Supplementary Information Supplementary Table 1

Supplementary information

Supplementary Table 1

Kinase Type Format N Average SD

(IC50) ALK Y Lantha 6 > 10 n.d. AURORA_A S/T Caliper 6 > 10 n.d. AXL Y Lantha 3 > 10 n.d. BTK Y Caliper 6 > 10 n.d. cABL Y Caliper 4 2.0 0.38 cABLT315 Y Caliper 6 > 10 n.d. CaMK2 S/T Caliper 3 > 10 n.d. CDK2A S/T Caliper 6 > 10 n.d. CDK4D1 S/T Caliper 3 > 10 n.d. CK1 S/T Caliper 3 > 10 n.d. cKIT Y Lantha 6 > 10 n.d. cMET Y Lantha 7 > 10 n.d. COT1 S/T Caliper 4 > 10 n.d. CSK Y Lantha 2 > 10 n.d. cSRC Y Lantha 3 > 10 n.d. EphA4 Y Lantha 6 > 10 n.d. EphB4 Y Lantha 6 > 10 n.d. ERK2 S/T Caliper 6 > 10 n.d. FAK Y Lantha 3 > 10 n.d. FGFR-1 Y Lantha 3 > 10 n.d. FGFR-2 Y Lantha 3 > 10 n.d. FGFR-3 Y Lantha 3 > 10 n.d. FGFR3K650E Y Lantha 6 > 10 n.d. FGFR-4 Y Lantha 6 > 10 n.d. FLT3 Y Lantha 3 > 10 n.d. FYN Y Lantha 3 > 10 n.d. GSK3beta S/T Caliper 6 > 10 n.d. HCK Y Lantha 3 > 10 n.d. HER1 Y Lantha 6 > 10 n.d. HER2 Y Lantha 6 > 10 n.d. HER4 Y Lantha 3 > 10 n.d. IGF1R Y Lantha 6 > 10 n.d. INS1R Y Lantha 6 > 10 n.d. IRAK4 S/T Caliper 3 > 10 n.d.

JAK1 Y Caliper 6 > 10 n.d. JAK2 Y Caliper 6 > 10 n.d. JAK3 Y Caliper 6 > 10 n.d. KDR Y Lantha 6 > 10 n.d. LCK Y Lantha 6 > 10 n.d. LYN Y Lantha 3 > 10 n.d. MK2 S/T Caliper 6 > 10 n.d. MK5 S/T Caliper 6 > 10 n.d. MNK1 S/T Caliper 2 > 10 n.d. MNK2 S/T Caliper 2 > 10 n.d. p38a S/T Caliper 6 > 10 n.d. PAK2-ATPactivated S/T Caliper 3 > 10 n.d. PDGFRa Y Lantha 6 > 10 n.d. PDK1 S/T Caliper 4 > 10 n.d. PIM2 S/T Caliper 3 > 10 n.d. PKA S/T Caliper 6 > 10 n.d. PKBa S/T Caliper 6 > 10 n.d. PKCalpha S/T Caliper 2 > 10 n.d. PKCtheta S/T Caliper 2 > 10 n.d. PKN1 S/T Caliper 2 > 10 n.d. PKN2 S/T Caliper 2 > 10 n.d. PLK1 S/T Caliper 3 > 10 n.d. RET Y Lantha 6 > 10 n.d. ROCK2 S/T Caliper 3 > 10 n.d. RON Y Lantha 3 > 10 n.d. S6K S/T Caliper 3 > 10 n.d. SYK Y Lantha 6 > 10 n.d. TYK2 Y Caliper 6 > 10 n.d. WNK1 S/T Caliper 3 > 10 n.d. YES Y Lantha 2 > 10 n.d. ZAP70 Y Lantha 3 > 10 n.d.

Supplementary Table 2

Kinase Inh. Kinase Inh. Kinase Inh. (%) (%) (%) CAMK1 (CaMK1) 14 ERBB2 (HER2) 7 NEK9 -2 CDK7/cyclin H/MNAT1 -8 ERBB4 (HER4) 9 NTRK1 (TRKA) 9 CDK9/cyclin T1 -15 FER 3 NTRK2 (TRKB) 10 CHUK (IKK alpha) -6 FES (FPS) 8 NTRK3 (TRKC) 10 DAPK1 -13 FGFR1 3 PAK2 (PAK65) 29 LRRK2 86 FGFR2 -2 PAK3 4 LRRK2 G2019S 61 FGFR3 13 PAK4 -6 PI4KB (PI4K beta) 98 FGFR3 K650E 2 PAK6 -24 PIK3C3 (hVPS34) -11 FGFR4 0 PAK7 (KIAA1264) -19

PIK3CA/PIK3R1 (p110 alpha/p85 alpha) 100 FGR 13 PASK -12

PIK3CD/PIK3R1 (p110 delta/p85 alpha) 100 FLT1 (VEGFR1) -1 PDGFRA (PDGFR alpha) 3 PIK3CG (p110 gamma) 92 FLT3 7 PDGFRA D842V 10 SPHK1 9 FLT3 D835Y 9 PDGFRA T674I 23 ABL1 78 FLT4 (VEGFR3) 14 PDGFRA V561D 10 ABL1 E255K 54 FRAP1 (mTOR) 61 PDGFRB (PDGFR beta) 5 ABL1 G250E 27 FRK (PTK5) 12 PDK1 6 ABL1 T315I 1 FYN 2 PHKG1 13 ABL1 Y253F 20 GRK4 12 PHKG2 -15 ABL2 (Arg) 39 GRK5 -2 PIM1 -12 ACVR1B (ALK4) -2 GRK6 9 PIM2 -10 ADRBK1 (GRK2) 1 GRK7 6 PKN1 (PRK1) 8 ADRBK2 (GRK3) -2 GSK3A (GSK3 alpha) 1 PLK1 15 AKT1 (PKB alpha) -4 GSK3B (GSK3 beta) 1 PLK2 6 AKT2 (PKB beta) -8 HCK 5 PLK3 4 AKT3 (PKB gamma) 5 HIPK1 (Myak) 12 PRKACA (PKA) 2 ALK -5 HIPK2 4 PRKCA (PKC alpha) -5 AMPK A1/B1/G1 9 HIPK4 64 PRKCB1 (PKC beta I) -7 AMPK A2/B1/G1 15 IGF1R 5 PRKCB2 (PKC beta II) -18 AURKA (Aurora A) 4 IKBKB (IKK beta) 4 PRKCD (PKC delta) 3 AURKB (Aurora B) 9 IKBKE (IKK epsilon) -4 PRKCE (PKC epsilon) -10 AURKC (Aurora C) 11 INSR 9 PRKCG (PKC gamma) -10 AXL 2 INSRR (IRR) 3 PRKCH (PKC eta) -15 BLK 6 IRAK4 9 PRKCI (PKC iota) 4 BMX 11 ITK 6 PRKCN (PKD3) 5 BRAF 12 JAK1 -3 PRKCQ (PKC theta) 4 BRAF V599E 45 JAK2 -9 PRKCZ (PKC zeta) -7

Kinase Inh. Kinase Inh. Kinase Inh. (%) (%) (%) BRSK1 (SAD1) 10 JAK2 JH1 JH2 -7 PRKD1 (PKC mu) -1 BTK 10 JAK2 JH1 JH2 V617F -6 PRKD2 (PKD2) 8 CAMK1D (CaMKI delta) -4 JAK3 0 PRKG1 2 CAMK2A(CaMKII alpha) -2 KDR (VEGFR2) 13 PRKG2 (PKG2) 15 CAMK2B (CaMKII beta) 0 KIT 11 PRKX 10 CAMK2D (CaMKII delta) 6 KIT T670I 25 PTK2 (FAK) 3 CAMK4 (CaMKIV) 6 LCK 8 PTK2B (FAK2) 8 CDC42 BPA (MRCKA) 3 LTK (TYK1) 4 PTK6 (Brk) 11 CDC42 BPB (MRCKB) 13 LYN A -7 RAF1Y340D Y341D 47 CDK1/cyclin B 15 LYN B 0 RET -1 CDK2/cyclin A 8 MAP2K1 (MEK1) 18 RET V804L 0 CDK5/p25 5 MAP2K2 (MEK2) -2 RET Y791F -9 CDK5/p35 2 MAP2K6 (MKK6) 2 ROCK1 -7 CHEK1 (CHK1) 16 MAP3K8 (COT) 12 ROCK2 16 CHEK2 (CHK2) 0 MAP3K9 (MLK1) 0 ROS1 9 CLK1 10 MAP4K2 (GCK) -2 RPS6KA1 (RSK1) -3 CLK2 91 MAP4K4 (HGK) 20 RPS6KA2 (RSK3) 13 CLK3 62 MAP4K5 (KHS1) -14 RPS6KA3 (RSK2) 7 CSF1R (FMS) 13 MAPK1 (ERK2) 8 RPS6KA4 (MSK2) 0 CSK -5 MAPK10 (JNK3) -13 RPS6KA5 (MSK1) 5 CSNK1A1 (CK1 alpha 1) 11 MAPK11 (p38 beta) -3 RPS6KA6 (RSK4) -8 CSNK1D (CK1 delta) 26 MAPK12 (p38 gamma) 6 RPS6KB1 (p70S6K) 6 CSNK1E (CK1 epsilon) 7 MAPK13 (p38 delta) 3 SGK (SGK1) -2 CSNK1G1(CK1gamma1) 4 MAPK14 (p38 alpha) 25 SGK2 -2 CSNK1G2(CK1gamma2) 2 MAPK14 (p38 alpha) Direct 0 SGKL (SGK3) 11 CSNK1G3(CK1gamma3) 9 MAPK3 (ERK1) 14 SNF1LK2 15 CSNK2A1 (CK2 alpha 1) -4 MAPK8 (JNK1) 38 SRC 5 CSNK2A2 (CK2 alpha 2) 15 MAPK9 (JNK2) 15 SRC N1 -5 DAPK3 (ZIPK) 8 MAPKAPK2 3 SRMS (Srm) 3 DCAMKL2 (DCK2) 6 MAPKAPK3 14 SRPK1 5 DYRK1A 28 MAPKAPK5 (PRAK) 4 SRPK2 3 DYRK1B 22 MARK1 (MARK) 10 STK22B (TSSK2) -3 DYRK3 39 MARK2 2 STK22D (TSSK1) -5 DYRK4 5 MARK3 21 STK23 (MSSK1) 15 EEF2K 6 MARK4 11 STK24 (MST3) 14 EGFR (ErbB1) 5 MATK (HYL) 2 STK25 (YSK1) -4 EGFR (ErbB1) L858R 6 MELK -2 STK3 (MST2) -11 EGFR (ErbB1) L861Q 3 MERTK (cMER) 11 STK4 (MST1) -7 EGFR (ErbB1) T790M 6 MET (cMet) 5 SYK -1 EGFR T790M L858R 4 MET M1250T 9 TAOK2 (TAO1) -3

Kinase Inh. Kinase Inh. Kinase Inh. (%) (%) (%) EPHA1 6 MINK1 -8 TBK1 5 EPHA2 15 MST1R (RON) 7 TEK (Tie2) -1 EPHA3 5 MST4 -15 TYK2 2 EPHA4 0 MUSK -3 TYRO3 (RSE) 1 EPHA5 12 MYLK2 (skMLCK) 9 YES1 4 EPHA8 2 NEK1 43 ZAP70 11 EPHB1 -5 NEK2 -18 EPHB2 4 NEK4 -2 EPHB3 -2 NEK6 3 8 EPHB4 6 NEK7

Supplementary Table 3

Kinase target NVP-BYL719 (% Control) AAK1 10 CLK1 7.7 CLK2 2.3 GAK 5.6 HIPK4 6.8 PIK3C2B 4 PIK3CA 0.05 PIK3CA (C420R) 0 PIK3CA (E542K) 0 PIK3CA (E545K) 0 PIK3CA (H1047L) 2.4 PIK3CA (H1047Y) 0.6 PIK3CA (I800L) 0.4 PIK3CA (M1043I) 0 PIK3CA (Q546Q) 0 PIK3CD 5.9 PIK3CG 0 PIK4CB 0

Supplementary Table 4

Kinase target NVP-BYL719

Kd (nmol/L)

AAK1 2‘800

CLK1 900

CLK2 1‘600

GAK 1‘100

HIPK4 540

PIK3C2B 510

PIK3CA 2.3

PIK3CB 2‘000

PIK3CD 510

PIK3CG 8

PIK4CB 120

Supplementary Table 5

Tumor xenografts Animal Dose Treatment T/C S473P-Akt PIK3CA

species (mg/kg) duration (%) IC80 (μmol/L) Genetic

(days) alterations

A2780 mouse 50 21 0.34 n.d. WT

A375 mouse 50 12 0.37 Not achieved WT

BT474 mouse 12.5 14 0.31 15 MUT

BT474 mouse 25 14 0.17 15 MUT

BT474 mouse 50 14 0.04 15 MUT

Colo 205 mouse 50 13 0.07 Not achieved WT

DU145 mouse 50 21 0.15 13 WT

EBC1 mouse 50 32 0.42 n.d. WT

HCC1954 mouse 50 21 0.11 8 MUT

HCT116 mouse 50 21 0.38 Not achieved MUT

HGC-27 mouse 50 14 0.03 3 MUT

KYSE-70 mouse 50 27 0.03 8 AMP

MCF-7 mouse 50 23 0.01 22 MUT

Mia PaCa-2 mouse 50 21 0.60 n.d. WT

NCI-H596 mouse 50 18 -0.06 10 MUT

NCI-H596 rat 20 12 0.25 10 MUT

NCI-H596 rat 40 14 -0.35 10 MUT

NCI-H596 rat 80 10 -0.80 10 MUT

NCI-N87 mouse 50 14 -0.01 15 WT

PANC-1 mouse 50 21 0.55 n.d. WT

PC3 mouse 50 21 0.67 n.d. WT

Rat1-myr-p110α rat 6.25 13 0.22 4 WT

Rat1-myr-p110α rat 12.5 13 0.05 4 WT

Rat1-myr-p110α rat 25 13 -0.35 4 WT

Rat1-myr-p110α rat 30 14 -0.60 4 WT

Rat1-myr-p110α rat 50 14 -0.80 4 WT

RT112 mouse 50 7 -0.01 n.d. WT

SJSA1 mouse 50 14 0.49 Not achieved WT

SKOV3 mouse 50 27 -0.24 n.d. MUT

SW48 mouse 50 16 0.28 Not achieved WT

SW620 mouse 50 21 0.70 Not achieved WT

T47D mouse 50 20 -0.62 8 MUT, AMP

TT mouse 50 21 0.23 Not achieved WT

U87MG-luc mouse 50 14 0.52 Not achieved WT

UACC812 mouse 50 22 0.08 n.d. WT

Supplementary Table 6

Amax EC PIK3CA Cell Line Primary Site 50 (%) (μmol/L) Genetic alterations 697 hem -56.94 1.52 WT 22Rv1 prostate -43.28 0.15 WT 639-V urinary tract -33.26 1.31 WT 769-P kidney -38.20 0.94 WT A-204 soft tissue -50.92 0.34 WT A2780 ovary -75.83 0.37 WT AN3 CA endometrium -46.57 2.37 WT AU565 breast -64.11 2.13 WT BDCM hem -76.31 2.35 WT CAL 27 upper aerodigestive tract -64.59 2.54 WT CHP-212 autonomic ganglia -34.30 0.002 WT CMK-86 hem -36.74 0.21 WT COLO 205 large intestine -48.67 1.96 WT COV504 ovary -36.13 0.49 WT Detroit 562 upper aerodigestive tract -65.21 1.02 MUT EFM-19 breast -81.76 0.32 MUT EM-2 hem -48.07 1.48 WT F-36P hem -55.70 0.59 WT FaDu upper aerodigestive tract -32.54 2.12 WT FU-OV-1 ovary -51.45 0.29 WT HARA lung -45.33 1.86 AMP HCC-15 lung -38.95 2.14 WT HCC1954 breast -85.76 2.03 MUT HCC-56 large intestine -37.38 1.52 WT HCT-15 large intestine -34.64 0.51 MUT HDQ-P1 breast -82.22 0.41 WT HEC-151 endometrium -56.66 2.33 MUT HGC-27 stomach -83.96 0.49 MUT HMC-1-8 breast -59.31 1.63 WT Hs 578T breast -40.32 2.67 WT Hs 746T stomach -40.46 1.64 WT Hs 852.T skin -39.10 0.64 WT HSC-2 upper aerodigestive tract -54.21 2.53 MUT HT-1080 soft tissue -30.28 2.25 WT HT-1197 urinary tract -40.75 0.16 MUT huH-1 liver -60.75 1.53 WT HUP-T4 pancreas -70.05 2.98 WT HuT 78 hem -61.29 1.07 WT IGROV1 ovary -73.33 0.52 WT IST-MES1 pleura -35.59 0.86 WT

JHH-5 liver -40.29 1.17 WT JHH-7 liver -40.51 1.67 WT JHUEM-2 endometrium -56.95 1.23 WT JMSU-1 urinary tract -68.43 1.31 WT KARPAS-620 hem -68.73 1.95 WT KASUMI-2 hem -61.27 0.84 WT KCL-22 hem -30.64 0.81 MUT KHM-1B hem -79.36 0.54 WT KMM-1 hem -56.64 1.67 WT KMS-11 hem -57.01 0.25 WT KMS-12-BM hem -68.36 2.12 WT KMS-34 hem -73.93 0.23 WT KP4 pancreas -41.00 2.67 WT KP-N-SI9s autonomic ganglia -54.24 0.61 WT KYSE-510 oesophagus -31.73 1.08 MUT KYSE-520 oesophagus -40.38 2.97 WT L3.3 pancreas -51.36 2.84 WT L-363 hem -82.16 0.13 MUT LC-1/sq-SF lung -36.03 2.09 WT LOX IMVI skin -32.19 2.92 WT LP-1 hem -56.62 0.002 WT LUDLU-1 lung -67.17 1.52 WT Malme-3M skin -35.13 1.16 WT MB 157 breast -31.35 0.37 WT MCAS ovary -36.51 2.64 MUT MCF7 breast -119.39 0.22 MUT MDA-MB-175- breast -64.68 2.59 WT VII MDA-MB-453 breast -81.22 0.45 MUT MEC-1 hem -55.50 0.55 WT MES-SA soft tissue -39.36 2.34 WT MFE-280 endometrium -78.97 1.81 MUT MHH-ES-1 bone -79.19 3.01 WT Mino hem -75.53 1.44 WT MOLP-8 hem -60.05 1.02 WT MOLT-16 hem -77.80 2.55 WT NCI-H1048 lung -79.81 0.20 MUT NCI-H1339 lung -36.52 2.29 WT NCI-H1341 lung -77.33 0.36 MUT NCI-H1648 lung -58.70 0.08 WT NCI-H1703 lung -48.08 0.79 WT NCI-H2087 lung -34.82 2.89 WT NCI-H2228 lung -52.25 0.95 WT NCI-H28 pleura -42.05 2.46 WT NCI-H322 lung -32.86 1.16 WT

NCI-H3255 lung -46.87 1.16 AMP NCI-H441 lung -35.16 0.19 WT NCI-H747 large intestine -55.61 2.01 WT NCI-N87 stomach -67.77 0.73 WT OCI-AML5 hem -49.92 2.38 WT OE21 oesophagus -69.98 2.59 WT ONCO-DG-1 ovary -44.70 2.31 WT OVMANA ovary -36.80 0.76 WT Panc 02.03 pancreas -58.16 1.29 WT Panc 04.03 pancreas -40.01 0.89 WT Panc 10.05 pancreas -37.78 0.19 WT PA-TU-8902 pancreas -37.17 2.97 WT Pfeiffer hem -56.16 0.99 WT PK-45H pancreas -36.52 1.05 WT Reh hem -52.95 2.99 WT RERF-GC-1B stomach -49.48 1.73 WT RERF-LC-MS lung -40.52 2.92 WT RKN soft tissue -37.53 1.18 WT SCC-25 upper aerodigestive tract -80.85 2.07 WT SIG-M5 hem -41.48 0.24 WT SJRH30 soft tissue -56.48 2.81 WT SK-ES-1 bone -51.75 0.54 WT SK-LU-1 lung -39.04 1.81 WT SK-MES-1 lung -62.58 0.002 WT SK-N-SH autonomic ganglia -44.63 1.62 WT SK-OV-3 ovary -59.16 1.19 MUT SU-DHL-4 hem -47.98 2.68 WT SW403 large intestine -68.28 1.46 MUT SW48 large intestine -66.48 0.002 WT SW480 large intestine -33.80 0.33 WT SW620 large intestine -37.20 0.36 WT T-47D breast -67.55 1.20 MUT, AMP TCCSUP urinary tract -45.45 1.69 MUT TE-11 oesophagus -35.52 2.94 WT TE-5 oesophagus -38.52 2.20 MUT TEN endometrium -40.40 0.77 WT UACC-812 breast -52.78 2.41 WT WM-983B skin -36.63 1.52 WT

Supplementary Table 7

Tumor volume change PDX PIK3CA (% relative to time 0) at last models Lineage Genetic alterations day of treatment PDX 1 Breast Mutation H1047R 70 PDX 2 Breast Mutation H1047R 6 PDX 3 Lung Mutation E545K -5 PDX 4 Lung Mutation E545K/Amplification -18 PDX 5 Lung Mutation M1043I -23 PDX 6 Breast Mutation C420R -36 PDX 7 CRC Mutation Q546K -38 PDX 8 CRC Mutation Q546K -53 PDX 9 Gastric Amplification -80

Supplementary Figure legends

Supplementary Figure 1: NVP-BYL719 was tested at 10 different concentrations (from 0.5 nmol/L to 10 μmol/L in 1 third dilution steps) against CLK2 (A) and LRRK2 (B). SelectScreenTM

Kinase Profiling Service uses XLfit from IDBS to determine the IC50 values (IC50 CLK2=621 nmol/L; IC50 LRRK2=3’220 nmol/L). The dose response curve is curve fit to model number 205

(sigmoidal dose-response model).

Supplementary Figure 2

Rat1-myr-p110α (blue), β (green) or δ (red) cells were treated with increasing concentrations of

NVP-BYL719 for 30 minutes. Levels of S473P-Akt in cell extracts were quantified by Reverse

Phase Protein Array as described in (18) and plotted as percentage of untreated control cells.

The graph illustrates n=3 independent experiments. IC50s ± SD and IC80s ± SD of n=3 independent experiments are reported.

Supplementary Figure 3

NVP-BYL719 does not inhibit mTOR and PIKKs involved in DNA damage-repair processes. A.

TSC1 -/- MEFs cells were grown in a 96-well format and treated for 1 h with increased concentrations of RAD001 or NVP-BYL719 (from 0.5 nmol/L to 10 µmol/L in 1 third dilution steps) and immediately fixed. S235/236P-RPS6 levels were measured and IC50 determined with the Excel module XLfit. Background (no primary Ab incubated); BL, Baseline. B: TSC1 -/- MEFs cells were treated with increasing concentrations of NVP-BYL719 as indicated or RAD001 at

500 nmol/L or an equivalent DMSO concentration for 30 minutes. Levels of S235/236P-RPS6 and total RPS6 in protein- normalized lysates were detected by Western blot analyzis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. C: 24 h post seeding, A549 cells were treated at the same time with Actinomycin D (Act D) at a concentration of 5 µmol/L (an agent used to induce DNA damage), and with increasing concentrations of NVP-BYL719 as indicated or with the vehicle control (DMSO) for 1 h. Levels of S15P-p53 and tubulin in protein-normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. D: 24 h post seeding, U2OS cells were pre-treated for 1 h with increased concentrations of NVP-BYL719 or KU55933 a specific small molecular mass inhibitor of ATM

(Supplementary reference 1) at a concentration of 10 µmol/L or with the vehicle control (DMSO).

The cells were then irradiated with 15 Gy and re-incubated at 37°C for 1 h and then lysed.

Levels of S1981P-ATM in protein- normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP- labeled secondary antibody and signal development by ECL.

Supplementary Figure 4

Linear correlation observed between tumor growth inhibition (% T/C) or tumor regression and the fraction of time over the in vivo S473P-Akt IC80 in different cancer cell line-derived tumor xenografts implanted in nude mice (represented as dots) and nude rats (represented as triangles) following NVP-BYL719 treatment (R2=0.77, p<0.001, n=27).

Supplementary Table Legends

Supplementary Table 1 Determination of NVP-BYL719 in vitro effects on human protein kinases. The kinase selectivity profile of NVP-BYL719 was examined in in vitro kinase assays using 65 recombinant human protein kinases (38 tyrosine and 27 serine/threonine-specific protein kinases). In vitro assays were conducted with the indicated recombinant protein kinases in the reported assay format in the presence of increasing concentrations of NVP-BYL719 as described in (18). Averages of IC50 values in µmol/L ± one SD are shown. N = Number of individual experiments, Y = Tyrosine-specific protein kinase, S/T = Serine/Threonine-specific protein kinase. The compound was inactive in 64 out of 65 kinase assays (IC50 >10 µmol/L) with only weak inhibitory activity found for cABL (IC50 = 2 μmol/L). In cellular assays however, NVP-

BYL719 showed no dose-dependent effect on proliferation and viability of BaF3-Bcr-ABL cells

(data not shown).

Supplementary Table 2 Activity profile of NVP-BYL719 in the Invitrogen Kinase panel.

NVP-BYL719 was tested against 254 kinases at a concentration of 10 µmol/L. Among all the kinases tested (excluding class I PI3K, PI4Kβ) only CLK2 and LRRK2 were inhibited by more than 80%. All kinases were tested at apparent Km [ATP], except for CAMK1, which was tested at 10 µmol/L and for BRAF, MEK1, MEK2, MKK6, COT, JNK1, JNK2, JNK3, PDK1 and cRAF,

14 which were all tested at 100 µmol/L. The % inhibition value represents the mean of two measurements.

Supplementary Table 3 Activity profile of NVP-BYL719 in the Ambit Kinase panel.

Ambit panel results of NVP-BYL719: hits found to be inhibited >90%. NVP-BYL719 was tested against 442 kinases (Ambit Inc.) at a concentration of 10 µmol/L. Among all the kinases tested

(excluding class I PI3K, PI4Kβ) only 6 kinases were inhibited by more than 90% compared to controls.

Supplementary Table 4 NVP-BYL719 Kd (nmol/L) determination in the Ambit Kinase panel for the hits found to be inhibited >90%.

Supplementary Table 5 NVP-BYL719 anti-tumor effects in diverse cancer cell line-derived xenograft models.

n.d: not determined; MUT: Mutation; AMP: Amplification (copy number >4); WT: Wild type.

Supplementary Table 6 CCLE cell lines responsive to NVP-BYL719

EC50 ≤ 3.04 µmol/L and Amax ≤ - 30%. MUT: Mutation; AMP: Amplification (copy number >4);

WT: Wild type.

Supplementary Table 7 Anti-tumor effect of NVP-BYL719 in patient-derived xenograft models carrying PIK3CA genetic alterations. Efficacy is reported as percentage change in tumor volume at last day of treatment relative to day 0 (start of treatment).

Supplementary References

1. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, Orr AI et al. Identification and

characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated

kinase ATM. Cancer Res. 2004; 64(24):9152-9.