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- (#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 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/ 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 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. (%) Kinase Inh. (%) Kinase Inh. (%) SBK1 17 ZAK 0 SgK085 0 ZAP70 4 SgK110 3 SIK 0 SIK2 0 SLK 0 SNARK 0 SRC 6 SRMS 0 SRPK1 0 SRPK2 27 SRPK3 0 STK16 0 STK33 0 STK35 0 STK36 0 STK39 2 SYK 0 TAK1 5 TAO1 2 TAOK1 0 TAOK3 10 TBK1 0 TEC 3 TESK1 0 TGFBR1 4 TGFBR2 24 TIE1 0 TIE2 0 TLK1 12 TLK2 13 TNIK 1 TNK1 10 TNK2 0 TNNI3K 0 TRKA 4 TRKB 0 TRKC 0 TSSK1B 5 TTK 7 TXK 0 TYK2(JH1) 0 TYK2(JH2) 12 TYRO3 0 ULK1 0 ULK2 0 ULK3 17 VEGFR2 0 2 WEE2 0 YANK2 11 YANK3 0 YES 0 YSK1 0 YSK4 21 The data set is shown in full. No entries have been removed. 7 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor

Supplementary Figure legends

Sup Figure 1: BKM120 activities on CSF1R, FGFR2 and EphA2 autophosphorylation assays. A. HEK293 cells that stably express CSF1R were plated in 96 well plates. 24h later the cells were incubated with a positive control compound NVP-BLZ945 (selective and potent

CSF1R inhibitor) and NVP-BKM120 at indicated concentrations for 90 min and stimulated with M-CSF for up to 10 min. The reduction of MCSF- induced CSF1R phosphorylation in the presence of compound is shown (with ± MCSF defining respectively 0 and 100% of signal, in the absence of compound). B. HEK293 cells were plated in 96-well clusters and transiently transfected with a FGFR2 expression vector. Twenty-four hours late, cells were then exposed for 40 min to increasing concentrations of BKM120. Cells were then lyzed and cell extracts subjected to a sandwich ELISA assay for the detection and quantification of phospho-tyrosine FGFR2 levels. Data are shown as percentage of untreated controls. C.

HEK293 cells were transfected (lanes 2-13) or not (lane 1) with an expression vector encoding the full-length form of EphA2. Forty-eight hours later, cells were washed and exposed or not (lanes 2, 6 and 10) for 1 h to the indicated concentrations of BKM120, in the absence (lanes 2-5) or presence of either EphA2-Fc agonist (lanes 6-9) or ortho-vanadate

(lanes 10-13). Cells were then lyzed, and cell extracts analyzed by western blot to detect

EphA2 phosphotyrosine levels, using the 4G10 antibody.

Sup Figure 2: Establishment of Rat1-myr-p110 mechanistic cell based models. Rat-1 cells were infected with various dilutions of retroviral supernatant containing the human PI3K class

I isoforms (p110α, p110β, p110δ) and subjected to puromycin selection. Pools of cells were

8 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor screened for the expression of the respective PI3K class I isoforms by Western-blot analysis with anti-HA antibodies. Phospho-Akt as well as total Akt levels were also characterized by

Western blot analysis. Pools with highest expression of the respective PI3K class I isoforms and the highest level of phospho-Akt shown here, were selected for NVP-BKM120 profiling studies (Figure 2A). Transduction with human p110γ expressing virus didn’t produced effective Akt activation, precluding the establishment of a Rat1-p110γ cell based assay.

Sup Figure 3: NVP-BKM120 inhibits the PI3K pathway, in a reversible manner. A. U87MG cells were treated with 1250 nM of NVP-BKM12 for 30 min, and then washed twice before addition of fresh medium in the absence of the compound. Cells were then lyzed at different times (0.5, 1, 2, 4 and 8 hours), and cell extracts analyzed by Western-blotting for either total or phosphorylated Akt levels.

Sup Figure 4: U87MG (A, C and D), or HT-29 (B) cells were first starved for 16 h and pre- treated for 30 min (A, B and C) with the indicated compounds and concentrations (1250 nM for B and C) of NVP-BKM120 or incubated for 24 h in presence of NVP-BKM120 and doxorubicin (D). Cells were then stimulated either with EGF (A), IL-4 (B), or anisomycin (C) for 10 min and lyzed. The cell extracts were analyzed by Western-blotting for the indicated proteins and phosphoproteins. NVP-BLJ197 is a Jak inhibitor.

Sup Figure 5: The presence of a KRAS mutation desensitizes PIK3CA mutant cells to NVP-

BKM120. A. Cell lines with different KRAS and PIK3CA genetic status were binned into four categories, PIK3CA/KRAS double mutants, PIK3CA mutant/KRAS WT, PIK3CA

9 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor

WT/KRAS mutant, and PIK3CA WT/KRAS WT. The total number of cell lines in each category was labeled on the plot as count. The percentages of growth inhibition under the treatment with 0.8 μM of NVP-BKM120 was plotted as box plots and compared between the four groups. p values were determined by Student's t test, medians of the group are labeled on each plot as the white bars. B. HCT116 cells were exposed for 48 h to the indicated amount of either NVP-BKM120 (left panel) or ZSTK474 (right panel) alone or in combination with the indicated concentration of the MEK inhibitor AZD6244. Cells were then lyzed and cell extracts were assayed by Western for the detection of PARP and cleaved PARP levels.

Sup Figure 6: PK/PD relationship and in vivo antitumor activity of BKM120 as a single agent. A. U87MG tumor bearing animals received a single dose of 30 mg/kg, p.o., of NVP-

BKM120. At the allotted times, the groups of mice (n=4) were sacrificed and blood and tissues were harvested and prepared for mass spectrometry analytics for NVP-BKM120 (right axis) and ELISA for S473P-Akt levels (left axis). B and C. U87MG (B) or MCF7 (C) cells were grown as subcutaneous (B) or breast orthotopic (C) xenografts, respectively in Harlan nude mice. Anti-tumor therapy studies (n=8) with BKM120 at the indicated dosage regimen were started when the tumors reached a size of 100 mm3 in average. The changes over time in tumor volume (left panels) and body weight (right panels) are shown. D and E. HCT116 (D) or BT-474 (E) cells were grown as subcutaneous (D) or mammary fat pad orthotopic (E) xenografts in Harlan nude mice. Anti-tumor therapy studies (n=7-8) with the indicated compounds were then started when the tumors reached a size of 100-150 mm3 in average.

NVP-BKM120 (D and E) and AZD6244 (D) were given orally once per day, whereas

10 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor

Herceptin (E) was given 3 times per week through the intra-peritoneal route (E). The changes over time in body weight are shown.

Sup Figure 7: In vivo antitumor activities of NVP-BKM120 in combination with targeted or cytotoxic agents. NCI-N87 (A), U87MG (B), or PC3M (C) cells were grown as subcutaneous xenografts in Harlan nude mice. Anti-tumor therapy studies were initiated (n=8) with the indicated compounds when the tumors reached a size of 100-150 mm3 in average. NVP-

BKM120 (A, B and C) was given orally, once per day. Herceptin (A) and Temozolomide (B) were given i.p., twice or 3 times per week, respectively. Docetaxel (C) was given i.v., once per week. The changes over time in tumor volume (upper panels) and body weights (bottom panels) are shown. The Combination Index (CI) as determined by the Clarke method (3) is: -

0.15 (A), -0.08 (B) and -0.13 (C), respectively. Negative CI values are reflective of in vivo synergism. *, p<0.05 (Holm-Sidak).

Sup Figure 8: NVP-BKM120 displays strong anti-angiogenic activities in vivo. A. Porous chambers containing PBS or VEGF (2 μg/mL) in 0.5 ml of 0.8 % w/v agar (containing heparin, 20 U/mL) were implanted subcutaneously in the flank of FVB mice. Animals were treated orally either with NVP-BKM210 at 25 and 50 mg/kg once a day or vehicle (5 ml/kg) starting 4-6 hours before implantation of the chambers and daily for 3 days. VEGF induces the growth of vascularized tissue around the chamber. The angiogenic response can be quantified by measuring the weight and Tie-2 levels of the tissue. Animals were sacrificed 4 days after the implantation. Values are mean ± SEM, n=5 to 6 per group.*, p < 0.05. B. Ratio of areas of Evans blue (TRITC/rhodamine filter) to FITC-dextran fluorescence measured in

11 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor

BN472 tumors after treatment with vehicle and BKM120 (left panel) are shown. Also shown are representative images of Evans blue (red) and FITC-dextran (green) fluorescence of treated BN472 tumors. The total number of animals per dose is given in parentheses in the graphs. Results are presented as means ± SEM. The significant level was set at p < 0.05.

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