 0 CSK 8 ERK3 0 AKT3 0 CSNK1A1L 0 ERK4 0 ALK 6 CSNK1D 27 ERK5 0 AMPK-alpha1 3 CSNK1E 7 ERK8 2 AMPK-alpha2 0 CSNK1G1 25 ERN1 5 ANKK1 0 CSNK1G2 0 FAK 21 ARK5 42 CSNK1G3 20 FER 0 ASK1 1 CSNK2A1 0 FES 0 ASK2 0 CSNK2A2 2 FGFR1 0 AURKA 0 CTK 28 FGFR2 98.4 AURKB 0 DAPK1 2 FGFR3 7 AURKC 0 DAPK2 13 FGFR3(G697C) 0 AXL 7 DAPK3 10 FGFR4 0 BIKE 0 DCAMKL1 7 FGR 0 BLK 0 DCAMKL2 30 FLT1 0 BMPR1A 0 DCAMKL3 51 FLT3 8 BMPR1B 0 DDR1 7 FLT3(D835H) 2 BMPR2 0 DDR2 19 FLT3(D835Y) 12 BMX 14 DLK 0 FLT3(ITD) 0 BRAF 18 DMPK 0 FLT3(K663Q) 0 BRAF(V600E) 17 DMPK2 0 FLT3(N841I) 6 BRK 0 DRAK1 0 FLT4 3 BRSK1 0 DRAK2 0 FRK 0 BRSK2 0 DYRK1A 11 FYN 0 BTK 6 DYRK1B 0 GAK 6 CAMK1 4 DYRK2 20 GCN2(Kin.Dom.2,S808G) 35 CAMK1D 0 EGFR 10 GRK1 0 CAMK1G 0 EGFR(E746-A750del) 0 GRK4 0 CAMK2A 2 EGFR(G719C) 4 GRK7 3 CAMK2B 18 EGFR(G719S) 10 GSK3A 42 CAMK2D 0 EGFR(L747-E749del, A750P 0 GSK3B 0 CAMK2G 0 EGFR(L747-S752del, P753S 1 HCK 0 CAMK4 50 EGFR(L747-T751del,Sins) 4 HIPK1 0 CAMKK1 0 EGFR(L858R) 16 HIPK2 0 CAMKK2 0 EGFR(L858R,T790M) 0 HIPK3 0 CDC2L1 10 EGFR(L861Q) 0 HIPK4 21 5 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) HPK1 0 MEK4 15 PHKG1 11 HUNK 18 MEK6 0 PHKG2 11 ICK 0 MELK 0 PIM1 2 IGF1R 0 MERTK 0 PIM2 0 IKK-alpha 29 MET 12 PIM3 5 IKK-beta 0 MET(M1250T) 20 PIP5K1A 0 IKK-epsilon 0 MET(Y1235D) 0 PIP5K2B 0 INSR 30 MINK 0 PKAC-alpha 0 INSRR 0 MKNK1 0 PKAC-beta 4 IRAK1 12 MKNK2 5 PKMYT1 0 IRAK3 0 MLCK 0 PKN1 0 ITK 0 MLK1 18 PKN2 0 JAK1(JH1) 38 MLK2 0 PLK1 30 JAK1(JH2) 0 MLK3 0 PLK2 0 JAK2(JH1) 12 MRCKA 0 PLK3 0 JAK3(JH1) 100 MRCKB 0 PLK4 1 JNK1 0 MST1 0 PRKCD 4 JNK2 0 MST1R 0 PRKCE 0 JNK3 17 MST2 0 PRKCH 0 KIT 0 MST3 5 PRKCQ 0 KIT(D816V) 3 MST4 0 PRKD1 36 KIT(L576P) 0 MUSK 0 PRKD2 38 KIT(V559D) 0 MYLK 12 PRKD3 11 KIT(V559D,T670I) 0 MYLK2 5 PRKG1 0 KIT(V559D,V654A 0MYO3A 7PRKG2 0 LATS1 4 MYO3B 1 PRKR 19 LATS2 19 NDR1 1 PRKX 0 LCK 0 NDR2 3 PRP4 4 LIMK1 0 NEK1 15 PYK2 0 LIMK2 0 NEK2 0 QSK 0 LKB1 0 NEK5 0 RAF1 7 LOK 4 NEK6 0 RET 8 LTK 0 NEK7 0 RET(M918T) 13 LYN 0 NEK9 0 RET(V804L) 12 LZK 0 NIM1 0 RET(V804M) 14 MAK 0 NLK 0 RIOK1 11 MAP3K1 0 OSR1 17 RIOK2 0 MAP3K15 0 p38-alpha 0 RIOK3 0 MAP3K2 0 p38-beta 0 RIPK1 0 MAP3K3 0 p38-delta 0 RIPK2 0 MAP3K4 1 p38-gamma 17 RIPK4 0 MAP4K2 17 PAK1 4 ROCK1 0 MAP4K3 0 PAK2 17 ROCK2 0 MAP4K4 9 PAK3 0 ROS1 0 MAP4K5 0 PAK4 13 RPS6KA1(Kin.Dom.1-N-term 0 MAPKAPK2 0 PAK6 16 RPS6KA1(Kin.Dom.2-C-term 23 MAPKAPK5 0 PAK7 0 RPS6KA2(Kin.Dom.1-N-term 2 MARK1 0 PCTK1 0 RPS6KA2(Kin.Dom.2-C-term 16 MARK2 0 PCTK2 26 RPS6KA3(Kin.Dom.1-N-term 0 MARK3 0 PCTK3 34 RPS6KA4(Kin.Dom.1-N-term 4 MARK4 0 PDGFRA 11 RPS6KA4(Kin.Dom.2-C-term 0 MAST1 0 PDGFRB 0 RPS6KA5(Kin.Dom.1-N-term 0 MEK1 0 PDPK1 0 RPS6KA5(Kin.Dom.2-C-term 50 MEK2 0 PFTAIRE2 0 RPS6KA6(Kin.Dom.1-N-term 0 MEK3 0 PFTK1 27 RPS6KA6(Kin.Dom.2-C-term 18 6 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Kinase Inh.
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